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V olum e 1
E m b ry o n a l S tem Cells: In tro d u c in g P la n n e d C h a n g e d in to th e A nim al G erm lin e
M artin L. H o o p e r
V olum e 2
M o lecu lar G en etics o f In h e rite d Eye D isorders
E d ite d by A lan F. W rig h t a n d B arrie Jay
A d itio n al v o lu m es in p re p a ra tio n
V olum e 3
M o lecu lar G en etics o f D ru g R esistance
e d ite d by C. R o lan d W olf a n d J o h n D. Hayes
This book is part o f a series. The publisher will accept continuation orders which may be cancelled at
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MOLECULAR GENETICS
edited by
Alan F. Wright
M RC Human Genetics Unit, Western General Hospital
Crewe Road, Edinburgh EH4 2XU, UK
Barrie Jay
Emeritus Professor o f Clinical Ophthalmology,
University o f London, UK
informa
healthcare
CRC Press
Taylor & Francis Group
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Boca Raton, FL 33487-2742
1994 by Taylor & Francis Group, LLC
CRC Press is an imprint of Taylor & Francis Group, an Informa business
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CONTENTS
Preface ix
C o n trib u to rs xi
(1) INVERTEBRATES
(2) VERTEBRATES
A. C H O R O ID AND RETINA
V
vi CONTENTS
12 C h o ro id e re m ia 303
F. P. M. Cremers and H.-H. Ropers
B. V IT R E O U S /L E N S
C. A N T E R IO R SEGM ENT
19 A n irid ia 445
I. H anson, T. Jordan and V. van Heyningen
D. M ETABOLIC
GLOSSARY 491
IN D EX 515
PREFACE T O THE SERIES
I I .J . Evans
PREFACE
T h e E ditors
IX
H E R E D IT Y
T h o m a s H ard y (1917)
To su p p o se th a t th e eye, w ith all its in im ita b le con triv an ces fo r ad ju stin g th e focus
to d iffe re n t distan ces, fo r a d m ittin g d iffe re n t a m o u n ts o f light, a n d fo r th e c o rre c
tio n o f sp h erical a n d c h ro m a tic a b e rra tio n , co u ld have b e e n fo rm e d by n a tu ra l se
le c tio n , seem s, I freely confess, a b su rd in th e h ig h est d e g re e. Yet rea so n tells m e,
th a t if n u m e ro u s g ra d a tio n s fro m a p e rfe c t a n d c o m p lex eye to o n e very im p e rfe c t
a n d sim ple, each g ra d e b e in g u seful to its possessor, can be show n to exist; if fu r
ther, th e eye d o e s vary ever so slightly, a n d th e v ariations b e in h e rite d , w hich is cer
tainly th e case; a n d if any v ariatio n o r m o d ific atio n in th e o rg a n be ever useful to
an an im al u n d e r c h a n g in g c o n d itio n s o f life, th e n th e difficulty o f believing th a t a
p e rfe c t a n d c o m p lex eye c o u ld be fo rm e d by n a tu ra l selectio n , th o u g h in su p e ra
ble by o u r im a g in a tio n , can h ard ly be c o n sid e re d real.
CONTRIBUTORS
XI
Xll CONTRIBUTORS
izygous (XY) state they lack a sec o n d , n o rm a l copy o f the affe cted g en e. T h e
e x p ressio n o f th e X c h ro m o so m e is also d iffe re n t fro m th e au to so m es in the
h o m o g a m e tic sex (X X ), th e fem ale in m am m als, because o f th e process o f ra n
d o m fu n c tio n a l in activ ation o f o n e o f th e two c h ro m o so m e s in each cell in early
em b ry o g en esis (X-inactivation). T his pro cess takes place at a stage o f em b ry o g en -
esis w h ere cell n u m b e rs a re sm all (e.g. <5 day o ld m o u se em bryos) so th at
c h a n c e flu c tu a tio n s in th e d istrib u tio n o f cells w ith o n e o r o th e r X ch ro m o so m e
active can have larg e effects o n th e e x p ressio n o f d e le te rio u s g e n es in heterozy
g o tes - p artic u la rly w h ere th e n u m b e r o f p rim o rd ia l cells giving rise to a tissue,
such as th e re tin a , a re relatively sm all. T his process is resp o n sib le fo r th e variable
a n d o fte n su b tle m a n ife statio n s o f disease in h eterozygous c a rrie rs o f X -linked
d iso rd ers.
A u to so m al g e n e s a re ex p ressed in every cell a lth o u g h it is now know n th a t th e
p a re n ta l so u rce (fa th e r o r m o th e r) o f th e c h ro m o so m e can have a p ro fo u n d effect
o n th e ex p ressio n o f c e rta in genes. T h is process has b e e n te rm e d im printing a n d ,
u n til recently, m ost ex am p les cam e fro m studies o f g en es ex p ressed in early m am
m alian d e v e lo p m e n t. R ecen t studies o n th e p a re n ta l o rig in o f ch ro m o so m e 15 d e
le tio n s associated w ith th e P ra d e r-W illi a n d A n g elm an sy n d ro m es in m an has now
esta b lish e d a sig n ifican t ro le fo r this process n o t only in early d e v e lo p m e n t b u t
also in th e e x p ressio n o f disease.
G e n e tic diversity is ach iev ed by two m a jo r m ech an ism s am o n g d ip lo id species.
T h e first is by m utation, w hich o ccu rs a t a slow b u t finite ra te th ro u g h o u t th e ge
n o m e . T h e e v o lu tio n a ry c o n stra in ts o n a lte rin g th e fu n c tio n o f g e n e s are such
th a t relatively few m u ta tio n a l d iffe re n c es survive in reg io n s th a t co d e fo r p ro te in s
o r c o n tro l g e n e ex p ressio n . T h e s ile n t re g io n s o f th e g e n o m e , on th e o th e r
h a n d , m a in ta in a h ig h level o f diversity. T h e ad vantage o f this h ig h po ly m o rp h ism
c o n te n t b ecam e ev id e n t to g en e m a p p e rs in th e early 1980s, w ho p ro p o se d th e use
o f such se q u e n c e varian ts as m a rk e rs to follow th e se g reg atio n o f g en es in k in
d red s. T h e v arian ts w ere id e n tifie d initially by th e ir ability to a lte r re stric tio n e n
d o n u c le a se cleavage sites (restriction fragm ent length polym orphism s, RFLPs),
w hich c o u ld b e d e te c te d by h y b rid isatio n o f specific ra d io la b e lle d p ro b e s ag ainst
g e n o m ic DNA a fte r d ig e stio n w ith th e a p p ro p ria te e n d o n u c le a se (F igure 1). Such
varian ts w ere fo u n d to o c c u r o n c e every 300 -5 0 0 base p airs in n o n -c o d in g re g io n s
o f th e g e n o m e a n d to b e p re s e n t as fre q u e n t a ltern ativ e fo rm s (alleles) in th e g e n
eral p o p u la tio n . T h ey have b e e n e x p lo ite d to th e full in m a p p in g g e n e tic traits,
w h ere only a lim ite d ra n g e o f b lo o d g ro u p a n d se ru m p ro te in p o ly m o rp h ism s ex
iste d previously fo r this p u rp o se.
In c o n tra st to th e n u c le a r g e n o m e , th e m itochondrial genom e consists o f a
sm all, c irc u la r DNA m o le c u le (16.5 x 10 3 base p airs long) th a t is m a in ta in e d in th e
cytoplasm as h u n d r e d s o r th o u sa n d s o f copies p e r cell. It en c o d e s several su b u n its
o f oxidative p h o sp h o ry la tio n enzym es th a t are vital to th e fu n c tio n o f re sp irin g
cells, th e o th e rs b e in g c o d e d by th e n u c le a r g e n o m e. T h e re are im p o rta n t differ
en ces betw een th e se two g e n o m es (C h a p te r 21), in c lu d in g a significantly h ig h e r
ra te at w hich m u ta tio n s a re in c o rp o ra te d in to th e m ito c h o n d ria l g e n o m e (fixa-
from the presence of a previous mutation within the restriction endonuclease cleavage site (G 'A) on H' thal results in failure to cleave al that p o in t.
The digested fragments are separated electrophoretically, d en atu red , transferred to a m em brane (Southern transfer) and hybridised with a single
stranded ~P labelled probe sequence that specifically anneals to the fragments containing the RFI The segregation of the two alleles are show n
within a small pedigree on the right. The RFI.P alleles do not segregate with the disease phenotype (shaded symbols) in this family.
MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 5
tio n ), som e o f w hich give rise to g e n e tic eye d iso rd ers. M u tatio n also arises as a
re su lt o f ch ro m o so m a l a b e rra tio n s su ch as tran slo catio n s, inversions a n d d e le
tions. T h ese a re im p o rta n t b o th in c re a tin g e v o lu tio n ary diversity a n d , in th e c o n
tex t o f g e n e m a p p in g , p ro v id e an im p o rta n t m ea n s o f id en tify in g g e n e s th a t have
b e e n d is ru p te d by tra n slo c a tio n o r d e le tio n events (C h ap te rs 8, 9, 10, 12, 13, 17,
1 9 ,2 1 ).
T h e se c o n d m e c h a n ism fo r g e n e ra tin g diversity occu rs d u rin g m eiosis w hen h o
m o lo g o u s p a re n ta l ch ro m o so m e s pair, e x ch an g e g e n e tic m a te rial by re c o m b in a
tio n a n d seg reg ate in to th e g e rm cells. T h e average n u m b e r o f ex ch an g e s p e r
ch ro m o so m e d u rin g m eiosis in a m am m a lia n cell is only 2 -4 , so th a t th e d a u g h te r
c h ro m a tid s a re c o m p o sites m ad e u p o f relatively lo n g seg m en ts o f e ith e r m a te rn a l
o r p a te rn a l ch ro m o so m es. T h e re c o m b in a tio n process e n su re s th a t th e p ro b a b il
ity o f an in d iv id u al in h e ritin g two g e n e s o r m ark ers fro m th e c h ro m o so m e o rig i
nally c o n trib u te d by a g ra n d p a r e n t decreases rapidly to th e level o f ch an ce,
d e p e n d in g o n how fa r a p a rt they are. T h e p aucity o f re c o m b in a tio n a l crossovers
m akes fo r a relatively efficien t m a p p in g p ro c e d u re in w hich th e ra te at w hich th e
two sites (genes, m ark ers) re c o m b in e is a m easu re o f th e g en e tic d ista n c e betw een
th e m (sectio n II). M istakes th a t o c c u r e ith e r d u rin g ch ro m o so m al re p lic a tio n o r
d u rin g m eio tic p a irin g can give rise firstly to d u p lic a tio n o f g en es a n d la te r to m u l
tip le co p ies o f g en es, p ro v id in g sco p e fo r th e d iv erg en ce o f g en es w ith re la te d
fu n ctio n s, as discu ssed in C h a p te r 9 o n co lo r vision.
T h e n u m b e r o f g e n e s in th e m am m a lian g e n o m e has b e e n e stim a ted to be in
th e re g io n o f 50,000-1 0 0 ,000. T h e average cell expresses o n e -q u a rte r to o n e h a lf
o f this n u m b e r a n d som e g e n e tra n sc rip ts a re very a b u n d a n t, o th e rs m ake only a
few co p ies p e r cell. T h is h ighly skew ed d istrib u tio n o f g e n e exp ressio n can b e an
im p o rta n t c o n sid e ra tio n in id en tify in g c a n d id a te g en es fo r g e n e tic d iso rd ers, p ar
ticularly w ith re g a rd to th e c o n stru c tio n o r sc re e n in g o f ex p re sse d se q u en ce
(cDNA) libraries. If an average m am m alian g e n e p ro d u c t c o n ta in s a b o u t 300 am i
n o acids, th e e q u iv a le n t a m o u n t o f D NA re q u ire d to co d e a n d express such a g en e
is only a b o u t o n e kilobase p a ir (kb; 103 base pairs). W hen e x tra p o la te d to th e size
o f th e w hole g e n o m e , it is readily a p p a r e n t th a t only 2 -4% o f th e g e n o m e actually
co des fo r p ro te in s. N u cleic acid re asso ciatio n studies show th a t as m u c h as 60% o f
m a m m a lia n DNA c o n ta in s u n iq u e o r low copy n u m b e r (<10) seq u en c es, w hile the
re m a in d e r is rep etitiv e. W ithin th e la tte r g ro u p , th re e classes o f re p e titiv e DNA
can b e d istin g u ish e d - h ig h , in te rm e d ia te a n d low a b u n d a n c e classes. T h e first
class o f highly re p e titiv e seq u e n c es in c lu d e s th o se w ith re ite ra tio n fre q u e n c ies
above a b o u t 100,000 copies, su ch as th e tan d em ly rep etitiv e sate llite seq u en c es
n e a r th e c e n tro m e re s o f som e ch ro m o so m e s. A n o th e r im p o rta n t ex am p le o f this
g ro u p is th e A lu rep etitiv e e le m e n t, w hich is 300 base p airs in le n g th a n d in te r
sp ersed th ro u g h o u t th e g e n o m e , o c c u rrin g on average every 3 -4 kb. T h ese a n d
o th e r in te rs p e rs e d re p e a ts a re useful fo r d istin g u ish in g th e DNA o f d iffe re n t m am
m alian species b u t in g e n e ra l are a g en eticists' n ig h tm a re . T h ey m ak e th e task o f
id en tify in g specific se q u e n ce s su ch as g e n e s con sid erab ly m o re difficult, so th a t
ra d io la b e lle d p ro b e se q u en c es o fte n re q u ire co m p e titio n w ith h ig h c o n c e n tra
tio n s o f rep etitiv e D NA in o rd e r to p re v e n t non-specific hy b rid isatio n signals.
6 MICHAEL B. GORIN AND AIAN F. WRIGHT
2
V .5
y (U r
a *>
ci -G
MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 9
II G EN ETIC MAPPING
A
B II X a
b
A
b
A
b
X a a
B
parental
chromosomes
A
BI a
b
A
b
a
B
gametes
coupling repulsion
Figu re 3 C o u p lin g a n d re p u lsio n . Two alleles a t e a ch o f two loci (A,a a n d B,b) a re show n, two
o f w hich a re in c o u p lin g (th e sam e c h ro m o s o m e ), two o f w hich are in re p u lsio n (o n o p p o site
ch ro m o so m es) a n d seg reg ate to g e th e r o r a p a rt in th e gam etes.
bI Os
U x s D D s DOT U U genotypes
A
1
---, I gametes
B
I i| sO 0
L
recombinants
Fig u re 4 R eco m b in a tio n . Two loci on th e sam e c h ro m o so m e are show n, each w ith two alleles
(A,a a n d B,b) w hich seg reg ate in th e g am e tes e ith e r as p a re n ta l (AB, ab) o r re c o m b in a n t (Ab
a n d aB) co m b in atio n s. Loci th a t a re fa r a p a rt o n th e sam e c h ro m o so m e will asso rt in d e p e n
d en tly o f each o th e r in th e g am etes (a n d offsp ring ) as a resu lt o f re c o m b in a tio n b etw een h o
m o lo g o u s c h ro m o so m e s in m eiosis.
0 1 ( e 4x - 1)
2 ( e 4 x + 1)
L (e) = ( 9 ) r ( i - 0 ) n ^ r p T (7 7 r 7 )- ! '
1There are exceptions to this, particularly towards the ends of some chromosomes.
2The total am ount of DNA per haploid genome is about 3 x 109 base pairs (bp) in mammals, these val
ues are therefore obtained by dividing the genetic length for each species by 3 x lO^bp.
MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 13
G enetic markers
B
ES cells containing Pronuclear injection of
targeted gene transgene
disruption
I
Blastocyst injection
I
Transgenic
mouse
Outcross
Selection of offspring
/
Intercross offspring
with germline transmission to produce homozygous
of transgene transgenic mice
Figu re 5 T ran sg en ic ex p erim e n ts. T ran sg en ic m ice can b e p ro d u c e d in two m ain ways. M ouse
e m b ry o n ic stam (ES) cells may b e ta rg e te d by h o m o lo g o u s re c o m b in a tio n in to a g e n e in or
d e r to in tro d u c e a specific m u ta tio n (e.g. k n o c k o u t). (A) T h e ta rg e te d ES cells are m icro in
je c te d in to a blatocyst, re im p la n te d a n d c h im aeric p ro g e n y id e n tifie d by m ean s o f c o a t c o lo u r
m arkers. T h e se can b e o u tc ro ssed to identify th o se cap ab le o f g e rm lin e tran sm issio n o f th e
tra n sg e n e a n d in tercro ssed to p ro d u c e h om ozygotes. A lternatively th e m ale p ro n u c le u s w ith
in fertilised oocytes is m ic ro in je c ted with th e tra n sg e n e a n d a fter re im p la n ta tio n in to a
p se u d o p re g n a n t fem ale, th e p ro g en y a re o u tc ro ssed to iden tify those show ing hem izygous ex
p ressio n o f th e tra n sg e n e a n d th e n in te rc ro sse d to p ro d u c e h o m ozygous e x p ressio n (B).
N u cleic acids a re ro u tin e ly se p a ra te d by gel e lec tro p h o resis. For relatively sm all,
lin e a r d o u b le -s tra n d e d (ds) DNA m o lecu les (<50 k b ), m ig ratio n w ithin an agar
ose gel in th e p re se n c e o f an electric field is a fu n c tio n o f m o le c u la r size ra th e r
th an c o n fo rm a tio n o r seq u en ce. T h e n u m b e r o f negatively c h a rg e d g ro u p s on
th e p h o s p h a te b a c k b o n e is p ro p o rtio n a l to th e n u m b e r o f base pairs, so th a t th e
ch a rg e density is co n sta n t. T h e d iffe re n t n u c le o tid e resid u es have little effect on
th e c h a rg e b e h a v io u r o f DNA, a lth o u g h this is less so fo r e le c tro p h o resis in poly
acry lam id e. DNA se q u e n c e analysis a n d h ig h re so lu tio n sep a ra tio n o f m o lecu les
u p to a b o u t 1000 n u c le o tid e s in le n g th (e.g. se q u e n c in g o r m icro satellite analy
sis) is g en erally c a rrie d o u t u sin g polyacrylam ide gel e le c tro p h o re sis u n d e r d e n a
tu rin g c o n d itio n s. In th e case o f sin g le-stran d ed (ss) DNA, th e d iffe re n t
n u c le o tid e s can have a m ajo r e ffect on th e c o n fo rm a tio n o f th e m o lecu le, w hich
can a lte r its b eh a v io u r in a gel unless d n a tu ra n ts are u sed to e lim in ate th e sec
o n d a ry stru c tu re . T h is is e x p lo ite d in th e te c h n iq u e o f single stra n d c o n fo rm a
tio n al p o ly m o rp h ism analysis (SSCP) in o rd e r to d e te c t single base ch a n g es fo r
m u ta tio n screen in g .
L arg e m o lecu les o f DNA (>50 kb) w ould n o rm ally be u n a b le to m ig rate w ithin
an ag aro se gel, b u t th e use o f p ulsed-field a n d field-inversion gel e le c tro p h o resis
(PFGE, FIGE) e x p lo it a c h a n g in g electric field to a lte r th e ir se c o n d a ry stru c tu re
so th a t they can m ig ra te at a ra te th a t is inversely p ro p o rtio n a l to size. In c o n trast
to yeast c h ro m o so m e s, h u m a n ch ro m o so m e s are m u c h too large to b e se p a ra te d
by PFGE a n d have first to b e cleaved w ith ra re c u tte r restric tio n e n d o n u c le a se s
in to fra g m e n ts below a b o u t 2 x 106 base pairs (2 Mb) in size. Yeast o r b a cte ria l ar-
MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 19
dficial chrom osom es (YACs, BACs) a n d PI vectors have been used to clone h u
m an, m ouse o r D rosophila DNA fragm ents in the size range th at can be separated
and purified by PFGE.
Flow cytom etry or sorting is based on a d ifferent principle than electrophore
sis since it distinguishes chrom osom es (or cells) on the basis of their ability to bind
fluorescent dyes (e.g. bisbenzim ide, DAPI) that are detected after laser excitation.
A dilute solution o f chrom osom es is passed th ro u g h a laser and the em itted fluo
rescent signal is d etected and a m agnetic field is activated to divert the electrically
charged water d ro p let containing the chrom osom e o f interest into a tube. T he
separation o f chrom osom es is th erefo re based on a com bination o f size an d the
structural properties th at allow the bin d in g of dyes.
Table 1 illustrates the d ifferent m ethods o f nucleic acid separation, the size
range an d sources o f the m olecules separated an d special applications of each
technique.
Specific pieces of DNA can be p ro d u ced in large quantities to allow for direct
study o r use as m olecular tools. This am plification process can be carried out by
chemical synthesis (as in the m anufacture o f oligonucleotides), by enzymatic
am plification using the polym erase chain reaction (PCR), or by cloning into vec
tors such as plasmids, cosmids, bacteriophage, PI or into yeast artificial chrom o
somes (YACs) (Table 3).
Chem ical synthesis of DNA is lim ited to m olecules o f less than ab o u t 100 nucle
otides in length an d is widely used for synthesising specific oligonucleotide se
quences (prim ers) for prim ing PCR and sequencing reactions. Small synthetic
oligonucleotides th at act as adaptors o r linkers for m odifying DNA ends are also
useful.
T he pro d u ctio n o f DNA by enzymatic am plification using PCR is central to a
wide range o f m olecular genetic experim ents. It is illustrated an d explained in Fig
ure 6. PCR synthesis o f DNA uses the o th er fundam ental tools, for exam ple an
nealing o f prim ers to the DNA tem plate, replication of DNA using heat-stable
polym erase, visual detection (as p art o f screening and analysis) an d purification
o f the p ro d u c t by electrophoresis but it is o f such fundam ental im portance that it
alm ost am ounts to a basic tool in itself.
20 MICHAEL B. GORIN AND ALAN F. WRIGHT
T ab le 2 Continued.
a -e II' *T3 -S 9
1 t 1 f e 'e l L ,
<
V ? bM
e p
~ ^ 2 J= 0 \_ V
z z< z< z< <
z <
Z Cu CLU &- >
<
.2
H bo .9 * g bc bo bo b
o G 0 C
0 G
.s G G
s Cu
*5
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Q 5 g 5
pC H 0 > -
(J c 0 0 0
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C/3 Cu G GGG
^ -O 4
M
M0
G 1C CM
in cm CM Tt< L i
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o G G c M
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cz Z
Q
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z
a u
-a
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cr Z o c
CJ a 2I
MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 23
O G
m
T
>- 3 ts
o
o <V g v g
u 3 C
^ Cfl
2 0
s O T3
3 t/J 3 c
O rt
-3 V5
.6
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ja
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3. V 3 W o bo
S-i o . 'I
0. ^
s
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O s 2 o
s Oh
b
o '
O- J3 8 u
T3 T3
O O
c 0
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be c tc fi
2 ^ <
^ o
CX On Q. u Z z
1 ? 1 .2 3
T3
2| S bI<IU
^ 'R.
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CM
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24 MICHAEL B. GORIN AND ALAN F. WRIGHT
d
be
C
'u
D
-d
c
o
d
B
bJD
.s
OJ
c
dC
26 MICHAEL B. GORIN AND ALAN F. WRIGHT
NONSPECIFIC DETECTION
Stains
N onfluorescent Chromosome banding Giemsa Light
photomicroscopy Cytogenetics
Nonspecific Silver Direct visualisation DNA/RNA on gels
Gold (very sensitive)
Direct Detection
Radioisotopes
End-labelled Dephosphorylated 5' end y' P-ATP Autoradiography Sequencing,
3' tailing reaction or*2PdNTP, Hybridisation
K S-dNTP
Fluorescent adducts
Chemically Oligonucleotide Fluorescein, Fluorescence Sequencing
incorp. svnthesis rhodam ine Hybridisation
derivatives
T ab le 4 Continued.
Secondary Detection
Enzyme adducts to
DNA
Chemically linked glutaraldehyde HRP, Stains, Chemilum Hybridisation
crosslink alkaline
amino-modified oligo phosphatase In situ hybrid
Avidin-conjugates
Biotinylated probe Internal incorporation avidin-FITC Fluorescence FISH
microscopy Hybridization
End-labelling avidin-HRP Stains, Chemilum
Photobiotinylation
An tibody-e nzyme
conjugates
Incorp. by synthesis PCR, nick translation, Alk. ptase-, Chemiluminescence, Hybridization
random prim er synthe HRP- colorimetric stains
sis in vitro transcription -galacto-
sidase-
conjugates
Digoxigenin digoxigenin-dUTP Substrates with,
Fluorescein fluor-dUTP chemilum.
or colored
products
T ab le 5 Continued.
In situ Cloned DNA Chromo Target Retention Glass slides Bound probe
hybridisation somes of probe
REFERENCES
Casey, D. (1992) Prim er on M olecular G enetics In: Human Genome 19919 2 Program Report. U.S.
D ep a rtm e n t o f Energy, W ashington, D.C.
Davies, K. E. and Read, A. P. (1988) Molecular Basis o f Inherited Disease. IRL Press, O xford, 77 pp.
King, R. C. and Stansfield, W. D, (1992) A Dictionary of Genetics, (Fourth ed. ) O xford University
Press, Inc, New York, 406 pp.
T hom pson, M. W., M clnnes, R. R., W illard, H. F. (1991) Thompson & Thompson Genetics in Medi
cine, (Fifth ed.) W. B. Saunders Co., Philadelphia, 500 pp.
2. RETINAL DEGENERATION MUTANTS
OF Drosophila
W ILLIAM L. PAR
Department o f Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA
ABSTRACT
I IN T R O D U C T IO N
cornea --------
pseudocone
Rl-6 photoreceptor
Rl-6 rhabdomere -
cone cell
R7 rhabdomere
~ r pigment cell
R4 R3
cone cell process R5 _ R2
R1
R8 rhabdomere----- R7
R8
10 \im
equator
a n d t e r t i a r y ( 3 ) p i g m e n t c e ll s . R l -6 p h o t o r e c e p t o r s r o u g h l y c o r r e s p o n d t o r o d s , R 7 / 8 p h o t o
r e c e p to r s to c o n e s , a n d th e r h a b d o m e r e s to th e o u te r s e g m e n ts o f v e rte b ra te p h o to r e c e p to r s .
A d a p te d fro m W o lff a n d R eady (1 9 9 3 ). R e p ro d u c e d w ith p e rm iss io n fro m th e C o ld S p rin g
H a r b o r P re ss.
RETINAL DEGENERATION IN DROSOPHIIA 33
a V3 t
& ^
o
h
S
S Q
& , C -O T3
5 C/3 cC/5 T5
u
g- 5 G
8 S 03
C/2 J5
wS cs/5 be ^ - (*<
n
^ C C OJ . g W
O c3 O 2
E ^ S .
*
3 .2 ^ 2 ^ 5 !
-G <u
3 C O.M T3
b a _ aj
* C/5 O QJ O QJ ^
Q.
^ I s QJ
V
V bo. ^S
^ C
G G ^ K
o
a | S QJ sO G ^
5 5 c G
5
U> Ou
^
c r c
U -C
RETINAL DEGENERATION IN DROSOPHILA 35
W IL L IA M L . PAK
Figure 3 Allele dependence of the time course of photoreceptor degeneration inDrosophila rhodopsin gene {ninaE) mutants and the mo
lecular defects carried by the mutants. Mean number of Rl-6 rhabdomeres per ommatidium, determined in light microscopy, is plotted
as a function of age. Adapted from Figure 1 and Table 1 of Leonard et al. (1992). Reproduced with permission from the Journal of Neu
robiology.
RETINAL DEGENERATION IN DROSOPHILA. 37
ly z in g t h e e x c h a n g e o f G T P f o r G D P . T h e G p r o te i n , in t u r n , a c tiv a te s p h o s p h o li p a s e C (P L C )
r e l e a s e d C a 2*, t h r o u g h a n u m b e r o f u n k n o w n s te p s , o p e n s a c la s s o f c a t i o n c h a n n e l s w ith r e l
a t i v e l y l o w C a 2+ p e r m e a b i l i t y . T h i s c l a s s o f c h a n n e l s is r e s p o n s i b l e f o r t h e tra n s ie n t p h a s e o f
th e r e c e p t o r p o t e n t i a l . I n th e m e a n t im e , t h e a c tiv a te d IP 3R a ls o o p e n s a d i f f e r e n t c la s s o f io n
c h a n n e l s w i t h h i g h C a 2+ p e r m e a b i l i t y t h r o u g h d i r e c t i n t e r a c t i o n . T h i s c l a s s o f c h a n n e l s i s r e
s p o n s ib le fo r th e s te a d y - s ta te phase o f th e re c e p to r p o te n tia l, a n d th e C a 2+ i n f l u x th ro u g h
6 M utants o f th e rdgC G en e
M u tatio n s in th e rdgC g e n e w ere iso lated fro m chem ical m u tag en e sis by lo o k in g
fo r th e ir d e g e n e ra tiv e effects o n th e d e e p p se u d o p u p il w ith age (Steele a n d
O T ousa, 1990). R h a b d o m e re s o f rdgC m u ta n ts a re n o rm a l w hen y o u n g b u t
u n d e rg o age- a n d lig h t-d e p e n d e n t d e g e n e ra tio n . As m o n ito re d by ex a m in in g th e
d e e p p se u d o p u p il, d e g e n e ra tio n is essentially c o m p lete by 8 d post-eclosion w hen
re a re d o n a 12 h r l i g h t / 12 h r d a rk cycle. R7 a n d R8 rh a b d o m e re s are m u c h m o re
re sista n t to d e g e n e ra tio n th a n R l-6 rh a b d o m e re s a n d ju s t b e g in show ing signs o f
d e g e n e ra tio n w h en th e la tte r have a lm o st com p letely d e g e n e ra te d . D e g e n e ra tio n
is n o t o b se rv e d if th e flies a re re a re d in c o m p lete dark n ess. U n lik e m o st o th e r
know n re tin a l d e g e n e ra tio n m u tan ts, p rio r to th e o n se t o f d e g e n e ra tio n , n o obvi
o us d e fe c t in th e re c e p to r p o te n tia l is ev id en t in rdgC m u tan ts. D e g e n e ra tio n
re q u ire s p h o to a c tiv a tio n o f rh o d o p sin b ec au se it d o e s n o t o c c u r if th e rh o d o p sin
level is re d u c e d by e ith e r vitam in A d e p riv a tio n o r th e use o f a rh o d o p sin ( ninaE)
g e n e m u ta tio n . H ow ever, u n like rdgB -induced d e g e n e ra tio n , rdgC-induced
d e g e n e ra tio n d o es n o t re q u ire th e norpA-en c o d e d PLC, su ggesting th a t it is m e d i
a te d by a rh o d o p sin activated pathw ay d iffe re n t fro m th e P L C -m ediated p h o
to tra n s d u c tio n pathway.
T h e g e n e was m a p p e d to 77B1 o f th e th ird ch ro m o so m e a n d was isolated by
ch ro m o so m a l w alking (S teele e t al., 1992). A DNA fra g m e n t th o u g h t to co n ta in
th e g e n e , iso lated fro m th e walk, was u se d to rescu e rdgC m u ta n t flies by g e rm line
tra n sfo rm a tio n , p ro v id in g p r o o f th a t th e g e n e h a d b e e n clo n e d in th e fra g m en t.
T h e g e n e e n c o d e s a p ro te in o f 661 a m in o acids w hich c o n ta in s a d o m a in h o m o l
o gous to th e catalytic d o m a in s o f types 1, 2A, a n d 2B s e r in e /th r e o n in e p ro te in
p h o sp h a ta se s (F igure 2). T h e p u tativ e catalytic d o m a in is follow ed by a d o m a in
c o n ta in in g several EF h a n d m otifs, o r p o te n tia l C a2+-b in d in g sites (F igure 2).
S teele e t al. (1992), th u s, su g g ested th a t th e rdgC p ro te in m ig h t be a novel type o f
s e r in e /th r e o n in e p h o sp h a ta se re g u la te d d irectly by C a2 r levels. T h e p ro te in is ex
p ressed in th e re tin a , ocelli, a n d m u sh ro o m b o d ie s o f th e b ra in . T h e fu n c tio n o f
th e p ro te in h as n o t yet b e e n e lu cid ate d .
IV DISCUSSION
VI REFERENCES
Scavarda, N .J ., O Tousa, J. an d Pak, W. L. (1983). Drosophila locus with gene dosage effects on
rhodopsin. Proceedings o f the National Academy of Sciences USA 80, 44414445.
Schinz, R. H., Lo, M.-V. C., Larrivee, D. C. a n d Pak, W. L. (1982). Freeze-fracture study o f the
Drosophila p h o to re c e p to r m em brane: m utatio n s affecting m em b ran e particle density. Journal
o f Cell Biology 93, 961-969.
Schneuwly, S., Burg, M. G., L ending, C., Pcrdew, M. H. a n d Pak, W. L. (1991). P roperties o f p h o
toreceptor-specific phospholipasc C en co d ed by the nurpA getie o f Drosophila m elanogaster.
Journal of Biological Chemistry 266, 2431424319.
Selinger, Z. an d Minke, B. (1988). Inositol lipid cascade o f vision studied in m u tan t flies, pp. 333-
341. The Molecular Biology o f Signal Transduction, Cold Spring Harbor Symposium on Quantitative
Biology, Vol. 53. Ed. J. D. W atson, M. W igler, a n d j. Feram isco, Cold Spring H arb o r Laboratory,
C old Spring H arbor, New York.
Smith, D. P., R anganathan, R., Hardy, R. W., M arx, J., Tsuchida, T. an d Zuker, C. (1991). Photo
recep to r deactivation and retinal d eg en e ra tio n m ed iated by a photoreceptor-specific p ro tein
kinase C. Science 254, 1478-1484.
Stahl, M. L., Fercnz, C. R., Kelleher, K. L., Kriz, R. W. and Knopf, J. L. (1988). Sequence similarity
o f phospholipasc C with the non-catalytic region o f src. Nature 332, 269-272.
Stark, W. S. a n d Carlson, S. D. (1982). U ltrastru ctu ral pathology o f th e co m p o u n d eye and optic
neu ro p iles of the retinal d eg en eratio n m utants (tu rdgBKs- 22) Drosophila melanogaster. Cell Tis
sue Research 225, 11-22.
Stark, W. S. and C arlson, S. D. (1983). U ltrastru ctu re o f the co m p o u n d eye and first optic n e u
ro p ile o f the p h o to re c ep to r m u tan t oraJK84 o f Drosophila. Cell Tissue Research 233, 305-317.
Stark, W. S. an d Sapp, R. (1987). U ltrastru ctu re o f the retin a o f Drosophila m elanogaster: the m u
tan t o ra (o u te r rh ab d o m eres absent) a n d its in hibition o f d eg en eratio n in rdgB (retinal de-
g en eratio n -B ). Jo u rn a l o f N eurogenetics 4, 227-240
Stark, W. S., C hen, D. -H., Jo h n so n , M. A. a n d Frayers, K. L, (1983), T he rdgB gene in Drosophila:
Retinal d eg en eratio n in d ifferen t m u ta n t alleles an d in hibition o f d eg en eratio n by norpA.
Journal of Insect Physiology 29, 123131.
Stark, W. S., Sapp, R. and C arlson, S. D. (1989). P h o to recep to r m ain ten an ce and deg en eratio n
in the norpA (no re ce p to r potential-A) m u tan t of Drosophila m elanogaster. Journal of Nevrug/'
neticsb, 49-59.
Stark, W. S., C hristianson, J. S., Maier, L. and C hen, D. -M. (1991). In h e rite d an d environm entally
induced retinal d eg en eratio n in Drosophila, pp. 6175. Retinal Degenerations. Ed. R. E. A nder
son, J. G. Hollyfield, an d IT. M. Lavail, CRC Press, Boca Raton.
Steele, F. and O Tousa, J. E. (1990). R hodopsin activation causes retin al d e g en eratio n in Droso
phila rdgC m utant. Neuron 4, 883-890.
Steele, F., W ashburn, T., Rieger, R. and O Tousa, J. E. (1992). Drosophila retinal d eg en eratio n C
(rdgC) enco d es a novel s e rin e /th re o n in e pro tein phosphatase. Cell 69, 669676.
S tephenson, R. S., O Tousa, J., Scavarda, N. J., Randall, L. L. an d Pak, W. L. (1983). Drosophila
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Cosens an d D. Vince-Price, C am bridge University Press, C am bridge, E ngland.
Suh, P. G., Ryu, S. H., M oon, K. H., Suh, H. W. and Rhee, S. G. (1988a). C loning and sequence
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Sung, C.-H., D avenport, C. M., Hennessey, J. C., M aum enee, I. H., Jacobson, S. G., Heckenlively,
J. R., et al. (1991). R hodopsin m utatio n s in autosom al d o m in a n t retinitis pigm entosa. Pro
ceedings o f the National Academy o f Sciences USA 88, 6481-6485.
52 WILLIAM L. PAK
Szuts, E. Z., Wood, S. F., Reid, M. A. and Fein, A. (1986). L ight stim ulates the rap id form atio n o f
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Vihtelic, T. S., H yde, D. R. an d 0 'T o u sa ,J. E. (1991a). Isolation and ch aracterization o f th e Droso
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Vihtelic, T., O Tousa, J. an d Hyde, D. (1991b). Localization o f the Drosophila rdgB p rotein: a phos
pholipid transfer pro tein . Molecular Neurobiology o f Drosophila (A bstract), Cold Spring H arb o r
Laboratory* Cold Spring H arbor, New York, p i 84.
Walz, B. (1982). C alcium -sequestering sm ooth endoplasm ic reticulum in retin u la cells o f the
blowfly. Journal of Ultrastructure Research 81, 240-248.
W ashburn, T. a n d O Tousa, J. E. (1989). M olecular defects in Drosophila rh o dopsin m utants. Jour
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Wolff, T. an d Ready, D. F. (1993). P attern form atio n in the Drosophila retina. Development o f Droso
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Wong, F., Schaefer, E. L., Roop, B. C., L aM endola,J. N .,Johnson-S eaton, D., an d Shao, D. (1989).
P ro p er fu n ctio n o f the Drosophila trp gene p ro d u c t d u rin g pupal d evelopm ent is im p o rta n t
for n o rm al visual tran sd u ctio n in th e adult. Neuron 3, 8194.
Wood, S. E., Szuts, E. Z. an d Fein, A. (1989). Inositol trisphosphate p ro d u ctio n in squid p h o to
receptors. Activation by light, alum inum fluoride, an d g u an in e nucleotides. Journal o f Biolog
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Yoshioka, T., In o u e, H. an d H otta, Y. (1985). A bsence o f diglyceride kinase activity in th e p h o to
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389-395.
Zinkl, G. M., Maier, L., Studer, K., Sapp, R., C hen, D.-M., and Stark, W. S. (1990). M icrophoto
m etric, ultrastru ctu ral, and electrophysiological analyses o f lig h t-d ep en d en t processes on vi
sual recep to rs in white-eyed wild-type and norpA (n o rece p to r potential) m u ta n t Drosophila.
Visual Neuroscience 5, 429439.
Zuker, C. S., Cowman, A. F. an d R ubin, G. M. (1985). Isolation and stru ctu re o f a rh o d o p sin gene
from D. m elanogaster. Cell 40, 851-858.
3. DIAGNOSIS IN INHERITED RETINAL DISORDERS
IN T R O D U C T IO N
Gmetic studies
R etinitis p ig m e n to sa m ay be in h e rite d as a n au to so m al d o m in a n t, an au to so m a l
recessive o r an X -linked trait. W h en transm ission has o c c u rre d th ro u g h several
g e n e ra tio n s th e re c o g n itio n o f X -linked o r au to so m al d o m in a n t in h e rita n c e is
usually sim ple. As w ith o th e r d iso rd ers, p a re n ta l co n san g u in ity may in d ica te th e
possibility o f au to so m a l recessive in h e rita n c e . W h en faced with a sp o ra d ic case,
how ever, th e p ro b le m o f id en tifying th e g e n e tic fo rm o f th e disease may be m o re
difficult. In th e larg e series o f cases a t M o o rfield s Eye H ospital, th e fre q u e n c y o f
sim p lex (sp o rad ic) cases in fam ilies w ith re tin itis p ig m e n to sa is at p re se n t 52 p e r
c e n t (Jay M, p e rso n a l c o m m u n ic a tio n ), a figure a little la rg e r th a n th a t p u b
lish ed 10 years ago (42% - Jay, 1982). O th e r a u th o rs have fo u n d a sim ilar h ig h
fre q u e n c y o f sim plex cases (63% - P e a rlm a n , 1979; 48% - H u , 1982; 50% -
B o u g h m a n 8c F ishm an, 1983; 42% - H eckenlively, 1987). U ntil fairly recen tly it
was co m m o n ly assu m ed th a t p a tie n ts w ith sim plex RP h a d au to so m a l recessive
disease b u t th e re is in c re a sin g clinical e v id en ce th a t m any o f th e se p a tie n ts do
n o t ex p ress th e disease in th e sam e m a n n e r as p a tie n ts with pro v en au to so m al
recessive RP (H eckenlively, 1988). T h e re is also g e n e tic evidence th a t som e cases
o f sim p lex RP c a rry a m u ta tio n o f th e rh o d o p sin g e n e (D ryja e t al, 1991). Sim
p lex cases m ay be X -linked if h etero zy g o u s fem ales are asym ptom atic o r only very
m ildly a ffected a n d affe c ted m ale relatives are n o t know n to th e p ro b a n d , o r th e
disease m ay b e au to so m a l d o m in a n t as a re su lt o f a new m u ta tio n .
T h e fre q u e n c y o f th e d iffe re n t g e n e tic fo rm s o f retin itis p ig m e n to sa varies in
d iffe re n t series a n d in d iffe re n t co m m u n itie s (Table 1).
F u rth e r g e n e tic in fo rm a tio n may b e o b ta in e d by e x a m in in g asym ptom atic re la
tives. In p articu lar, it is u sefu l to ex am in e th e m o th e rs o f severely affe cted m ales
b e c a u se they fre q u e n tly show m ild retin al involvem ent, in d ic a tin g th e h etero zy
g o u s state o f X -linked disease (B ird, 1975).
T h e severity o f th e disease m ay b e u sed as a g u id e to th e likely in h e rita n c e (Jay
& Bird, 1973). In g e n e ra l X -linked a n d a u to so m a l recessive retin itis p ig m e n to sa
cause severe d isease o f early o n set, w hile d o m in a n t disease is m ilder. Severe dis
ease in a fem ale usually in d ic a tes a u to so m a l recessive a n d m ild disease in a m ale
au to so m a l d o m in a n t disease. M ild disease in a fem ale is c o m p atib le w ith e ith e r th e
DIAGNOSIS IN INHERITED RETINAL DISORDERS 55
T a b le 1
Author No of AI) AR XL Sp
Gases % % * %
Functional studies
field (F led eliu s & S im o n sen , 1970), b u t rarely th e disease affects only th e su p e rio r
(R ag n etti, 1962), nasal (V ukovich, 1959) o r te m p o ral fu n d u s (A lezzandrini,
1965). In m o st re p o r te d cases w ith lim ited secto r involvem ent th e in h e rita n c e was
au to so m al d o m in a n t a n d th e re g io n a l d istrib u tio n o f disease was c o m m o n to af
fe c te d m e m b e rs o f th e fam ily (H om m er, 1959; K uper, 1960; Lisch, 1960; H aase &
H e lln e r, 1965; F led eliu s & S im o nsen, 1970). However, sim ilar cases also a p p e a r in
fam ilies w ith relatives w ho have inv o lv em en t o f th e w hole fu n d u s (Krill e t al.,
1970) a n d som e a re h e tero zy g o u s fo r X -linked re tin itis p ig m e n to sa (Krill e t al.,
1970; B ird, 1975). By flu o re sc e in angiography, m in o r ch a n g e s are o fte n fo u n d to
be m o re w id esp read th a n m ig h t b e a p p re c ia te d by o p h th a lm o sc o p y with w hite
light, a n d th e secto r o f retin itis p ig m e n to sa may b e an a rea o f m ax im u m disease
r a th e r th a n o f exclusive in v o lv em en t (Krill et al., 1970; B ird, 1975). In typical sec
to r RP, ro d a n d c o n e ERGs show m ild re d u c tio n in a m p litu d e w ith n o rm a l co n e
im p licit tim es (B erson 8c H o w ard, 1971; M assof 8c F inkelstein, 1979; F u lto n &
H a n se n , 1988). T h is p a tte rn o f disease is co m m o n to all a ffected m e m b e rs w ithin
a fam ily irresp ectiv e o f age, su g gesting th a t th e disease, in c o n tra st to o th e r fo rm s
o f au to so m a l d o m in a n t RP, is n o n -p rogressive o r p ro g resses very slowly.
In som e fam ilies th e re is slow recovery fro m b leach (A lex an d er & F ishm an,
1984). C o n e a d a p ta tio n a n d th e early p a rt o f th e reco v ery o f ro d sensitivity follow
th e n o rm a l tim e co u rse, b u t th e la te r p h a se o f ro d a d a p ta tio n is m ark ed ly p ro
lo n g ed .
U n ila te ra l re tin itis p ig m e n to sa was r e p o rte d as early as 1865 by P e d ra lg ia a n d
th e o p h th a lm o sc o p ic o b serv atio n s w ere c o rro b o ra te d by histological e x a m in a tio n
25 years later. In 1952, F ran o is a n d V erriest review ed th e 56 u n ila te ra l cases re
p o r te d u p to th a t tim e a n d c o n c lu d e d th a t only 10 cases h a d th e typical o p h th a l
m o sco p ic a p p e a ra n c e o f g en etically d e te r m in e d retin itis p ig m e n to sa a n d w ere
strictly u n ila te ra l. S u b se q u e n t re p o rts (K olb & Galloway, 1964; C a rr & Siegel,
1973) have show n th a t, in som e cases, m in o r c h an g es can be d e m o n stra te d in th e
u n a ffe c te d fellow eye by so p h istica te d e x a m in a tio n . F u rth e rm o re , C a rr a n d Siegel
(1973) th o u g h t th a t th e lesions in cases d e sc rib e d by th e m a n d by som e prev io u s
a u th o rs w ere d u e to vascular disease a n d w ere n o t h e ritab le . To illu strate th e dif
ficulty in assessing th e sig n ificance o f such cases, P e arlm an a n d colleagues (1976)
d e sc rib e d a p a tie n t w ith m ild b u t w id e sp rea d u n ila te ra l o u te r re tin a l disease, b u t
they p re s e n te d n o ev idence as to th e in h e rita n c e o f th e disease. It sh o u ld b e e m
p h a sise d th a t in n o case o f u n ila te ra l retin itis p ig m e n to sa has a fam ily h isto ry o f
eye disease b e e n id e n tifie d a lth o u g h , in o n e p a tie n t, th e p a re n ts w ere c o n sa n g u in
e o u s (C o rd ie r e t al., 1966). M ost w orkers take th e view th a t th e re is n o g o o d evi
d e n c e th a t u n ila te ra l retin itis p ig m e n to sa re p re se n ts a d istin c t h e rita b le disease.
T h e expressivity in som e fam ilies is hig h ly variable su ch th a t a b o u t 70% o f
m e m b e rs w ith th e a b n o rm a l g en o ty p e have m o d e ra te to severe disease w hile
th e re m a in d e r a re asym ptom atic, b u t w ith m ild fu n d u s a b n o rm a litie s a n d electro-
physiological c h a n g e s in d ic a tin g th e p re se n c e o f th e a b n o rm a l g e n e (E rn st &
M o o re, 1988). In th ese fam ilies, th e re a p p e a rs to b e b im o d a l e x p ressio n o f disease
a lth o u g h th e factors th a t m o d u la te g e n e ex p re ssio n are un k n o w n .
DIAGNOSIS IN INHERITED RETINAL DISORDERS 51
C h o ro id erem ia
e t al., 1987; C re m e rs et al., 1989; M erry e t al., 1989), b u t d esp ite th e size o f th e
d e le tio n , th e c h o ro id e re m ia rem ain s rem a rk ab ly c o n sta n t in clinical exp ressio n .
It is likely th a t th e p ro m in e n t c h o ro id a l a tro p h y is a se c o n d a ry resp o n se to p ig
m e n t e p ith e lia l cell loss. C h o ro id a l a tro p h y is n o t u n iq u e to c h o ro id e re m ia , since
it h as b e e n id e n tifie d in a b o u t h a lf th e eyes w ith retin itis p ig m e n to sa su b ject to his-
to p a th o lo g ic a l study, b u t p ro fo u n d a tro p h y early is u n lik e re tin itis p ig m e n to sa a n d
signifies a p a th o g e n ic p ro cess d iffe re n t fro m th o se in o th e r o u te r re tin a l diseases.
R e fs u m d ise a se
A b e ta lip o p ro te in a e m ia
T his ra re au to so m a l recessive d iso rd er, w hich was first d e sc rib e d in 1950 (Bassen
& K ornzw eig, 1950), p re se n ts initially w ith m a la b so rp tio n sy ndrom e a n d fat in to l
e ra n c e , a n d has a p ig m e n ta ry re tin o p ath y , progressive ataxic n e u ro p a th y an d
64 ALAN C. BIRD AND BARRIE JAY
O th e r c e n tra l d y stro p h ie s
F u n d u s flav im ac u latu s
T h e re is c o n sid e ra b le d o u b t as to w h e th e r th e fu n d u s ch an g es o f fu n d u s flavi-
rn acu laiu s a n d b u lls eye d ystrophy in d ic a te th a t th e d iso rd e rs a re clearly se p a ra t
e d in to two b ro a d g ro u p s. S tudies o f fu n d u s a p p e a ra n c e show th a t in som e fam ilies
th e ch an g es are c o n sta n t, w hilst in o th e rs b u lls eye dystrophy m ay b e seen in early
disease a n d flavim aculatus lesions seen later.
C o n e d e f e c ts
Mono chromatism
R o d d e fe c ts
Oguchi's disease
Fundus albipunctatus
C O N C LU SIO N
even tu ally b e possible to o ffe r effective tre a tm e n t to th e p a tie n t with re tin itis pig
m en to sa.
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4. HUMAN ALBINISM AND MOUSE MODELS
ABBREVIATIONS USED
AS A n g elm an S y n d ro m e
c m o u se albino locus
CH S C h e d ia k H ig ash i S yndrom e
H PS H erm an sk y P u d la k S yndrom e
OA O c u la r A lbinism
OA1 X -L inked OA
O CA O c u lo c u ta n e o u s A lbinism
OCA1 T y ro sin ase-related OCA
OCA2 T yrosinase-positive OCA
p m o u se pink-eyed dilution locus
P H u m a n P g e n e locus w hich is h o m o lo g o u s to m o u se pink-eyed
dilution locus
PWS Prader-W illi S y n d rom e
TRP1 T yrosinase re la te d p ro te in 1; gp75; brown locus
TRP2 T yrosinase re la te d p ro te in 2; d o p a c h ro m e ta u to m e rase ; slaty locus
TYR H u m a n tyrosinase g e n e locus
IN T R O D U C T IO N
M ELANIN SYNTHESIS
B io c h e m ic a l P ro c e s s
M elan in biosynthesis has b e e n stu d ied fo r over seventy years (R aper, 1928;
M ason, 1948; L e rn e r a n d F itzpatrick, 1950), a n d fo r m o re th a n 50 years was
th o u g h t to be re g u la te d solely by th e enzym e tyrosinase w ith all su b se q u e n t reac
tio n s o c c u rrin g non-enzym atically (C o lem an , 1962). In 1978, L ogan a n d W eath-
e rh e a d re p o r te d th a t h a ir follicle tyrosinase activity o f th e C h in ese h a m ste r was
h ig h w ith th e w in te r a n d su m m e r m olt, w hile p ig m e n te d h airs w ere p ro d u c e d
only w ith th e su m m e r m o lt (L ogan a n d W ea th e rh ea d , 1978). T his show ed th a t
tyrosinase activity in itself wras n o t su fficien t fo r p ig m e n ta tio n a n d o th e r m e c h a
n ism s fo r c o n tro l o f th e p ro cess m u st exist. T his o b serv a tio n led to a se arch fo r
o th e r enzym es a n d facto rs th a t re g u la te m e la n in synthesis, a n d several have now
b e e n id e n tifie d , in c lu d in g tyrosinase re la te d p ro te in 1 o r gp75 (TRP1) (Shiba-
h a ra e t al., 1986; Jack so n , 1988; Jac k so n e t al.,1990), tyrosinase re la te d p ro te in 2
o r d o p a c h ro m e ta u to m e ra se (TRP2) (Ja c k so n , 1988; Jac k so n et al.,1992; Tsuka-
m o to e t al., 1992) a n d Pm el 17-1 (Kwon e t al., 1987). T h e specific ro le o f th ese
newly d e sc rib e d facto rs in n o rm a l m e la n o g e n esis is u n k n o w n b u t a g re a t d e a l o f
w ork is b e in g d e v o te d to this p ro b le m , a n d h u m a n a n d m ouse m u ta n ts are p ro
v idin g im p o rta n t insights.
T h e biosynthesis o f m e la n in starts w ith th e o x id a tio n o f tyrosine a n d e ith e r red-
yellow p h e o m e la n in o r black-brow n e u m e la n in can fo rm (P rota, 1988a; P ro ta a n d
T h o m so n , 1976; P ro ta, 1986; P ro ta, 1988b; P ro ta, 1980). T h e c o m m o n steps to
b o th types o f m e la n in are th e o x id a tio n o f tyrosine to d o p a a n d th e d e h y d ro g e n a
tio n o f d o p a to d o p a q u in o n e , th e two steps b e in g catalyzed by tyrosinase (L e rn e r
a n d F itzpatrick, 1950; P o m e ra n tz a n d W arner, 1967). If sulfhydryl c o m p o u n d s are
p re se n t, they re a c t w ith d o p a q u in o n e a n d c o m m it th e process to p h ae o m elan iza -
tio n th ro u g h a series o f cysteinyldopa a n d b en z o th ia z in e in te rm e d ia te s th a t poly
m erize to acid-soluble p h e o m e la n in (Je rg il e t al., 1983; A g ru p et al., 1979, 1982;
H a n sso n e t al., 1980; R o rsm an e t al., 1 9 7 3 ;J a ra e t al., 1988). T h e p ro d u c tio n o f
p h e o m e la n in a p p e a rs to be c o n tro lle d by th e availability o f sulfhydryl c o m p o u n d s
in th e m e la n o so m e ( J a r a et al.,1988). In th e ab sen ce o f sulfhydryl c o m p o u n d s,
d o p a is c o n v e rte d to d o p a c h ro m e (C ab an es et al., 1987) w hich is th e n c o n v e rte d
to 5,6-dihydroxy indole-2-carboxylic acid by th e catalytic actio n o f d o p a c h ro m e
HUMAN ALBINISM AND MOUSE MODELS 91
M o le c u la r B iology
Tyrosinase
TRP1
TRP2
N y stag m u s
A lm ost all in d iv id u als w ith albinism have nystagm us (Collew ijn e t al., 1985; van
D o rp , 1987; C reel et al., 1990). Several recen tly re p o rte d fam ilies co n ta in individ
94 RICHARD A. KING ET AL.
P h o to p h o b ia
V isu al A cuity
Iris P ig m e n t
R e tin a l P ig m e n t a n d F o v eal D e v e lo p m e n t
O p tic T ra c t D e v e lo p m e n t
All individuals w ith alb in ism have excessive decu ssatio n o f th e o p tic fibers at the
chiasm (C reel e t al., 1974, 1990; G uillery e t al., 1975). T h e ipsilateral p ro je ctio n s
fro m th e re tin a to th e la te ra l g e n ic u la te are re d u c e d a n d the s tru c tu re o f th e lat
eral g e n ic u la te is a b n o rm a l (G u illery e t al., 1975). T h e d e m o n stra tio n o f optic
tra c t m is ro u d n g by visual evoked p o te n tia l studies is th e critical diag n o stic p ro c e
d u re fo r q u e stio n a b le cases (C reel e t al., 1974, 1978, 1981, 1990; A p k arian e t al.,
1990). M isro u tin g o f th e o p tic fib ers can be d e m o n s tra te d w ith visual evoked
p o te n tia ls re c o rd e d w ith m o n o c u la r stim u latio n (L ev en th al a n d C reel, 1985;
C reel e t al., 1974, 1978, 1981; A p k arian e t al., 1990). T h e a lte re d d e v e lo p m e n t o f
th e o p tic tracts c o n trib u te s to th e strab ism u s a n d d im in ish e d stereo acu ity th a t is
usually p re se n t.
O v erv iew
O C U L O C U T A N E O U S A L B IN IS M (O C A )
Unclassified types
Brown OCA
R ufous/red OCA
Primary Defect not Specific for Melanin Synthetic Pathway
Hermansky-Pudlak Syndrome
Chediak-Higashi Syndrome
OCULAR ALBINISM
OA1 X-Linked OA
OA2 Autosomal Recessive OA
Clinical phenotype
Molecular phenotype
>
t M t - O C S S S O < =Ot> 2 H O Z O >
+
>+ >
O Z O O 3 3 o O > > ^ - l q - ro ) ro O cow w w u h & ft H O
r o -* cn '"J co co > - T ^ <> o o *nI 'J -i co <x> o j g o o - 1^
r o - v jc n _ .c d c o - * - ^ 03 { o i\> ct> 05 cn -* -w c o ro c o & M co o ?5 c o c n c o co <
co o2
cn O Z < D r H r a c n S x iS W - i S S D x l -n T o D ia m O m m z x CO .
rim r h i m M n 5\
2 1 3 H 4 -CD i
u
Cu (A)
u
Cu (B)
tyrosinase neg ativ e OCA1 (su p p o sed ly an allele associated with n o tyrosinase
fu n c tio n ). S ince th e yellow QCA1 p h e n o ty p e varies in fam ilies c o n ta in in g individ
uals hom ozy g o u s fo r th e P406L m u ta tio n as well as in individuals w ho are co m
p o u n d h etero zy g o tes, it a p p e a rs th a t o th e r fam ilial p ig m e n t g en es can in flu e n c e
this p h e n o -ty p e (G iebel e t al., 1991d). In th e case o f re d u c e d tyrosinase activity,
re d /y e llo w p h e o m e la n in b iosynthesis is favored over b la c k /b ro w n e u m e la n in
biosynthesis.
In o n e in d iv id u al, a te m p e ra tu re-sen sitiv e fo rm o f tyrosinase w7as o b se rv ed (see
above) (K ing et al., 1 9 9 1 b ). T his in d iv id u al was fo u n d to have a m u ta tio n at c o d o n
422 (R 422Q ) re su ltin g in an a rg in in e to g lu ta m in e su b stitu tio n w ith a tyrosinase
n egative OCA1 m u ta tio n o n th e h o m o lo g o u s allele (G iebel et al., 1991c). E xpres
sion o f th e 422 c o d o n m u ta tio n p ro d u c e d a tyrosinase th a t was in activ ated above
35C. It is in te re s tin g th a t th e m o u se H im alayan m u ta tio n , also a te m p e ra tu re sen
sitive tyrosinase m u ta tio n , is lo c a te d in th e sam e reg io n o f th e p e p tid e , c o d o n 420
(h istid in e to a rg in in e ) (Kwon e t al., 1989). T h e re has also b e e n re p o rte d a tem
p e ra tu re sensitive m u ta tio n a t c o d o n 402 (R 402Q ) (T rip ath i et al., 1991). It is now
obvious th a t th e re is a c o n tin u o u s sp e c tru m o f re d u c e d tyrosinase activities re su lt
in g fro m m u ta tio n s o f th e tyrosinase g e n e (K ing e t al., 1988; K ing a n d O e ttin g ,
1992). T h ese a lte re d tyrosinase m o lecu les p ro d u c e p h e n o ty p ic v ariatio n fro m a
co m p le te ab se n c e o f p ig m e n t to a p ig m e n t p h e n o ty p e in d istin g u ish a b le fro m n o r
m al. In all th ese cases o f OCA1 th e re is o c u la r involvem ent. T his sp e c tru m o f pig
m e n t has also b e e n o b se rv e d in th e c-Iocus m u ta tio n s o f th e m o u se (Silvers, 1979).
Several n o n p a th o g e n ic p o ly m o rp h ism s have also b e e n d e te c te d . Two o f these
re su lt in a m in o acid su b stitu tio n s. A p o ly m o rp h ism a t c o d o n 192 c o n tain s e ith e r
a serin e (TAT) o r tyrosine (TC T) a n d affects a Mbo I re stric tio n e n d o n u c le a se site
(G iebel a n d Spritz, 1990). It is in te re stin g th a t this m u ta tio n at c o d o n 192 is w ithin
th e c o p p e r A b in d in g site b u t d o es n o t a p p e a r to affect th e p h e n o ty p e o f th e in d i
vidual. T h e d istrib u tio n o f this p o ly m o rp h ism is eq u al in C aucasian p o p u la tio n s,
b u t only th e T C T c o d o n is fo u n d in th e O rie n ta l p o p u la tio n . T h e seco n d polym or
p h ism is at c o d o n 402 a n d is e ith e r a n a rg in in e o r g lu tam in e. T h is poly m o rp h ism
d o es affect tyrosinase activity by re d u c in g enzym atic activity by 75% w hen ex
p ressed in c u ltu re d cells b u t th e clinical p h e n o ty p e is n o t a lte re d (T rip ath i e t al.,
1991). A th ird p o ly m o rp h ism is a t th e C C A \T T bo x o f th e p ro m o te r a n d affects a
Taq I restric tio n e n d o n u c le a se site (O e ttin g et al., 1991). A n o th e r po ly m o rp h ism
lo c a te d in th e p ro m o te r reg io n is a G A -cluster 300 bp u p stre a m o f th e sta rt site;
fo u r alleles have b e e n d e te c te d u sing PC R am p lificatio n (M orris e t al., 1991). Two
o th e r p o ly m o rp h ism s are fo u n d by m e a n s o f R estriction F ra g m e n t L e n g th Poly
m o rp h ism s (RFLP) analysis w ith e ith e r Taq I o r Bgl II a n d u sin g th e tyrosinase
cDNA as a p ro b e (Spritz e t al., 1988; Spritz a n d S tru n k , 1990). H a p lo ty p e analysis
can be p e rfo rm e d w ith analysis o f th e se p o ly m o rp h ic sites. T h e m issense m u tatio n
a t c o d o n 47 (G47D) has b e e n o b se rv e d in th re e d iffe re n t p o p u la tio n s, U n ite d
States, P u e rto Rico a n d th e C a n ary Islands (O e ttin g e t al., 1993b). All individuals
h a d th e G 47D m u ta tio n asso ciated w ith h a p lo ty p e 1 show ing a c o m m o n fo u n d e r
fo r this m u ta tio n . O n th e o th e r h a n d , th e m u ta tio n a t c o d o n 81 (P81L) was fo u n d
102 RICHARD A. KING ET AL.
T yrosinase Positive O C A (O C A 2)
Overview
Clinical phenotype
Molecular Phenotype
U nclassified Types
O CU LA R ALBINISM
T h e re a re very few m ouse m u ta tio n s w h ere eye p ig m e n ta tio n is affe cted w ith o u t
h a ir a n d skin involvem ent. O n e m u ta n t allele o f ruby eye-2, maroon, has b e e n d e
scrib ed in w hich som e m u ta n t anim als can have lig h te n e d eyes b u t a n o rm a l coat
co lo r; a lth o u g h affe c ted litte rm a te s m ig h t have m u c h lig h te n e d coats. In su p p o rt
o f th e id ea th a t OA2 h u m a n s m ig h t have tyrosinase m u tatio n s, th e d iffe re n t m u
ta n t alleles o f m o u se albino, th e tyrosinase g en e , can have d iffe ren tial effects on
h a ir versus eye p ig m e n ta tio n . T h e extreme dilution m u ta tio n has a very pale coat,
b u t d a rk eyes, w hereas th e ruby-eyed dilute allele gives a d a rk e r coat w ith ru b y eyes.
O n th e w hole, however, th e reverse is tru e ; m u ta tio n s w hich have lig h te n e d h a ir
b u t d a rk eyes o c c u r m u ch m o re frequently.
CLINICAL APPLICATION
D iagnosis o f A lbinism
T re a tm e n t
FUTURE
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5. INHERITED RETINAL DEGENERATIONS
IN TH E MOUSE
Jules Stein Eye Institute and Department of Ophthalmology, UCI-A School of Medicine, Los
Angeles, California 900241; and Loyola-Marymount University, Los Angeles,
California 90045, USA2
U ntil very recently, none o f the genes responsible for the several in h erited
m urine retinal deg en eratio n s1 had been identified. However, in 1989 and 1990
the defective genes responsible for two o f these diseases were isolated and charac
terized. In each case, a candidate gene was cloned by subtractive an d differential
hybridization and m apped to a particular m ouse chrom osom e by somatic cell
hybrid analysis. B oth candidate genes were localized to the m urine chrom osom e
known to contain the respective disease locus. O ne o f the genes identified is the
retinal degeneration or rd, which causes a rapidly developing, early retinal degener
ation m apping to m ouse chrom osom e 5 (Sidm an an d G reen, 1965) an d encodes
the P subunit of the rod-specific enzyme cyclic GM P-phosphodiesterase (Bowes
et al., 1989; 1990). T h e o th er gene isolated is responsible for the retinal degenera
tion slow or rds m utation, which m aps to m ouse chrom osom e 17 (D em ant et al.,
1979; Travis et al., 1989). T he rds gene encodes a rod p h o to rec ep to r o u ter seg
m en t m em brane p rotein known as rd s /p e rip h e rin (C onnell et al., 1991). Fur
ther, the abortive developm ent o f the ro d o uter segm ents in rds is understandable
in term s o f a defective disc m em brane p rotein. Similarly, the degenerative p ro
cess in rd m ouse photoreceptors closely correlates with the finding of increased
levels o f cyclic GMP d u e to a defective cyclic G M P-phosphodiesterase.
In this chapter, the studies that led to the identification o f the rd gene will be
discussed in detail; the work involved in the identification o f the rds gene will be
sum m arized and the use o f the m ouse as a m eans of identifying an d characterizing
hum an disease genes will be discussed.
'T h e following m ouse m utants show signs o f retinal d eg en eratio n (th eir symbols a n d chrom osom al lo
cations, w here known, are shown in parentheses: retinal d eg en eratio n (rd, 5; rd-3, l:/M-4.4), retinal de
generation slow {rds, 17). p urkinje cell d e g en e ratio n (pal, 13), cribriform d eg en e ratio n (cn, 4),
nervous (nr, 8), vitiligo (vit, 6) (G reen, 1989; Heckenlively et al., 1992; C hang et al., 1993).
124 DEBORA B. FARBER AND MICHAEL DANCIGER
THE rd MOUSE
Retinal degeneration in the m ouse (rd m utation) was first described in wild ani
mals caught in different areas of E urope and is p resen t in several inbred labora
tory strains (Sidm an an d G reen, 1965). Over the years, these m ice have been the
object of many genetic, m orphological, electrophysiological, biochem ical, im m u
nological, m olecular biological and behavioral studies.
T he rd disease is in h erited in an autosom al recessive fashion and results in the
rapid and selective degeneration o f the retinal p h o to rec ep to r cells (Sidm an and
G reen, 1965; LaVail and Sidm an, 1974). T he m orphology of the norm al m ouse
retina is shown in Figure 1. T he rd visual cells appear to form norm ally during the
prenatal an d early postnatal period and to achieve some degree o f differentiation,
as indicated by the developm ent of ru d im en tary rod o u ter segm ents and a synap
tic ribbon com plex (LaVail and Sidm an, 1974; Blanks et al., 1974). T he m orphol
ogy o f a rod p h o to rec ep to r is shown in Figure 2. By 10 days of age, the
p h otoreceptors of both the rd/rd m ouse a n d the rd /+ heterozygote (which has a
m orphologically n orm al retina) look alike at the light m icroscope level, b u t by day
20, virtually all the rd/rd rod visual cells have deg en erated (Noell, 1965). T he sin
gle row o f nuclei left from the o u ter nuclear layer at this time in the rd retin a cor
responds to cone nuclei (Carter-Dawson et al., 1978). Most o f these nuclei
disappear in the following few m onths (Figure 3). At the electron m icroscope lev
el, the first signs of ultrastructural pathology in the rd p hotoreceptors are ob
served on the 7th to 8th postnatal day as 1) fewer ribosom es and swelling o f the
m itochondria o f the in n er segm ents, followed by 2) slower developm ent a n d dis
organization o f the o u ter segm ent m em branes and 3) absence of triad configura
tions at the p h o to recep to r term inals (S onohara and Shiose, 1968; Caley, Jo h n so n
an d Liebelt, 1972; Sanyal and Bal, 1973; Blanks, Adinolfi and Lolley, 1974).
T he retinal pigm ent epithelium as well as the cells o f the in n er re tin a rem ain
m orphologically and functionally norm al d u rin g the course of the dissolution of
the o u ter nuclear layer (LaVail an d Sidm an, 1974). However, subtle an d slow
changes occur in the ganglion cells and in the in n er nuclear and in n er plexiform
layers after several m onths o f life (Grafstein, M urray an d Ingoglia, 1972).
T he rd retina is responsive to high intensity illum ination and betw een 12 and 18
postnatal days it displays an atten u ated electroretinogram which becom es com
pletely flat by 28 postnatal days (Noell, 1965). T he rate o f visual cell degeneration
is n o t accelerated by the level o f environm ental light in which the mice are reared.
T he first clues regarding the cause o f the rd m utation cam e from studies o f cyclic
nucleotide m etabolism . Cyclic GMP levels becom e elevated in the rd retin a when
co m pared to control retina by the 6th postnatal day, at least two days p rio r to the
onset o f p h o to rec ep to r cell degeneration, an d they are several-fold h ig h er than
norm al by day 14 (Farber an d Lolley, 1974). Cyclic GMP levels decline thereafter,
correlating with the loss o f visual cells from the rd retina. Analysis o f microdis-
I N H E R I T E D R E T I N A I . DEC. E N E R A T K ) N S IN T H E M O l SI- 115
Choriocapillaris
RPE
Outer segments
Inner segments
Photoreceptor
Outer nuclear layer
layer
Outer plexiform
layer
Inner nuclear
layer
Inner retinal
layers
Inner plexiform
layer
Ganglion cell
layer
Pigment Epithelium
F ig u re 2 D ia g ra m o f a v e r te b r a te r o d p h o t o r e c e p t o r cell. T h e o u t e r s e g m e n t c o n ta in s sta ck s
o f m e m b r a n o u s disks f o r m e d b y lip id b ila y e rs w h ic h p ro v id e a m a trix fo r p r o te in m o le c u le s.
M o st o f th e s e d isks a r e n o t c o n tin u o u s w ith th e p la s m a m e m b r a n e o f th e p h o t o r e c e p t o r a l
th o u g h th e y a r e c o n n e c te d by f ila m e n to u s s tru c tu r e s . T h e visual p ig m e n t rh o d o p s in , th e m a
j o r p r o t e i n o f t h e r o d o u t e r s e g m e n t, is a n in tr in s ic g ly c o p r o te in w h ic h s p a n s th e d isk
m e m b r a n e th ic k n e s s c ro ssin g th e m e m b r a n e i n te r f a c e sev eral tim e s. A m o d ifie d c iliu m c o n
n e c ts th e o u t e r s e g m e n t w ith th e i n n e r s e g m e n t, w h e re th e c ell sy n th e siz e s p ro te in s , p r o d u c e s
th e e n e rg y -ric h n u c le o s id e tr ip h o s p h a te s a n d a sse m b le s th e d isk m e m b ra n e s . B elow t h e n u c le
us, th e p h o t o r e c e p t o r a x o n p r o c e e d s to th e s y n a p tic te r m in a l, w h ic h fo rm s sy n a p se s w ith th e
n e u r o n s o f th e i n n e r r e tin a a n d c o n ta in s th e s p e c ia liz e d b io c h e m ic a l m a c h in e r y n e e d e d fo r
n e u r a l sig n a lin g .
INHERITED RETINAI, DEGENERATIONS IN THE MOUSE 127
Choriocapillaris
RPE
Inner nuclear
layer
Inner plexiform
layer
G anglion cell
layer
sected samples consisting o f the p h o to rec ep to r layer and the in n er layers o f the
rd retina showed th at cyclic GMP accum ulates exclusively in the p h o to rec ep to r
cells (Farber and Lolley, 1974). This elevation in cyclic GMP levels is the result o f
a deficiency in the activity o f the ro d p h o to recep to r cyclic GM P-phosphodi-
esterase, the enzyme th at hydrolyzes cyclic GMP to 5'-GMP (Farber and Lolley,
1976). Cyclic GMP is synthesized norm ally by the rd visual cells via the guanylate
cyclase reaction from the onset of differentiation (Farber and Lolley, 1976).
Cyclic G M P-phosphodiesterase activity could never be d etected when it was as
sayed in hom ogenates o f freshly dissected retinas obtained from rd m ice of any
age. But if kinetic studies were carried out using freeze-dried retinas o f 12-day-old
rd mice, the enzyme was found to have a M ichaelis constant (Km) sim ilar to that
of p h o to recep to r cells o f control retina, although its m axim um velocity (Vmax)
was well below norm al (Farber and Lolley, 1977). T he abnorm ality in cyclic nucle
otide hydrolysis seems to be restricted to the retinal photoreceptors. Phosphodi
esterases in o th er tissues of the rd m ouse such as m uscle, skin, brain and several
blood com ponents - plasma, serum , lymphocytes and erythrocytes - have activities
com parable to those present in the corresponding tissues o f control mice (Lolley
and Farber, 1980).
128 DEBORA B. FARBER AND MICHAEL DANCIGER
during all the developm ental period in which the p h o to rec ep to r cells are viable
(Farber et al., 1988). However, m easurem ents of cyclic GM P-phosphodiesterase
levels by radioim m unoassay showed th a t the concentration o f the enzyme in the
rd retina is already lower than norm al at 6 postnatal days, w hen no signs of degen
eration are discernible, an d th at it rem ains at about half the norm al level until the
p hotoreceptors begin to die (Farber et al., 1988). These results suggested that rd
cyclic G M P-phosphodiesterase is abnorm ally labile and that it gets degraded very
soon after it is m ade. In agreem ent with this idea is the finding th at peptides from
im m ature rd retinas that are recognized by antibodies against cyclic GMP-phos
phodiesterase sedim ent on sucrose gradients m ore slowly than the corresponding
proteins from norm al m ouse retina. T hese proteins m igrate within the gradient
with an a p p a ren t m olecular mass o f 105 kDa, w hereas the proteins from the n o r
mal retina form a com plex with an a p p a ren t m olecular mass o f 170 kDa. T he 170
kDa com plex is never d etected in extracts from rd retina (Lee et al., 1988). Thus,
the subunits of cyclic G M P-phosphodiesterase do n o t assemble into a functional
com plex in rd photoreceptors. This w ould m ake them susceptible to proteolysis
once they are synthesized.
D uring the course o f the studies focused on d eterm in in g w hether the proteins
involved in the activation/deactivation of cyclic G M P-phosphodiesterase in the rd
retin a were functional, a defect in the process o f dephosphorylation of rd rh o d o p
sin was identified. A lthough the co n cen tratio n o f the visual pigm ent is com parable
in norm al an d rd ph o to recep to rs until 10-11 postnatal days (Caravaggio an d Bon-
ting, 1963; Shuster an d Farber, 1986), rd rhodopsin cannot be phosphorylated in
vitro at any age (Shuster an d Farber, 1986). This lesion is n o t caused by an abnor
mality in rhodopsin kinase (the enzyme which catalyzes the transfer o f phosphate
from ATP to rh o d o p sin ), since the activity of the rhodopsin kinases from norm al
and rd retinas is com parable during all the developm ental period in which the rd
photoreceptors are viable (Palczewski, Farber an d H argrave, 1991). N or does the
lesion result from a defective rhodopsin m olecule, since at the mRNA level and in
im m unocytochem ical reactions, the rhodopsins from norm al an d rd retinas are
identical (Bowes a n d Farber, 1988; Bowes, van Veen an d Farber, 1989). Lack of
phosphorylation o f rd rho dopsin stem s from abnorm al activity of protein phos
phatase 2A, the enzyme that dephosphorylates rhodopsin (Palczewski, F arber and
Hargrave, 1991). P rotein phosphatase 2A activity is twice as high in rd than in nor
mal retina betw een 5 an d 10 postnatal days, although the rrf enzyme seems to be
structurally norm al. Apparently, some regulatory co m p o n en t p resen t in the rd ret
inal extract may be responsible for its increased activity. In previous studies carried
ou t in o u r laboratory, rhodopsin phosphorylation in o u ter segm ents o f norm al
rod photoreceptors was shown to be inhibited by high concentrations o f cyclic
GMP (Shuster and Farber, 1984). A possible explanation for this p h en o m en o n is
that raised cyclic GMP could en h a n ce the activity of protein phosphatase 2A,
which would remove the phosphate from rhodopsin as soon as the visual pigm ent
is phosphorylated. O u r findings with the rd retin a are consistent with this hypoth
esis. T he abnorm ality in the rd p ro tein phosphatase 2A activity w ould then be sec
130 DEBORA B. FARBER AND MICHAEL DANCIGER
C oncom itant with the work described above, the search for the rd gene began by
following a totally different strategic plan. This plan was based on the assum ption
th at the mRNA encoded by the rd gene would be eith er absent o r expressed in
h ig h er o r lower levels in the rd ph o to recep to rs com pared to norm al visual cells.
T he approach was to obtain the cDNAs present exclusively in the norm al p h o to
receptors an d then to com pare these cDNAs with the same pool o f cDNAs
p resen t in rd visual cells in retinas of 9- to 11-day-old mice. N ine to eleven day-old
rd/+ an d rd/rd litterm ates were used for the experim ents, since the m axim um
am o u n t o f age-m atched retinal tissue can be obtained at this age, before the rd/rd
cells suffer advanced degeneration.
T he adult m utant retin a appeared to be the perfect starting m aterial, since it is
devoid of all the ph o to recep to rs and is constituted solely by the in n er retinal lay
ers. A dult rd/rd retin a cDNAs were subtracted from those o f 9- to 11-day old nor
mal, rd/+ m ouse retina, and the photoreceptor-enriched, subtracted cDNAs were
used to probe a library o f the 9- to 11-day-old rd/+ retina. From approxim ately
100,000 clones tested, a pool o f 588 p h otoreceptor-enriched cDNAs was obtained.
These clones were screened by differential hybridization with single-stranded
cDNAs from 9- to 11-day-old rd/+ and rd/rd retinas and from adult rd/rd retina.
C om parison o f the positive clones hybridizing to the young an d the adult retinal
cDNAs confirm ed the p h o to rec ep to r specificity o f 400 clones; the rest could be
ru led out, since any clone able to hybridize to adult rd/rd m ouse cDNAs had to be
long to the in n er retinal layers. O f these 400 clones, 3 clones hybridized differently
with the 9- to 11-day-old rd/+ and rd/rd cDNAs. However, only one o f these candi-
INHERITED RETINAL DEGENERATIONS IN TH E MOUSE 131
T a b le 1 C h r o m o s o m a l lo c i f o r th e g e n e s o f p r o t e i n s in v o lv e d in th e v isu al cycle o f r o d p h o
to r e c e p to r s .
Gene Mouse
Protein symbol Chromosome Reference
date clones for the rd gene, zr.408, gave a different hybridization signal on N orth
ern blots containing the mRNA o f young rd/rd retinas com pared with the mRNA
of age-m atched, rd/+ control retinas (Bowes et al., 1989).
discussed below, the second a n d third possibilities have been shown to occur. In
addition, differences betw een the rd/rd and + /+ m ouse DNAs revealed by
genom ic S outhern blot analysis, confirm ed th at structural differences in d eed
exist between the m u tan t an d norm al genes hybridized by zr.408. Tissue specific
ity o f the candidate cDNA was d eterm in e d by hybridization on slot blots to RNA
from several m ouse tissues. Only retinal RNA gave a strong signal, w hereas brain,
kidney, liver, lung and spleen RNAs were negative (Bowes et al., 1989).
T he chrom osom al location o f the gene corresponding to the zr.408 cDNA was
established by hybridization o f the candidate cDNA with the DNA o f ham ster-
m ouse somatic cell hybrids. T he zr.408 clone m apped, w ithout any discordancy, to
m ouse chrom osom e 5, the same chrom osom e to which the rd locus h ad b een as
signed. Expression of the mRNA encoded by the putative rd cDNA was detectable
on slot blots on postnatal day 1 in both norm al and rd/rd retinas, an d it continued
to increase steadily during developm ent. However, the concentration o f zr.408
mRNA was always lower in the diseased than in the + / + retina. By postnatal day 14,
following the degeneration p attern o f the rd retina, the rd zr.408 mRNA level d e
creased sharply and, as expected, it was n o longer detectable at 31 days o f age.
T herefore, the expression of the candidate rd cDNA is abnorm al in the rd/rd reti
n a from postnatal day 1, several days before cyclic GMP levels becom e elevated in
the p h o to rec ep to r cells. This is the earliest detected m olecular lesion in the rd dis
ease (Bowes et al., 1989).
In o rd e r to fu rth e r confirm that zr.408 was a p art o f the rd gene, two d ifferent
investigations were carried out. T he aim o f the first was to establish w hether the
gene corresponding to the zr.408 cDNA a n d the rd gene m ap p ed to the same
location on chrom osom e 5. T he second study was to verify if there was cosegrega
tion o f the candidate rd gene with the expression o f the rd disease, p h o to rec ep to r
degeneration, in m ouse crosses. Interspecies backcrosses were used for both
projects, since the mice resulting from these crosses have been shown to give
restriction fragm ent length polym orphism s (RFLPs) m ore frequently than those
resulting from in b red crosses.
To establish the location o f the gene corresp o n d in g to the zr.408 cDNA within
chrom osom e 5, the RFLPs d etected by hybridization to the zr.408 cDNA were first
analysed in the DNAs from N F S /N an d Mus musculus m ice. These RFLPs were
th en identified in DNAs from 62 m ice which were the backcrossed progeny o f the
cross (N FS/N x Mus musculus)F 1 xM us musculus. This allowed us to d eterm in e how
m any o f the 62 progeny were hom ozygous or heterozygous for zr.408. T he same
type o f analysis was p erfo rm ed for RFLPs th at were hybridized by Afp and Gits, two
o th e r genes that lie on opposite sides o f the rd gene on chrom osom e 5. From a
com parison o f the in h eritan ce p attern o f the three genes, we established the nu m
b er of recom binants present, an d from the rate o f recom bination, the distance in
centiM organs o f each gene relative to the others (D anciger et al., 1990). In this
INHERITED RETINAL DEGENERATIONS IN THE MOUSE 133
way, zr.408 was placed betw een Afp an d Gus at distances sim ilar to those established
for the rd gene (C hapm an et al., 1975).
To d eterm in e w hether the zr.408 cDNA cosegregated with the retinal degener
ation trait, we classified 72 mice, the backcrossed progeny o f the cross (C57BL/6J
rd/rd x Mus spretus)FI x C57BL/6J rd/rd, eith er as hom ozygous (rd/rd, affected with
retinal degeneration) o r heterozygous (rd/+, m orphologically norm al) by two dif
feren t m ethods. In the first m ethod, backcross progeny were scored by RFLP
S outhern blot analysis to find ou t if they were hom ozygous or heterozygous for
zr.408 hybridizing fragm ents co rresponding to the C57BL/6J strain. In the second
m ethod, we p erfo rm ed a histological exam ination o f each o f the retinas o f the 72
progeny mice. W hen the results o f both approaches were com pared, zr.408 was
found to cosegregate with the rd disease with no recom binants; that is, each of the
assignm ents to rd/rd or rd/+ d eterm in e d by S outhern blot hybridization with
zr.408 corresp o n d ed exactly to the histologic identification (D anciger et al.,
1990).
T he m olecular genetic results from these two d ifferent studies su pported very
strongly the n otion th at zr.408 was a p a rt o f the rd gene. As described below, this
has since been confirm ed in studies o f transgenic mice.
T he initial search for hom ologies betw een zr.408 an d cDNAs encoding the a and
P subunits o f ro d cyclic G M P-phosphodiesterase an d the cone a 1 subunits o f the
enzyme showed strong cross-hybridization with the ro d (3 subunit cDNA. Since
zr.408 had been isolated from a cDNA library p rep ared with RNA from rd/+ reti
nas, this clone was used to screen n orm al m ouse (+/+) retinal cDNA libraries.
Sequencing o f several o f the isolated clones an d o f PCR-derived fragm ents, estab
lished th at zr.408 is a tru n ca ted clone which corresponds to 2 /3 o f the total
sequence o f the p subunit o f cyclic G M P-phosphodiesterase (from base 1,114 to
base 2,706). T here is only one nucleotide changed in the zr.408 sequence, which
may be due to a polym orphic difference in the m ouse strains th at were used for
the generation o f the libraries (Bowes et al., 1990).
In o rd e r to fu rth e r confirm that zr.408 is a p art o f the P subunit o f cyclic GMP-
phosphodiesterase, a com parison o f the patterns o f hybridization o f poly (A)+
RNA an d genom ic DNA from rd/rd, rd/+ an d + /+ m ouse tissues with zr.408 cDNA
an d with several m urine an d bovine cDNA probes o f the P subunit o f cyclic GMP-
phosphodiesterase was carried out. T he same transcripts as well as RFLPs were ob
served on the N o rth ern and S outhern blots with all d ifferent cDNA probes tested.
These observations, along with the m apping of zr.408 to the rd region on m ouse
chrom osom e 5, su p p o rted the conclusion th at the norm al co u n terp art of the rd
gene encodes the P subunit o f cyclic G M P-phosphodiesterase (Bowes et al., 1990).
134 DEBORA B. FARBER AND MICHAEL DANCIGER
ence o f Xmv-28 at this site and the rd m utation in 16 m ouse strains, while n o n e of
36 non-rd strains contained Xmv-28 (Bowes et al., 1993). T he Xmv-28/rd allele has
also been found in strains derived from wild mice caught recently in Asia, USA and
Britain, which raises questions ab o u t the evolutionary significance o f such a wide
spread and apparently ancient m utation. As m en tio n ed before, the levels o f [3-sub-
u n it mRNA are greatly red u ced in rd mice as well as abnorm al in size. It is
therefore unclear w hether the ab e rra n t transcription or the stop codon or both
give rise to the rd phenotype. We are currently investigating the m echanism s that
lead from cyclic G M P-phosphodiesterase deficiency to death o f rd photoreceptors.
A variety o f o th er anim al m odels o f retinal degeneration have been identified,
one o f which also appears to have a m utation in the P subunit of ro d cyclic GMP
phosphodiesterase (Farber et al., 1992). T he Irish setter dog suffers from an early
onset rod-cone p h o to rec ep to r deg en eratio n d u rin g the period o f o u ter segm ent
elongation. T he retinas o f these dogs are deficient in cyclic GM P-phosphodi
esterase activity and have greatly elevated levels o f cyclic GMP that precede m or
phological signs o f degeneration, sim ilar to rd/rd mice (Aguirre et al., 1978).
While the p h o to recep to r cells o f the diseased retinas are viable, the mRNAs for op
sin, transducin oc^ an d pj, arrestin, phosducin an d the a an d y subunits o f cGMP-
phosphodiesterase have norm al transcript sizes an d levels. In contrast, the cyclic
G M P-phosphodiesterase p-subunit mRNA is p resen t in levels lower than norm al
from early life (Farber et al., 1992). Since this abnorm ality is observed prior to any
signs o f arrested p h o to rec ep to r developm ent an d p rio r to the described biochem
ical defect (Aguirre et al., 1982), o u r results indicate a specific involvem ent o f cy
clic G M P-phosphodiesterase P-subunit in the Irish setter disease. Recently, Suber
et al.(1993) showed that this is associated with a nonsense m utation in the canine
hom ologue of the rd gene which is pred icted to elim inate 49 am ino acid residues
from the C term inal e n d o f the P subunit o f cyclic GM P-phosphodiesterase, includ
ing an isoprenylation addition site. This isoprenylation site o f the norm al p-sub-
unit may facilitate its interaction e ith e r with the disk m em branes or with o th er
protein subunits of the enzyme.
Finally, several m utations have recently been re p o rted in the hum an hom ologue
of the rd gene in patients with autosom al recessive retinitis pigm entosa (ARRP).
M cLaughlin et al. (1993) studied 7 o f the 22 exons of the hum an cyclic GMP-phos-
phodiesterase p-subunit gene in 99 un related patients. They fo u n d 4 m utations
that were n o t observed in 100 control individuals w ithout ARRP an d that co-seg-
regated with the disease in families. Two o f these were nonsense m utations (Gin
298X an d Arg 513X), one was a missense m utation (His 557 Tyr) an d one was a
one bp deletion (Pro 496). A lthough fu rth e r work is req u ired to establish the fre
quency o f these m utations in ARRP, these exciting findings provide fu rth e r valida
tion o f the rd m ouse as a m odel for hu m an retinal degeneration.
136 DEBORA B. FARBER AND MICHAEL DANCIGER
In the late 1970s, a new retinal d egeneration was described in the 020/A inbred
m ouse strain which was found to be due to a single defective gene in h erited as an
autosom al recessive (van Nie et al., 1978). T he gene was localized distal to and
n ea r the H-2 locus in the m iddle region o f m ouse chrom osom e 17 (D em ant
et al., 1979). Due to the relatively slow rate o f degeneration o f the retinal ph o to
receptors in the 020/A mice when com pared with the rate o f d egeneration of
these cells in hom ozygous rd mice, the m utation was nam ed retinal degeneration
slow or rds (van Nie an d D em ant, 1978).
D egeneration in hom ozygous rds mice is characterized by absence of the o uter
segm ents o f ro d p h otoreceptors and the gradual and com plete loss o f the rest of
these visual cells over time. T he rod o u ter segm ents, which norm ally begin to d e
velop during the first week of life, simply fail to form in the rds m ouse even though
the o th er retinal layers appear to develop norm ally. In place o f the rod o u ter seg
m ent, a bulb o f plasm a m em brane w ithout any disk structures accum ulates on top
o f the cilium. T he o u ter segm entless p h o to rec ep to r cells then gradually d egen
erate over a p erio d of 7 m onths to a year u n til they are no longer found in the ret
ina (for reviews of the rds phenotype, see Sanyal et al., 1985; Hawkins et al., 1985;
C ohen, 1983). A lthough the rds m utation was first decribed as a recessive, rds/+
heterozygotes show rod o u ter segm ents th at are reduced in length an d contain ir
regular, swollen and vacuolated disks. T he ph o to recep to rs also u n d erg o very slow
degeneration, so th at the rds m utation is sem i-dom inant.
Over a period of several years, m any im m unocytochem ical studies were per
form ed which com pared the am o u n t an d localization o f several p h o to rec ep to r
proteins in rds and norm al retina. T he proteins investigated included the inter-
p h o to rec ep to r retinoid binding protein (IRBP), opsin, arrestin, transducin,
actin, an d sulfated proteoglycans (van Veen et al., 1988; Carter-Dawson and Bur
roughs, 1989; Schalken et al., 1990; Navon e t al., 1987; Jansen et al., 1990; C haitin
et al., 1988; Tawara an d Hollyfield, 1990; C antera et al., 1990). A lthough each of
these studies co ntributed to o u r un d erstan d in g o f the m echanism o f p h o to recep
tor degeneration, n o n e was able to specifically p o in t to the defective gene in the
rds retina. In fact, the rds gene was identified by a com pletely different approach,
again utilizing the techniques of m olecular biology (Travis et al., 1989).
Retinal cDNA was m ade from adult rd/rd mice which, as described earlier, do
n o t have any photoreceptors. This pool o f photoreceptorless cDNAs was used to
subtract non -p h o to recep to r messages from cDNAs isolated from norm al retina.
T he rem aining cDNAs from norm al retina, highly en rich ed for p h o to recep to r
cDNAs, were cloned an d screened with single stranded cDNAs from developing
norm al ds/rds retinas. Twelve clones were selected which seem ed to give d ifferent
hybridization signals with m u tan t an d norm al probes. N o rth e rn blot analysis and
INHERITED RETINAL DEGENERATIONS IN TH E MOUSE 137
chrom osom al localization by m eans o f som atic cell hybrids revealed a single cDNA
clone that hybridized m ore weakly with rds/rds retinal mRNA (com pared to n o r
mal retinal mRNA) th at is increased in size an d m apped to m ouse chrom osom e
17. F u rth er study o f the rds m utation dem onstrated that the gene was defective
due to disruption of the coding region by insertion o f a 2.7 kb m ouse repetitive
elem ent at nucleotide position 899 (the entire coding sequence is 1038 bp, coding
for 346 am ino acids; Travis et al., 1989).
T he norm al pro d u ct o f the rds gene is a p h o to rec ep to r disk protein called rd s /
p erip h erin to distinguish it from an u n re la ted neurofilam ent protein called pe-
rip h erin (C onnell et al., 1991; Travis et al., 1992a; Arikawa et al., 1992). T he
bovine hom ologue o f rds/p e rip h e rin h ad been identified independently of the rds
study (C onnell and Molday, 1990). R d s/p e rip h e rin is a 39 kDa glycoprotein o f u n
known function, localized by im m unocytochem istry to the rims o f p h o to rec ep to r
disks (Molday et al., 1987) which is pred icted to have four m em brane-spanning
a-helices an d is expressed exclusively in the ro d an d cone p h o to recep to r disks.
T he rds m utation would be expected to tru n cate the protein p ro d u c t by 116 am ino
acids with loss o f the glycosylated region o f the intradiscal D2 loop, th o u g h t to be
im p o rtan t in interaction with o th er proteins, an d o f the fourth m em brane-span
ning segm ent and the cytoplasmic C3 segm ent (Travis et al., 1989).
C om plete rescue o f the m u tan t rds phenotype has recently b een achieved in
transgenic m ice th at h ad in co rp o rated norm al copies o f the rd s /p e rip h e rin cDNA
(Travis et al., 1992b). T he transgene contained upstream regulatory sequences
from the m ouse opsin gene fused to a norm al rd s /p e rip h e rin m ini-gene with the
SV40 early splice an d polyadenylation signals. T he construct was m icroinjected
into fertilized m ouse eggs from rds/+ m utants. T hree transgenic lines were ob
tained an d crossed with hom ozygous rds m utants to observe the effects o f the
transgene in a hom ozygous rds/rds background. Two high rd s/p e rip h e rin express
ing lines showed retinas th at were indistinguishable from the wild-type, with nor
mal o u ter segm ent m orphology an d no p h o to rec ep to r degeneration. An
interm ediate phenotype was seen in a low expressing line, with about 10% o f wild-
type rd s/p e rip h e rin levels, which showed whorls o f disorganized disks in the sub-
retinal space, representing dysplastic o u ter segm ents. N on-transgenic litterm ates
showed the typical rds/rds m u tan t phenotype. These results indicate th at the semi
d om inant rds phenotype probably results from haplo-insufficiency - lack o f suffi
cient norm al gene p ro d u ct even in the heterozygous state - ra th e r than a delete
rious effect o f the m u tan t p ro d u ct per se (dom inant negative m utation). This
would be consistent with the rds m utation being a null allele, with little or no ex
pressed product, as predicted from the m utation analysis.
T he introduction of upstream p ro m o ter sequences from the m ouse opsin gene
into transgenic mice coupled to a lacZ re p o rte r gene had previously been shown
to drive rod-specific transgene expression with onset shortly after the appearance
of o u ter segm ent structures (around the first postnatal week) (Lem et al., 1991;
Zack et al., 1991). T he transgenic rescue o f rds m utants would be expected to re
verse rod bu t n o t cone p h o to rec ep to r degeneration, since rds/p e rip h e rin is ex
138 DEBORA B. FARBER AND MICHAEL DANCIGER
pressed in both cell types and the opsin p ro m o ter is rod-specific. However, only
ab out 3% o f photoreceptors in mice are cones (Carter-Dawson et al., 1978) an d it
was not possible to confirm this prediction (Travis et al., 1992b).
T he m ain difference betw een the course o f scientific discovery o f the rhodopsin
m utations and o f the R D S /p erip h erin m utations in m an was the direct involve
m en t o f the rds anim al m odel o f retinal degeneration in the latter case. H um an
m apping an d pedigree linkage analysis p u t rhodopsin and ADRP together. Since
rhodopsin is the m ost ab u n d a n t p rotein in ro d photoreceptors, it was a logical
choice for the site o f a gene defect. Interest in the hum an p erip h erin gene as a
candidate for RP m utations occu rred after it was dem onstrated that its m urine h o
m ologue was responsible for the rds disease, one o f several m ouse m odels o f RP.
This is a straightforw ard exam ple o f a case w here the hum an hom ologue o f a gene
responsible for an in h erited disease in an anim al is responsible for a sim ilar h u
m an disease. As discussed above, the hum an co u n terp art of the rd g ene also now
appears to cause retinal d egeneration in m an. It may well be that, w hen the m o
lecular basis o f the o th er rem aining m ouse retinal degeneration m utants becom es
known, fu rth e r hum an disease genes will be identified and explained.
T he gene for rhodopsin (Rho) was localized to distal m ouse chrom osom e 6 in the
vicinity of the m icroophthalm ia (mi) locus (Elliott et al., 1990), an in h erited
m urine defect which results in a sm aller eye. A nother gene expressed in the re t
ina has recently b een m apped to the same chrom osom al region, a third G p ro
tein p-subunit, Gnb-3 (D anciger et al., 1992a). F u rth er studies will n eed to be
carried ou t with both Rho and Gnb-3 now that they are potential candidates for mi
based on their chrom osom al location.
T he gene for the a-subunit of calciu m /calm o d u lin -d ep en d en t p rotein kinase II
(Camk2a ) has also recently been m ap p ed to m ouse chrom osom e 18 in the vicinity
of the bouncy (be) neurological m u tan t (D anciger et al., 1992b). T he Camk2a gene
p ro d u ct is p resen t in brain, re tin a a n d o th er tissues. Since the only published in
form ation about the be m u tan t is related to its neurological symptoms (bouncy gait
and trem or) or its breed in g capabilities (Lane et al., 1981; G reen, 1989), it is pos
sible that pathology involving o th er tissues (such as retina) may occur. T herefore,
by virtue of its chrom osom al location and expression in brain a n d o th er tissues,
the Camk2a gene is a potential candidate for the be m utation.
In the course o f m apping the transducin p-subunit gene (Gnb-1) to m ouse chro
m osom e 4, we localized two o th er hom ologous sequences to m ouse chrom osom es
5 and 8 (D anciger et al,, 1990a). C hrom osom al m apping was done by S outhern
blot analysis o f C hinese ham ster-m ouse som atic cell hybrid DNAs th at were
probed with the X TB112 cDNA for the Gnb-1 gene (provided by Dr. Melvin I. Si
m on [Sugim oto et al., 1985; Fong et al., 1986]; Figures 4 an d 5 an d Table 2 ). Link
age analysis with the m arker genes Afp an d Gus (Table 2) was used to localize the
chrom osom e 5 sequence (Gnb-2) to a region that was hom ologous to the region of
hum an chrom osom e 7 w here a second G p rotein p-subunit gene (GNB2) had
been m apped (Blatt et al., 1988; Hillyard et al., 1992; Lyon and Kirby, 1992; Searle
140 DEBORA B. FARBER AND MICHAEL DANCIGER
et a l , 1987). Linkage data was collected prim arily by m eans o f allelic RFLP analysis
o f S outhern blots (Figure 6 and Table 3). T he G n b -2 gene was in the vicinity of the
rd locus, but m ore than 10 cM distal to it (Hillyard e t al., 1992; Lyon an d Kirby,
1992). T herefore, fine m apping dem onstrated th at G n b -2 is n o t the ratgene - which
was later confirm ed by the identification o f the rd site as the cyclic GM P-phospho-
diesterase p-subunit gene - avoiding the n eed for fu rth e r effort on the G n b -2 gene.
1 2 3 4 5 6 7 8 9
- 23.1 kb
-- 9.4 kb
- 6.6 kb
8
5 -> -- 4.4 kb
4
S
2.3 kb
~ 2.0 kb
1 33 50 38
2 46 58 39
3 38 38 33
4 0 36 24
5 33 0 29
6 42 62 35
7 50 77 65
8 20 40 4C
9 23 35 23
10 27 15 19
11 33 11 22
12 42 74 47
13 38 58 46
14 32 32 24
15 59 88 71
16 30 26 39
17 46 58 39
18 29 38 29
19 32 52 24
X 36 44 36
a T h e chrom osom e c o n te n t o f the som atic cell hybrids was d e te rm in e d by trypsin-Giem sa b an d in g fol
lowed by staining with H oechst 33258, o r by typing for specific m ark e r loci.
b T h e n u m b e r o f som atic cell hybrids th a t had the p a rticu la r m ouse chrom osom e b u t did n o t show the
m ouse hybridizing sequence o f th e gene, plus th e n u m b e r o f hybrids th a t did not have th e c h ro m o
som e b u t d id show the hybridizing sequence o f th e g en e, divided by the total n u m b e r o f hybrids eval
u a te d for th a t p a rticu la r chrom osom e.
c T h e 4% discordancy was d u e to o n e hybrid w hich c o n ta in ed Chr.8 , but did not show th e 5.9-kb scor
ing m ouse fragm ent. T h e discrepancy can be exp lain ed by the fact th at this hybrid line had a low
percentage o f cells c o ntaining C hr.8 (low copy n u m b er). F urther, the C hr.8 assignm ent was co rro b
o rated by m ultilocus linkage analysis o f backcross progeny (D anciger e t al., 1990a).
INHERITED RETINAL DEGENERATIONS IN THE MOUSE 143
1 2 3 4 5
#
mm
F igu re 6 A u to ra d io g r a m o f a S o u th e r n b lo t o f te s t c ro ss p ro g e n y D N A s fro m th e c ro ss (N F S /
N x Al. m. m usculus)F I x M . in. musculus. D N A s w e re d ig e s te d w ith H in d lll a n d h y b rid iz e d w ith
X T B 1 12 cD N A ; e a c h la n e h a s 10 |ig o f D N A . L a n e 1, p a r e n ta l N F S /N DN A; la n e s 2-5, r e p r e
s e n ta tiv e b a c k c ro ss p ro g e n y . T h e a rro w s p o in t to a 14 k b A l m. m usculus f r a g m e n t ( A l m. m.)
p r e s e n t in all te s t c ro ss p r o g e n y a n d a 4.5 k b f r a g m e n t r e p r e s e n tin g th e u n i q u e N F S /N a lle le
(fo r Gnb-2 ) p r e s e n t o n ly in h e te ro z y g o te s . D N A s w e re e x tr a c te d fro m m o u s e livers, c le a v e d
w ith H in d lll, r u n o n 0.4% a g a ro s e gels, t r a n s f e r r e d to n y lo n m e m b r a n e s a n d h y b rid iz e d to
la b e le d p r o b e as d e s c rib e d by I l o g g a n e t al. (1 9 8 8 ).
144 DEBORA B, FARBER AND MICHAEL DANCIGER
parentals
+b + + 21
- - - 23
single recombinants
+ - - 9
- + + 6
+ + - 3
- - + 0
double recombinants
- + - 1
% R ecom bination
(Afp, Gnb-2) = 16/6 3 = 25 5.5C
(Gnb-2, Gus) = 4 /6 3 = 6 3 .i
a Afp a n d Gus alleles were scored by S outhern blot hybridization with ap p ro p ria te cDNA probes. DNA
was digested with EcoRI for Afp, and llirullil for Gus. A unique 6.0-kb N F S/N restriction frag m en t hy
bridized with a 960-bp cDNA p ro b e released from the pBR322-AFPl plasm id (provided by Dr. Shirley
T ilghm an, Princeton University [D Eustachio et al., 1981]). A u n iq u e 3.7-kb N F S/N restriction frag
m en t hybridized with a 1.45-kb cDNA probe c o rresp o n d in g to the Gus gene, released from the pGA l
plasm id (provided by Dr. G ordon W atson, UC Berkeley [Watson e t al., 1985]).
b T he symbol refers to progeny th at are heterozygous.
'P e rc e n t recom bination and standard e rro rs were calculated according to G reen (1981); all recom bi
n a n t fractions are significant to the 0.05 level.
O nce a gene has been localized in the m ouse genom e, it is an easy m atter to
check the chrom osom al m aps to see if the hom ologous region in the hum an con
tains any m utations. For exam ple, N orth C arolina m acular dystrophy has recently
been localized to hum an chrom osom al region 6q by linkage analysis of a single
large family (Small et al., 1992). H um an 6q has hom ology with small regions of
m ouse chrom osom es 4, 9 and 17 an d with a larger region o f m ouse chrom osom e
10. Thus, any m urine retinal gene m apping to these regions becom es a potential
candidate for this disease. T here are o th er in h erited retinal diseases that have
been linked to regions o f the hu m an genom e an d to which the same logic can be
applied: U sher syndrom e type II (Lewis et al., 1990; K im berling et al., 1990); sev
eral form s o f X-linked retinitis pigm entosa (M usarella et al., 1991; 1989; O tt et al.,
1990; Levy et al., 1989) an d the RP1 form o f autosom al d o m in an t retinitis pigm en
tosa (B lanton et al., 1991).
In summary, the advances in the fields of m olecular biology a n d m olecular ge
netics that have o ccu rred in the last few years, together with the availability o f an
imal m odels of retinal deg en eratio n - eith er in h erited or created by the
integration of m u tan t genes into the m ouse genom e - have o p en ed up trem en
dous possibilities for the u n d erstan d in g o f genetic diseases affecting the retina.
We are encouraged by the re cen t findings an d hope that in the n ear future no t
only the causes will be unravelled bu t also ways to prevent or cure the devastating
disorders that lead to blindness.
ACKNOWLEDGEMENTS
We wish to thank Dr. Cathy Bowes for h er co ntinued contribution to the study o f
the rd m ouse an d Dr. J o h n F lannery for providing us with the figures of the n o r
mal an d rd m ouse retinas. This work was supported by the N ational Institutes of
H ealth grants EY02651 an d EY08285 and Core g ran t EY0331; a C enter g rant from
the N ational Retinitis Pigm entosa F oundation; and a g ran t from the G eorge
G und F oundation. D.B.F. is the recipient o f a Research to Prevent Blindness
Senior Scientific Investigator Award.
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6. AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA
SUMMARY
Retinitis pigm entosa (RP) is the collective term for a group o f in h erited diseases
of the eye involving a degeneration o f the retina, the light sensitive m em brane
carrying the ro d an d cone photoreceptors. As these cells die off, symptoms of
night blindness develop, usually followed by a gradual constriction o f the
patient's field o f vision. In addition, pigm entary deposits may build up in the
re tin a as a result o f d egeneration o f the neural epithelial cells, which has given
rise to the nam e retinitis pigm entosa. RP is a prevalent genetic disorder, affecting
approxim ately one in every four thousand persons (probably ab o u t one an d a
half m illion people th ro u g h o u t the w o rld ). It can be in h erited in an X-linked, an
autosom al d o m in an t or a recessive m ode. Approxim ately fifty p ercen t of cases
have no family history and are presum ed to be the result o f autosom al recessive
disease, reduced penetrance, new X-linked o r autosom al d o m in an t m utations or
phenocopies. It is in the autosom al d o m in an t form of the disease th at m uch
recen t progress has been m ade in o u r u n d erstanding o f the m olecular pathology
of RP. H ere, the genetic aetiology has been shown to have its origins in defects
either o f the rhodopsin o r rd s/p e rip h e rin proteins, both o f which are com po
nents of the rod p h o to rec ep to r o u ter segm ent disc m em branes. Recently rh o d o p
sin m utations have also been identified in patients with an autosom al recessive
form RP. G enetic linkage studies have identified the location of fu rth e r loci
responsible for adRP on chrom osom es 8, 7p, 7q an d 19q indicating th at fu rth e r
genes rem ain to be identified. An intriguing picture o f the m olecular basis for
the degenerative processes involved in RP is now em erging.
INTRODUCTION
The group o f degenerative conditions o f the eye term ed retinitis pigm entosa
(RP) represents the m ost prevalent retinopathy (disease o f the retina) showing
clear-cut M endelian inheritance. T he disease is at p resen t incurable, appears to
be p resen t in all of the w orlds populations an d currently afflicts up to 1.5 million
people. Pathological changes associated with the disease include the progressive
death of ro d p h o to recep to r cells. O f the two types o f p h o to rec ep to r within the
retina, rods an d cones, the rods are by far the m ost light-sensitive, containing the
visual pigm ent rhodopsin. R hodopsin is too sensitive to function in norm al day
light, b u t enables the eye to perceive light in dimly lit conditions (at night, for
154 PETER HUM PHRIES ET AL.
exam ple). As the rod cells die off, night blindness, or nyctalopia, develops. In
some cases, the progression o f the disease is rapid, n ight vision problem s devel
oping within the first decade o f life. In o th e r instances, such symptoms may not
m anifest until the fifth decade o r beyond. Variability o f clinical expression is a
classical feature in some genetic types of RP, particularly the d o m in an t form . T he
d eath of the rod p h otoreceptors is followed by m ore extensive pathological
changes in the retina. C one cells, responsible for the perception o f colour, begin
to die off an d there is a gradual constriction o f the patient's visual fields (Figure
I)-
A
90
T he retin a visibly thins an d the blood vessels supplying the retinal tissue begin
to attenuate. Gradually, black pigm entary deposits build up in the neural retina,
the result of dam age to the p h o to rec ep to r and pigm ent epithelial cells, which
AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 155
leads to m igration o f pigm ent laden cells into the o u ter and in n er retinal layers.
Many patients eventually lose all o f th eir sight. Pathological changes in the retinal
fundus typical of RP are illustrated Figure 2.
V i f '
* I -V
"V j %
, 4 ; ' '
While the symptoms o f RP clearly originate in the death o f rod cells, nothing
was known, until very recently, o f the m olecular events associated with this d egen
erative process. Rod cells carry a sophisticated battery of proteins form ing com po
nents o f the visual transduction cycle, the process through which light is absorbed
and converted into neu ro n al signals (C habre and D eterre, 1989; C habre and
Vuong, 1992). T he disease could, in some way, be associated with defects in this
cycle. Alternatively, defects could lie in structural com ponents o f the retinal cells
or even in cells of the underlying pigm ent epithelium . Possibly, the problem could
be one o f vascular supply. A gene, o r genes associated with the m aintenance o f the
fine netw ork of capillaries suffusing the re tin a could be defective, resulting in
gradual cell death through ineffectual blood supply. With no clues, the search for
the defective protein, or proteins, m ight seem virtually impossible. Fortunately, a
powerful technique is available to enable the localization o f defective genes, p ro
vided th at sufficiently large families can be located in which the disease is
156 PETER HUM PHRIES ET AL.
A B
tO
2
3
long arm o f chrom osom e 3 were sim ultaneously analysed, the rhodopsin m arker
was shown to m ap to the disease locus with zero recom bination and a lod score of
approxim ately 20. These results very strongly suggested th at a m utation o f the
rh odopsin gene m ight be the underlying defect which caused RP in the Irish farn-
ily.
An intensive search for m utations w ithin the rhodopsin gene in patients suffering
from autosom al d om inant RP (adRP) was m o u n ted as a result of the above obser
vations. A m utation was rapidly found in an Am erican p atien t (Dryja et al., 1990).
This was a single am ino acid substitution (a C ^ A transversion) at codon 23 of
the p rotein, resulting in the removal o f proline and its substitution by histidine.
T his m utation has now been found in up to 15% o f all d o m in an t cases o f RP in
the U nited States. Interestingly, the m utation is eith er totally absent within the
p o pulation o f Europe, or present at a very low frequency (F arrar et al., 1990).
Using a variety of new techniques, the rapid screening of DNA for disease-relat-
ed m utations is now possible. A com bination of such m ethodologies, including
single strand conform ational polym orphism (SSCP), d irect sequencing, h etero d u
plex analysis an d allele-specific oligonucleotide analysis have been used in screen
ing the rhodopsin gene from patients with RP. Using the first technique, SSCP
analysis (O rita et al., 1989), small sectors o f the gene (up to a few h u n d red s o f base
pairs) are am plified from genom ic DNA using the polym erase chain reaction. The
AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 159
A B
1 2
P oint m utations are identified as double bands at the same position in the se
quencing gel. T he presence of a m utation may then be confirm ed by the use of
allele-specific oligonucleotides (ASOs). S hort prim ers specific eith er for the n o r
m al or m u tan t DNA are synthesised a n d allowed to hybridise to DNA from m em
bers o f the pedigree. DNA is usually im m obilised on filters in the form of dot
160 PETER HUM PHRIES ET AL.
blots. N orm al DNA hybridises only to the norm al ASO, while m utant DNA (be
cause it carries both norm al an d m u tan t alleles) will hybridise to both norm al and
m u tan t ASOs. An exam ple o f the detection o f a p erip h erin /R D S in m utation seg
regating in DNA from an RP pedigree using the ASO technique is illustrated in
Figure 5.
H i M
Using a com bination o f these, and sim ilar techniques, at least 60 m utations with
in the rhodopsin gene have been identified in patients with d om inant RP over the
last three years (reviewed by H um phries et al., 1992). T he positions of these m u
tations within the p rotein are illustrated in gure 6. In o rd e r to gain some insight
into how such m utations m ight cause RP, an u n d erstanding o f the function of
rhodopsin within the visual transduction cycle is req u ired (C habre and D eterre,
1989; C habre and Vuong, 1992)
AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 161
P e rip herin/R D S
P e ripherin/R D S
F ig u re 7 In th is d ia g ra m , b o th th e r h o d o p s in a n d rd s p r o te in s a re se e n e m b e d d e d in th e p h o
to r e c e p t o r o u t e r s e g m e n t disc m e m b r a n e . B o th p r o te in s twist in a n d o u t o f th e lip id bilayCi
h a v in g 7 a n d 4 tr a n s m e m b ra n e s e g m e n ts re sp ectiv ely .
F ig u re 8 T h e r o d c ell o u t e r s e g m e n t d isc h a s r h o d o p s in m o le c u le s e m b e d d e d in th e m e m
b r a n e . P h o to a c tiv a te d rh o d o p s in m o le c u le s stim u la te tr a n s d u c in , w h ic h a c tiv a te s cG M P p h o s
p h o d ie s te r a s e a n d le a d s to lo w e re d levels o f cG M P in th e c y to p la sm h e n c e r e s u ltin g in c lo s u re
o f c G M P -g a te d io n c h a n n e ls . T h e re s u ltin g lo w e re d c a lc iu m levels le a d s to th e s tim u la tio n o f
re c o v e rin a n d g u a n y la te cyclase a ctiv ities a n d re su lts in cG M P p r o d u c tio n a n d r e o p e n i n g o f
th e c h a n n e ls .
AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 163
Olsson et al. (1992) have constructed a transgenic m ouse containing the Pro-23-
His m utation that is com m on in N orth America. T he en tire rhodopsin gene,
including 4-5 kb o f upstream and 6-8 kb o f dow nstream regulatory sequences, was
isolated from a p atien t with adRP who was heterozygous for this m utation. T he
no rm al and m u tan t genes were m icroinjected into the m ale pronuclei o f single
cell m ouse em bryos an d five lines o f transgenic m ice were studied from the
resultant progeny (two carrying the wild-type and four the m u tan t allele). All
th ree m u tan t lines developed p h o to rec ep to r degeneration, the degree o f d egen
eration correlating with the extent o f m u tan t allele expression. T he retinas o f
affected mice showed atten u ated vessels a n d geographic patches o f retinal pig
m ent epithelial hyper- or hypopigm entation, contrasting with the dendritic
clum ps o f pigm ent found in the hum an disorder. A m onoclonal antibody specific
to the hu m an ro d opsin detected expression of the m utant opsin in o u ter seg
m ents an d failed to find accum ulations in the endoplasm ic reticulum . This con
trasts with the findings of Sung et al. (1991) who showed a failure o f the Pro-23-
His opsin to be in corporated into plasm a m em branes in vitro and re te n tio n in
th e endoplasm ic reticulum . Olsson et al. (1992) unexpectedly fo u n d abnorm ally
located m u tan t opsin at the synaptic en d o f rods (outer plexiform layer), raising
the possibility o f disturbed intracellular transport.
Surprisingly, one of the two transgenics containing the wild-type allele also d e
veloped a very sim ilar retinal degeneration. T he ratio of hum an to m ouse ro d op
sin mRNA expressed in this line was 5:1 com pared to the unaffected wild-type
transgenic which expressed equal am ounts o f hum an an d m ouse transcript. T he
d egeneration was th o u g h t therefore to be related to overexpression o f the (hu
m an) opsin gene.
These experim ents show firstly that this m utation confers a degenerative p h en o
type, even in the presence o f two norm al m ouse genes, which excludes the possi
bility that it is a null allele which gives rise to adRP as a result o f a deficiency of
norm al gene product. Secondly, some proposed explanations for the d eg en era
tion, such as failure of RPE cells to phagocytose rod o u ter segm ents or inability of
the m utant protein to be in corporated into o u ter segm ent discs, ap p eared less
likely in the light o f these findings. Finally, the finding that overexpression even of
the norm al hum an gene p ro d u ct causes a severe retinal d egeneration perhaps
AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 165
em phasises how finely tu n ed the retin a is for m aintaining norm al structure and
function.
R hodopsin m utations account for only about 25% of all d o m in an t cases of RP.
Following the establishm ent o f the initial rhodopsin linkage, we u n d erto o k addi
tional linkage work in a second adRP pedigree o f Irish origin which showed
exclusion o f a locus from the long arm o f chrom osom e 3 (F arrar et al., 1990). In
this family the disease was o f m uch later onset than that in the first pedigree. RP
is clinically divisible into two m ajor form s, so called types 1 and 2, the form er
being the m ore severe (C hapter 3). T h e disease is also classifiable as eith er a dif
fuse o r a regional retinopathy, d e p e n d in g on the n ature and ex ten t of pathologi
cal changes in the retina. Diffuse a n d regional form s o f the disease usually
correlate with type 1 and 2 form s respectively. M em bers of the second Irish family
had a regional (type 2) form of RP with night blindness and field loss n o t usually
m anifesting until the fo u rth decade. By the tim e o f this second linkage study, a
new generation o f genetic m arkers had becom e available. Such m arkers are
called m icrosatellites (W eber an d May, 1989; Litt and Lutty 1989). H um an DNA
contains many (one h u n d re d thousand o r m ore) short blocks o f dim er repetitive
sequence o f the type, d(C A )n, interspersed th ro u g h o u t the genom e. T he num
bers o f CA repeats at any given locus usually varies on each hom ologous chrom o
some. O ligonucleotides representing the sequence o f DNA im m ediately flanking
such repeats can be synthesized a n d used as prim ers in the polym erase chain
reaction to amplify the segm ent o f the genom e spanning the block o f repetitive
DNA. Am plified DNA may th en be analysed by electrophoresis in acrylam ide
gels, sim ilar to the type used for DNA sequencing. Variation in the length o f the
repetitive block, even if this is as small as a single CA unit, is readily detectable.
M icrosatellites have two m ajor advantages over conventional RFLPs. Firstly, they
can be used w ithout the n eed for enzymatic digestion o r S outhern blotting, in
which lengthy gel separations, DNA transfer to m em branes, hybridisations and
autoradiography are required. Secondly, m icrosatellites are generally o f m uch
higher inform ation c o n te n t than conventional m arkers. This m eans that a pro
portionately greater n u m b er o f genetic crosses are inform ative for linkage in any
particular pedigree.
C ontinued linkage studies, based largely on the use o f such m arkers, resulted in
the localisation of a causative gene (F arrar et al., 1991; Jo rd a n e ta l., 1991; K um ar-
Singh et al., 1991). T he gene resided close to the HLA locus on th e short arm of
chrom osom e 6. Interestingly, the gene for an additional p rotein co m p o n en t o f the
rod o u ter segm ent discs, p erip h erin /R D S , h ad recently been m apped to the same
region (Travis et al., 1991). Co-segregation of the disease locus with the m icrosat
166 PETER HUMPHRIES ET AL.
ellite m arker at the 3 en d of the p erip h erin /R D S gene is illustrated in Figure 3B.
This was o f special significance, since the rd s /p e rip h e rin protein had previously
been im plicated in the cause of the naturally occurring retinopathy o f mice called
retinal degeneration slow (rds) (C hapter 5). T h e protein takes its nam e from this
ro d en t disease which, in many respects, is sim ilar to RP in man. T he rd s/p e rip h -
erin p rotein thus becam e a prim e candidate for hum an adRP and a m utation with
in the gene was soon fo u n d in a patient from an Irish adRP pedigree (Farrar
et al., 1991). T he m utation was a trinucleotide deletion o f codons 118/119, rem ov
ing o n e o f a pair o f conserved cysteine residues.
T he hum an p erip h erin /R D S gene is highly conserved in mice, cattle, rats and
hu m ans an d contains three exons, coding for a predicted 193, 83 an d 70 am ino
acid residues respectively. T he p rotein p ro d u c t is a 39 kDa glycoprotein b u t m uch
less is known o f its structure and function com pared with rhodopsin. It is n o t be
lieved to be a com ponent o f the visual transduction cycle but, like rhodopsin, it
resides within the m em branes o f o u ter segm ent discs (Figure 7). U nlike rh o d o p
sin, it is present in rod and cone p h o to rec ep to r o u ter segm ents (Arikawa et al.,
1992). To date, ab o u t th irteen m utations within the p erip h erin /R D S gene have
b een re p o rted in cases of adRP (F arrar et al., 1992; Kajiwara et al., 1992; S. Bhat
tacharya, A. Gal, personal com m unications). T he original Irish pedigree in which
linkage was established contains an am ino acid substitution at codon 212 (F arrar
et al., 1992). In addition, a n u m b er o f silent polym orphic am ino acid substitu
tions have b een detected which are n o t associated with a disease phenotype
(J o rd a n et al., 1992). T hree of these polym orphism s cluster toward the carboxyl
term inus o f the m olecule. (Interestingly, these silent polym orphic variations oc
cur at residues h ith erto considered to be conserved am ong m am m alian rd s /p e
rip h erin p ro te in s). C u rre n t m odels suggest th at the rd s /p e rip h e rin p rotein has
fo ur m em bane-spanning a-helical dom ains with both the carboxyl an d am ino acid
term inals facing the cytoplasmic side of the disc m em brance (Figure 9). T he pro
tein is believed to have two intradiscal loops, one m uch larger than the other. It is
possible that the protein may dim erise via at least o n e disulphide bridge, and it
may have a role in m aintaining disc structure.
It is too early to say what p ro p o rtio n o f adRP is caused by m utations within the
RDS gene. T he figure may well be sim ilar to the frequency o f rhodopsin-associated
RP. However, a n u m b er o f m utations in the p erip h erin /R D S gene have recently
been identified in families segregating dom inantly in h erited disorders o th e r than
RP. Kajiwara et al.(1993) screened over 350 patients with a variety o f hereditary
retinal disorders for p erip h erin /R D S gene m utations using SSCP analysis. Exons
1 an d 2 together with som e flanking DNA (intron or 5' u ntranslated) were
screened, om itting exon 3 which contains freq u en t polym orphism s in norm al in
dividuals. Only one patient, with autosom al d om inant retinitis punctata albescens,
was found to have a m utation. T he clinical phenotype consisted o f m acular hyper
pigm entation and atrophy associated with w idespread yellow-white flecks u n d er
the retina.
AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 167
family with autosom al d om inant adult vitelliform m acular dystrophy. This may
also be a null allele, similar to th at in the p atien t with retinitis punctata albescens,
although the phenotypes are ra th e r different. Finally, Nichols et al. (1993)
described a family with autosom al d om inant butterfly-shaped pigm ent dystrophy
of the fovea associated with a missense m utation in p erip h erin /R D S codon 167,
which would substitute an aspartate for a highly conserved glycine residue. T he
m utation segregated with the disease (lod score = 4 at a recom bination fraction
o f zero) and was no t present in 100 control chrom osom es. T he disorder is char
acterized by accum ulations o f yellowish or pigm ented m aterial at the level o f the
retinal pigm ent epithelium (RPE), central visual loss apparently related to the
ex tent of accom panying RPE atrophy, n orm al dark adaptation thresholds and
electroretinogram s. H ence, like rhodopsin, m u tan t rd s /p e rip h e rin proteins show
a wide range o f phenotypic expression. Indeed, m ost recently families with
digenic adRP have been identified, in which only patients who in h erit m utations
in both the rd s/p e rip h e rin gene an d the gene encoding the rod o u ter m em
b ra n e (ROM) protein are affected (T. Dryja, T h e Association for Research in
Vision and O phthalm ology M eeting, 1994). Such families segregating with
digenic adRP show autosom al d o m in an t inheritance with reduced p e n e tra n c e .
A dditional RP families have been identified in which n eith er rhodopsin n o r
rd s /p e rip h e rin is involved. O ne such family form s the large adRP pedigree
UCLA1 from the U nited States. H ere, the causative gene has been localised by ge
netic linkage analysis to the pericentric region o f chrom osom e 8 (B lanton et al.,
1991). However, no potential candidate gene has, as yet, been identified. Most re
cently, two fu rth e r genes have been identified in large adRP families o n opposite
arm s o f chrom osom e 7. Inglehearn et al. (1993) found linkage to a gene on the
short arm of chrom osom e 7 (7p) in a large English adRP kindred, close to the
m arker D7S460 (Z = 5.60 at 0 = 0). A n o th er large kindred was fo u n d b y jo rd a n
et al. (1993) to have a disease gene linked to m arkers on the long arm o f ch ro m o
some 7, close to the m arker D7S480 (Z = 7.20 at 0 =0). Most recently an adRP
gene has been localised to the long arm o f chrom osom e 19 close to the m arkers
D19S180 an d D19S214 (Al-Maghteh et al., 1994). In addition, at least o n e o th er
adRP family currently u n d e r study in o u r own laboratory shows no evidence for
linkage on chrom osom es 3, 6, 7 o r 8, indicating the presence o f at least one m ore,
as yet unlocalised, adR P gene (Kumar-Singh et al., 1992, 1993;Jordan et al., 1992).
T hus in the d o m in an t form o f RP, a m inim um of six genes are responsible for
th e cause o f the disease in different families. Possibly about one third o f all dom
in ant cases can be accounted for by known m utations in the rhodopsin an d pe
rip h erin /R D S genes. It is o f interest that both of these genes code fo r proteins
which are located within the m em branes o f the o uter segm ent discs. W hether all
adRP proteins are located within these structures rem ains to be determ ined.
AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 169
CONCLUSIONS
In this review we have lim ited our coverage to the d o m in an t form o f RP, in which
two o f the first proteins responsible for RP were identified. Perhaps the most sur
prising finding in adRP has been the very extensive genetic heterogeneity. T here
now ap p ear to be a m inim um o f six loci responsible for this disorder, on chrom o
somes 3, 6, 7p, 7q, 8 an d at least o n e o th er as yet unidentified site. D ifferent
m utations in the rhodopsin gene on chrom osom e 3 give rise to very different
phenotypes, from a very early onset and severe degeneration in som e types of
adRP to the clinically norm al carriers o f the tru n cated m olecule associated
with autosom al recessive disease. In the case o f the p erip h erin /R D S gene on
chrom osom e 6, the phenotypic variation is proving even m ore diverse. D ifferent
m utations are associated eith er with predom inantly m acular o r p eripheral re tin o
pathy. Families with m acular dystrophy, adult vitelliform m acular dystrophy,
butterfly-shaped pigm ent dystrophy, retinitis pigm entosa and retinitis punctata
albescens have all been associated with R D S /p erip h erin m utations. In some
cases, m ore than one phenotype has been fo u n d within the same family. T he
clinical heterogeneity is as striking as the genetic heterogeneity.
D evelopm ents are rapidly taking place in o th er form s of retinitis pigm entosa
and in related conditions. T he locations of two X-linked RP genes have been
known for some tim e (O tt et al., 1990). In autosom al recessive form s o f the dis
ease, genes for U sher syndrom es type 1 an d 2 (RP associated with deafness) have
also been localised by genetic linkage analysis (Kim berling et al., 1990) (C hapter
15). M oreover, a m utation w ithin the rhodopsin gene an d several m utations within
the (3 subunit o f cyclic GMP phosphodiesterase have recently b een identified in
families displaying autosom al recessive RP (Rosenfeld et al., 1992; M cLaughlin
et al., 1993). O u r own studies o f a large family o f D utch origin indicate that there
is an additional autosom al recessive locus on lq close to the m arker F13B an d the
gene encoding the retinal protein phosducin (Bleeker-W agemakers et al., 1992;
van Soest et al., 1994). In addition, genes responsible for two o th er dom inantly
heritable form s of m acular deg en eratio n - N orth C arolina m acular dystrophy and
Bests disease - have been localised on chrom osom es 6 and 11 respectively (Small
et al., 1992; Stone et al., 1992) (C hapter 15). T he availability o f additional pedi
grees suitable for genetic linkage analysis, coupled with the now well developed
techniques for positional cloning an d rapid m utational screening, will ensure that
the many o f these genes will be characterised within the n ext few years.
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A u to so m a l d o m in a n t re tin itis p ig m e n to sa (A D R P ): L o ca lisatio n o f a n A D RP g e n e to th e lo n g
a rm o f c h ro m o s o m e 3. Genomics 5, 619622.
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172 PETER HUMPHRIES ET AL.
INTRODUCTION
A nu m b er o f epidem iologic studies have shown that diseases affecting the retinal
m acula cause a significant p ro p o rtio n of severe visual loss in o ld er individuals
(Sorsby, 1966; Kahn an d M oorhead, 1973; Kahn et al., 1977; Leibowitz et al.,
1980; Ganley an d R oberts, 1983; Klein and Klein, 1982; M artinez et al., 1982;
G hafour et al., 1983; Som m er et al., 1991). For practical reasons, such studies
usually consider m acular disease in patients over about 50 years of age to be a sin
gle clinical entity now known as age-related m acular degeneration (AMD).
U nfortunately, the creation of a single epidem iologic pigeon h o le for older
patients with m acular disease, and the assignm ent of a single term to refer to it
have tended to obscure two im p o rtan t facts:
1) AMD is n o t a single disease at the clinical, cellular, o r biochem ical level; and
2) the predisposition to AMD is in h erited as an autosom al d o m in an t trait in a sig
nificant percentage o f affected patients.
In this chapter, we will review the published data that support these two asser
tions an d pu t fo rth o u r view o f th eir im portance to the design o f studies aim ed at
elucidating the pathogenesis o f - and developing a b etter treatm en t for - the dis
eases that m ake up AMD. We will then discuss the ways in which m olecular genet
ics can be used to exploit the heritable n atu re o f these disorders to identify the
precise m olecular m echanism s involved in their pathogenesis an d provide some
exam ples of recent progress that has been m ade with this approach.
DEFINITION OF AMD
abnorm ality o f the m acular retinal pigm ent epithelium (Kahn et al., 1977; Klein
an d Klein, 1982; Hym an et al., 1983). In m ost such patients, drusen are present,
and in 5-10% choroidal neovascularization eventually develops (Leibowitz et al.,
1980, Hym an et al., 1983). Even w hen signs o f a specific m acular disease are
present (old central serous retinopathy, the presum ed ocular histoplasm osis syn
drom e, angioid streaks, etc.) the diagnosis o f age-related m acular d egeneration
is often applied to patients over 50, w hereas the m ore specific diagnoses are m ore
likely to be used in patients u n d er 50.
HETEROGENEITY OF AMD
However pragm atic the foregoing definition is for the clinic, it poses some diffi
culties for investigators trying to work o u t the pathophysiology o f the diseases
that m ake up AMD. First, it is ap p a ren t from the designs of the two population-
based epidem iologic studies (Leibowitz et al., 1980, Ganley an d Roberts, 1983)
th at a variety o f d ifferent pathophysiologic entities were g ro u p ed u n d er this sin
gle heading. T he fraction o f AMD caused by each distinct m acular disorder can
n o t be deduced from such studies. This is im p o rtan t because if one assum es that
AMD is a single pathophysiological entity, an d designs experim ents to try to id en
tify a single disease m echanism , one is likely to fail if the study population actu
ally consists o f patients with clinically similar bu t biochem ically different
disorders.
For exam ple, suppose that 5% o f AMD is caused by a genetically d eterm in e d ab
norm ality o f a p h o to rec ep to r p rotein that increases the eyes vulnerability to ultra
violet light. If two groups o f AMD patients with different light exposure histories
are studied, the ap p a ren t effect of LTV light on the entire high ex posure group
m ight be insignificant even though a very significant effect is p resen t in the 5% of
the population with high UV vulnerability. An exam ple from the literature in
which the study p articipants specific diagnosis dram atically affected the apparent
effectiveness o f a treatm ent fo r m acular disease is the juxtafoveal arm o f the m ac
ular photocoagulation study (Figure 1). In this study, patients who had laser treat
m en t for juxtafoveal choroidal neovascular m em branes secondary to the
presum ed ocular histoplasmosis syndrom e (POHS) h ad a 31% rate o f m em brane
persistence o r recu rren ce over five years (M acular P hotocoagulation Study G roup,
1989) while patients with juxtafoveal m em branes secondary to non-PO H S m acu
lar d egeneration had a five-year rate o f persistence or recu rren ce o f 79% (M acular
P hotocoagulation Study G roup, 1990).
Over the years, a num ber o f d ifferent structural an d functional abnorm alities,
physical exposures and dietary deficiencies have been proposed to play a role in
the pathophysiology of m acular degeneration, including:
1) abnorm ality of the neurosensory retin a (G artner an d H enkind, 1981; Bird
and Marshall, 1982; Katz et al., 1986);
THE M OLECUIAR GENETIC APPROACH TO MACUIAR DEGENERATION 175
<1>
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18 24 30 36 42
Months of Follow-Up
F ig u re 1 C o m p a r is o n of m e m b r a n e r e c u n v t u e fo llo w in g la s e r tr e a tm e n t o f ju x ta fo v e a l c h o
ro id a l n e o v a s c u la r m e m b r a n e s s e c o n d a r y to th e p r e s u m e d o c u la r h isto p la sm o sis sy n d ro m e
(P O H S ) a n d a g e r e la te d m a c u la r d e g e n e r a tio n . T h e fra c tio n o f tr e a te d p a tie n ts s u ffe rin g a
p e r s is te n c e (less th a n 3 m o n th s a fte r tr e a tm e n t) o r r e c u r r e n c e o f a c h o r o id a l n e o v a s c u la r
m e m b r a n e fo llo w in g la s e r tr e a tm e n t is g iv e n o n th e Y axis w h ile m o n th s fo llo w in g t r e a tm e n t
is g iv e n o n th e X axis. W ith in five y e a rs o f t r e a tm e n t, th e to ta l fra c tio n o f k ry p to n -tre a te d
P O H S p a tie n ts w h o h a d a p e r s is te n t o r r e c u r r e n t m e m b r a n e was 31% w h ile 7 9 % o f A M D p a
tie n ts sh o w e d p e rs is te n c e o r r e c u r r e n c e o f t h e i r n e o v a s c u la riz a tio n d u r i n g th is s a m e in te rv a l.
T h is f ig u re w as d ra w n u s in g d a ta f ro m tw o i n d e p e n d e n t r e p o r ts o f th e M a c u la r P h o to c o a g u
la tio n S tu d y G r o u p (se e t e x t ) .
2) abnorm ality o f the retinal pigm ent epithelium associated with a co-factor de
ficiency (Newsome et al., 1988), o r an inborn e rro r o f m etabolism (Deut-
m an an d Jansen, 1970);
3) abnorm ality of B ru ch s m em brane associated with a faulty prim ary structure
(e.g. pseudoxanthom a elasticum a n d A lport syndrom e) or by deposition o f an
abnorm al substance from an RPE o r vascular source (Bird, 1986; Newsome et
al., 1987);
4) abnorm ality of the choriocapillaris (Friedm an et al., 1963; Tso, 1985);
5) im m une system activation including developm ent of anti-retinal antibodies
(G urne et al., 1991) an d attraction o f m acrophages (Penfold et al., 1986);
6) abnorm al scleral rigidity (Friedm an et al., 1989);
176 EDWIN M. STONE AND VAL C. SHEFFIELD
M i I 1 1 I
100
1-20 years
21-60 years
80
61-100 years
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Severity
Figure 3 P re v a le n c e o f u ltras!) tu t.urai a b n o rm a litic .s in B ru c h 's m e m b r a n e in p a tie n ts o f d if
f e r e n t ages. T h e f ig u re is d ra w n u s in g d a ta r e p o r t e d bv F e e n e y -B u rn s a n d E lle rsie c k (1 9 8 5 ).
T h e Y ax is gives th e f ra c tio n o f p a tie n ts In e a c h a g e g r o u p a ssig n e d to e a c h o f six g ra d e s o f
u ltr a s tr u c tu r a l sev erity th a t a rc d is tr ib u te d a lo n g th e X axis. W h ite b a rs in d ic a te p a tie n ts 1 -2 0
y e a rs o f ag e; g re y b a rs, 2 1 -6 0 y e a rs o f ag e; a n d , b la c k b a rs 61-100 y e a rs o f ag e. N o te t h a t 10%
o f t h e y o u n g e s t p a tie n ts h av e th e s a m e d e g r e e o f a b n o r m a lity (g ra d e 3) as 5 4 % o f th e o ld e s t
p a tie n ts .
n o t discuss the possibility o f varying susceptibility to drusen form ation within the
population, it is interesting that o ne patient (10%) from their youngest group had
the sam e ultrastructural severity as 20 patients (54%) from their oldest group. Pos
sible explanations for this observation are that Feeney-Burns and Ellersiecks
age-related sequence o f changes is 1) a com m on m anifestation o f a variety of dif
ferent biochem ical processes with d ifferent ages of clinical onset; o r 2) tru e age-
related p h en o m en a are superim posed on a background o f variable genetically-de
term in ed susceptibility.
A dditional insight into this possibility was provided by Coffey and Brown stein
(1986) who co unted drusen in serial sections of 23 pairs of postm ortem eyes. T he
unusual and im p o rtan t feature of this study is that th eir data were not averaged -
THE M O LECUIA R GENETIC APPROACH TO MACUIAR DEGENERATION 179
HERITABILITY OF AMD
An im portant req u irem en t for a m olecular approach to any hum an disease is the
existence of families affected with a heritable form . T he way in which such fami
lies are used for the study o f disease will be discussed m ore fully below, b u t at this
ju n c tu re it m ight be reasonable to ask w hether there is any evidence that a subset
o f AMD is heritable.
T h ere are a n u m b er of dystrophies th at affect the m acula whose heritability has
been un d isp u ted for decades. Some o f these are clinically similar enough to AMD
that distinction from the latter is often difficult in affected patients over the age
of fifty, especially if the family history is n o t known. These include Bests disease
(Best, 1905), the p attern dystrophies (M arm or and Byers, 1977; Hseih et al. 1977;
Watzke et al., 1982; D ejong and D ellem an, 1982), Sorsbys m acular dystrophy
(Sorsby et al., 1949), and do m in an t d ru sen (Pearce, 1968; D eutm an an d Jansen,
1970). T he familial n atu re o f the latter entity was first described nearly 100 years
ago (Doyne, 1899), and at least o n e au th o r feels that it is clinically indistinguish
able from typical AMD (Gass, 1973). Bests, Sorsbys and the p attern dystrophies
also have some AMD-like features including an accum ulation o f abnorm al m ateri
al at the level of B ru ch s m em brane a n d the RPE as well as the eventual develop
m ent o f choroidal neovascularization o r geographic atrophy in a subset of
patients. N orth C arolina M acular Dystrophy can also mimic AMD ophthalm oscop-
ically b u t differs from the o th er dystrophies in that it is usually n o t progressive
(Small et al., 1991). T he specific genetic defects th at cause each o f these diseases
are o f great interest because the affected genes may be involved in a sizeable pro
portion of typical AMD. At the very least, identification o f a gene th at causes any
m acular dystrophy has the potential to greatly im prove o u r u n d erstanding o f the
cascade o f m olecular events th at occurs in AMD.
If m utations in m acular dystrophy genes play an im p o rtan t role in AMD, one
would expect to find evidence o f heritability in a significant p ro p o rtio n o f typical
AMD patients. However, it is im p o rtan t to realize that even if all AMD has a pri
marily genetic basis, this would n o t necessarily be clinically obvious. Since the dis
ease does n o t usually becom e sym ptom atic until the seventh o r eighth decade, the
parents o f affected patients are no t usually living and their siblings may also be de
ceased or otherw ise unavailable for exam ination. Also, since only a m inority o f af
fected patients experience severe visual loss, it is im possible by history alone to
determ ine w hether a siblings 2 0 /5 0 vision is due to AMD or some o th er cause;
and conversely, w hether a p aren t with excellent vision actually had num erous as
ym ptom atic drusen. C ertainly if one assumes that AMD is a natural aging p h en o m
enon an d does no t even question patients about their family, one is unlikely to
180 EDWIN M. STONE AND VAX. C. SHEFFIELD
discover evidence to support a genetic basis for the disease. To restate this im por
tant point: failing to look for evidence of heritability is not equivalent to carefully
looking for it w ithout success.
For exam ple, a recently published study exam ined AMD risk factors in over 400
patients and 600 controls (Eye Disease Case C ontrol Study G roup, 1992). T he sub
jects were questioned about cigarette use, alcohol an d coffee consum ption, sun
light exposure, m arital status, physical activity, the presence o f diabetes or
cardiovascular disease, p rio r hysterectomy, an d the use o f estrogen or birth con
trol pills. No questions regarding AMD in family m em bers were asked and it is thus
n o t surprising that these authors did not even m ention the possibility o f genetic
risk factors, m uch less find any evidence to su p p o rt them .
In contrast, w hen evidence for genetic factors has been looked for, it has been
found. In 1973, Gass re p o rted the results o f his study o f 200 patients with m acular
d egeneration who had been followed for an average o f fo u r years. He stated: al
though it was my initial im pression that few patients with d rusen an d disciform d e
tachm ent had a positive family history, m ore careful questioning o f the patients
and investigation o f the few relatives available during this study has revealed a sig
nificant incidence of familial involvem ent. Specifically, he obtained a positive
family history of central visual loss in 38 of the 200 patients (19%) an d was able to
confirm the diagnosis o f AMD in the relatives o f 10 o f these patients by personal
exam ination. In a case-control study by Hym an et al. (1983) the presence o f AMD
in parents and siblings of the study subjects was investigated via questionnaires dis
tributed to the subjects, th eir siblings and th eir siblings eye exam iners. Inclusion
o f the latter group was revealing because the eye exam iners o f the case siblings re
p o rted the presence of AMD alm ost twice as often as the case siblings themselves,
suggesting that patients are often not told that they have m acular degeneration
unless they have experienced visual loss. A ccording to the eye exam iners respons
es, 19.9% o f the siblings o f AMD cases had AMD themselves in com parison to 7.9%
of the siblings o f controls. T he age and sex distribution o f the case siblings were
the sam e as those o f the controls. W hen the parental and sibling data were com
b ined by defining a positive family history as eith er a p a re n t or a sibling with a his
tory o f the disease, the data were similar with 21.6% of the cases having a positive
family history as opposed to 8.6% of the controls.
T here are a few reports of identical twins with AMD com plicated by choroidal
neovascularization (Melrose et al., 1985; Meyers an d Zachary, 1988; Dosso and
Bovet, 1992). In all th ree of these reports, the second twin developed symptoms of
choroidal neovascularization within 16 m onths o f the first. T he twins re p o rte d by
Meyers and Zachary belonged to a sibship o f 13, and four of the o th e r 11 siblings
were also affected.
At least three studies have suggested that AMD is significantly m ore com m on in
white patients than in blacks (Chumbley, 1977; G regor an d Joffe, 1978; Som m er
et al., 1991). A lthough it is possible that increased pigm entation is directly respon
sible for the protective effect, it is also possible that AM D-predisposing genes are
less com m on in the black population.
THE MOLECULAR GENETIC APPROACH TO MACUIAR DEGENERATION 181
U nfortunately, n eith er the F ram ingham study (Kahn et al., 1977; Leibowitz et
al., 1980) n o r the National H ealth a n d N utrition Exam ination Survey (Ganley and
R oberts, 1983) collected data regarding a family history of AMD. However, these
studies were already underw ay when Gasss 1973 paper was published an d were
com plete before the re p o rt o f Hyman et al. in 1983. It is less understandable why
m ore recent studies (W eiter et al., 1985; Bressler et al., 1989; Klein et al., 1991; Eye
Disease Case C ontrol Study G roup, 1992) have chosen to ignore the possibility of
genetic predisposition.
In any case, th ere seems to be sufficient evidence in the literature to support the
assum ption th at a significant fraction o f AMD is heritable and therefore am enable
to m olecular genetic attack. Moreover, the evidence that AMD is pathophysiolog-
ically h eterogeneous is also com pelling and we believe that som e m ethod of reli
ably dividing patients according to disease m echanism is essential for new
therapies to be rationally designed, tested and adm inistered.
Identification of genes that cause in h erited diseases has been accom plished with
a variety of approaches. T he first genetic disease genes were identified based on a
knowledge of the specific cellular proteins involved in the pathogenesis of the
diseases. Exam ples o f this are the globin genes involved in the thalassem ias and
sickle cell anem ia (Ingram , 1956; W eatherall a n d Clegg, 1981; B unn and Forget,
1986). For many genetic diseases, including hereditary eye diseases, inform ation
concerning proteins involved in the pathogenesis o f the disease is lacking or has
failed to aid in the identification o f the disease-causing locus. For such disorders,
the use o f genetic linkage analysis with polym orphic DNA probes to identify the
genetic location of the gene has proven valuable.
Figure 4 provides an overview of a stepwise genetic approach to m acular disease.
T he ideal starting m aterial is a large family affected with an autosom al dom inant
disease which can be used for linkage analysis. Linkage analysis does no t require
any specific hypothesis ab o u t the pathogenesis of the disease. It requires only the
availability o f a sufficient n u m b er o f correctly diagnosed individuals to allow a sta
tistically significant relationship between m arker alleles and the disease p h en o
type to be dem onstrated. Linkage data are expressed as lod scores which are
derived and in terp re ted as discussed in C hapter 1.
T he first m acular disease to be successfully linked to a genetic m arker was an
atypical vitelliform dystrophy linked to the m arker GPT-1 (Ferrell et al., 1983)
which is now known to lie on the long arm of chrom osom e 8 (Rocha et al., 1988).
More recently, Small and co-workers (1992) m apped N orth C arolina M acular Dys
trophy (Figure 5) to chrom osom e 6. Two m icrosatellite m arkers located in chro
m osom al region 6ql3-q21 (MFD171, MFD97) were found to be linked to the
disease locus with lod scores of 8.4 a n d 13.1 at recom bination fractions o f 0.004
and 0.017 respectively. A patient with a cone dystrophy and m ental retard atio n as-
182 EDWIN M. STONE AND VAL C. SHEFFIELD
r' i
\ i / DGGE
TEGEN
CANDIDATE GENES
\ G A TC GATC
>
SINGLE AFFECTED PATIENTS
TRANSGENIC ANIMAL
DNA
(with or without family history)
Sequencing
/ \
SSCP
MUTATION SCREENING
sociated with a translocation breakpoint in the same region has been re p o rted by
T ranebjaerg et al. (1986). These rare chrom osom al rearrangem ents are potential
ly im p o rtan t for the task o f identifying the gene directly. Also in 1992, we (Stone
et al., 1992) an d an o th er g roup (Forsm an et al., 1992) independently m apped
Best's Vitelliform Dystrophy (Figure 6) to the long arm of chrom osom e 11.
This disorder has been studied histopathologically and found to be associated
with lipofuscin-like deposits within and beneath the retinal pigm ent epithelium .
O u r group m apped a large five-generation family with this early-onset autosom al
do m in an t form of vitelliform m acular dystrophy to the 11 q 13 region, between
m arkers INT2 and D11S871 (Zmax = 9.3 at 3.9 cM from m arker D11S871) (Stone
et al., 1992). T he o th er group m ap p ed the Best gene in a large Swedish family to
the same chrom osom al region (11 q 13) with a lod score of 15.12 at a recom bina
tion fraction o f 0.01 for the closest marker, I S'12 (Forsm an et al., 1992). A possible
candidate gene is the ROM-1 gene (Bascom et al., 1992) which m aps to same chro
m osom al region and shows similarity to the p erp h erin /R D S gene (see below).
T he gene product, rom-1, is a m em brane-associated protein o f rod o u ter segm ents
th at is o f sim ilar size and am ino acid sequence as rd s/p e rip h e rin .
O th er m acular diseases th at should be am enable to linkage analysis include
dom inant drusen (both typical and cuticular), Sorsbys fundus dystrophy, various
p attern dystrophies, an d S targardts disease.
IIS4 EDWIN M. STONE AND VAL C. SHEFFIELD
O nce a disease is m apped to a region, one may find that a candidate gene has
been previously m apped to the same location. This was the case for autosom al
d o m inant retinitis pigm entosa in which the rhodopsin gene had been localized to
the long arm o f chrom osom e 3 (Sparkes et al., 1986) before linkage analysis sug
gested that a disease-causing m utation m ight exist at the same locus (McWilliam
e ta l., 1989 and C hapter 6).
An alternative m ethod used to identify disease-causing genes is the candidate
g e n e approach. Genes that are likely candidates for playing a role in a given ge
netic disease are selected and tested for th eir involvem ent in the disease eith er by
m utation analysis o r by linkage studies that use polym orphism s previously identi
fied w ithin the gene sequence. A candidate gene can be selected based on its func
tion, its chrom osom al location, or the tisstie in which it is prim arily expressed.
Successful identification o f a gene involved in hum an retinal disease was recently
accom plished using the candidate gene approach. Specifically, the m urine RDS
gene was shown to be m utated in anim als with retinal degeneration (Travis et al.,
1989) and this finding m ade the hum an hom ologue (Travis et al., 1991) a good
candidate gene for retinitis pigm entosa. This was confirm ed when m utations in
THE MOLECULAR GENETIC APPROACH TO M ACITAR DEGENERATION 185
the hum an RDS gene were identified in patients with autosom al do m in an t retini
tis pigm entosa (F arrar et al., 1991; Kajiwara et al., 1991; Wells et al., 1993). Using
the same reasoning, we recently identified two different m utations in the RDS
g en e (Nichols et al., 1993a and 1993b) in families affected with an autosom al dom
in an t p attern dystrophy (Figures 7 and 8) resem bling D eu tm an s butterfly dystro
phy of the fovea (D eutm an et al., 1970). T he first was a G to A substitution at
codon 167 th at substitutes an aspartate for a highly conserved glycine residue. T he
m utation segregated with the disease in eleven affected and fo u r unaffected (>55
yrs) family m em bers (Zmax = 4.0 at 6 - 0). T he glycine1*^ residue is also conserved
in the peripherin-related p rotein rom-1, which may associate non-covalently with
p erip h erin in rod p h o to recep to rs (Bascom et al., 1992). T he phenotype in this dis
o rd e r involves accum ulation o f yellow m aterial within and beneath the retinal pig
m en t epithelium in the perifoveal region. T he parallels with the rds/+ m ouse are
interesting, since the latter shows dysplastic o u ter segm ents which appear to be u n
stable and to shed at an increased rate. T he underlying pigm ent epithelium con
tains large and abnorm al phagosom es which may result from im paired digestion
o f the abnorm al o u ter segm ents (Sanyal and Hawkins, 1988). It is possible that
accum ulation of such phagosom es in the m acular pigm ent epithelium gives rise
F ig u re 8 D e n a tu r in g g r a d ie n t g el e le c tr o p h o r e s is o f PC R p r o d u c ts c o n ta in in g R D S e x o n 1 se
q u e n c e s . C lin ically a ffe c te d in d iv id u a ls a r e in d ic a te d by c lo s e d sy m b o ls w h ile c lin ically u n a f
fe c te d in d iv id u a ls a re in d ic a te d by o p e n sym bols. S p o u ses a re in d ic a te d by sy m b o ls w ith o u t
n u m b e r s w h ic h a re c o n n e c te d to a n a ffe c te d p a tie n t's sym bol by a h o riz o n ta l lin e . E ac h gel
la n e c o n ta in s a s a m p le fro m th e in d iv id u a l w h o se p e d ig r e e sy m b o l is d ire c tly ab o v e. A ffe cte d
in d iv id u a ls h a v e m u ltip le b a n d s o n th e g el (b o th h o m o d u p le x e s a n d h e te r o d u p le x e s ) w h ile
n o r m a l in d iv id u a ls h a v e o n ly o n e b a n d c o r r e s p o n d in g to n o r m a l h o m o d u p le x m o le c u le s.
F ro m N ic h o ls, e t al., N ature Genetics (1 9 9 3 ); u s e d w ith p e rm iss io n .
to the deposits of yellowish m aterial seen in the butterfly lesions (Nichols et al.,
1993a). The second m utation seen in a family with butterfly-shaped pigm ent dys
trophy of the fovea was caused by a 2 base pair deletion in the RDS gene overlap
ping codons 299 and 300 that results in a translational frameshift. This m utation
THE M OLECUIAR GENETIC APPROACH TO MACULAR DEGENERATION 187
onstrating th a t the prim ary expression o f the genetic defect is actually in the neu-
rosensory retina.
Some caution should be introduced at this point: no t all sequence changes in
candidate genes are disease-causing m utations. W hen h u n d red s o f patients are
screened for changes in alm ost any gene sequence, some variation is likely to be
fo und. A change is likely to be a disease causing m utation if it has the following
characteristics.
1) It results in an altered am ino acid (this is especially convincing if the m utation
results in a stop codon, a fram eshift, o r a dram atic change in charge, size or
hydrophobicity o f a single am ino a c id ).
2) It is not found in norm al individuals to any m easurable extent. For exam ple,
if screening fifty n orm al controls (100 chrom osom es) does n o t reveal a similar
sequence change, it is less likely to be a clinically silent polym orphism .
3) T he m utation segregates with the disease phenotype in a statistically signifi
cant fashion (Figure 8 ) .
T he latter criterion is im p o rtan t because if a heterozygous polym orphism is de
tected in an affected p atien t the probability th at a second affected sibling o r par
en t will h arb o r the same polym orphism ju s t by chance is 50%; the probability that
a third would have the polym orphism is 25%, and so on. Thus, pedigrees contain
ing only one or a few affected patients can n o t by themselves prove the participa
tion o f a given candidate gene in the disease. It is rem otely possible for a DNA
sequence change to satisfy all three of the above criteria w ithout actually causing
any disease. For exam ple, suppose the RDS sequence change illustrated in Figure
8 was actually a silent polym orphism , an d that the true disease-causing m utation
was in a n o th er gene a centiM organ o r two from RDS. If the polym orphism s were
in phase with the disease-causing m utation, o n e would get exactly the sam e result
as shown in Figure 8, even although the RDS gene was n o t really involved in the
disease. O f course, the likelihood of this hypothetical situation occurring (espe
cially in two d ifferent families) is so low th at statistically significant segregation o f
an am ino-acid-changing m utation is usually accepted as tentative p ro o f o f the m u
tation's involvem ent in the disease. In fact, once a given phenotype has b een sig
nificantly associated with a m utation in a given gene, it is probably sufficient to
satisfy only the first two criteria. Each new family with a given phenotype that har
bors a d ifferent m utation in the same candidate gene lends additional support to
the hypothesis that the m utations are the prim ary cause o f the disease.
T he ultim ate p ro o f th at a given m utation actually causes the disease is the cre
ation o f the disease in a transgenic anim al by the introduction of a specific m uta
tion initially identified in an affected hum an. T he existence o f such anim als also
provides a platform for studying a disease in ways that are im possible in hum ans.
For exam ple, no hum an eyes with butterfly dystrophy have ever been studied his-
topathologically, and if such specim ens do eventually becom e available they will
likely be from an elderly p atien t with advanced atrophic disease. T he existence of
a transgenic anim al m odel would allow the m orphological features o f the disease
THE MOI .EClI.AR GENETIC APPROACH TO MACULAR DEGENERATION 189
to be worked out in great detail, including the tem poral changes from birth to ad
vanced disease. U ltrastructural, biochem ical, and cell biological studies using
transgenic anim als would have a m uch greater chance o f elucidating the m olecu
lar sequence of events that cause the clinically evident disease than would similar
studies in the hum an population. In addition, once the disease m echanism was
well worked out, physical, dietary o r pharm acologic interventions could be de
vised and tested m uch m ore readily in a transgenic anim al m odel than in the h u
m an population.
Figure 4 dem onstrates two o th er im p o rtan t features o f the m olecular genetic
approach to hum an disease; the d ep e n d en ce at nearly every level on the availabil
ity o f actual hum an patients affected with various disorders an d the n eed for
skilled clinicians to correctly diagnose these patients and carefully define their
phenotypes. It is the clinician who initially discovers a family that is suitable for
linkage analysis. M ore im portantly, if such an analysis is to be successful, each pa
tient m ust be diagnosed with a high degree o f accuracy. For exam ple, it could be
very dam aging to a linkage study to incorrectly diagnose a p atien t with the pre
sum ed ocular histoplasmosis syndrom e as affected in a family with p attern dys
trophy. In a fully inform ative sibship o f 11 sibs and 2 living parents, such a
m isdiagnosis could decrease the lod score from 3 to 1.3. If the nearest genetic
m arker was 10 centiM organs from the disease gene, a single m isdiagnosis could
result in a m axim al lod score o f only 0.5 which m ight not even result in additional
intensive searching in th at area.
At the candidate gene level, clinicians can participate by co ntributing patients
with various phenotypes fo r m utation searches. W hen such m utations are found,
clinical ophthalm ologists can attem pt to correlate specific features o f the disease
with individual m utations. Such correlations have the potential to m ake m olecular
diagnosis a very useful clinical tool. For exam ple, if certain m utations are found
to be associated with a high risk o f exudative m acular degeneration while others
ten d to be associated with slowly progressive geographic atrophy, such inform a
tion could be used to counsel patients as well as to help plan follow-up visits so that
the greatest am o u n t o f scrutiny could be focused upon the patients at greatest risk.
Two recent advances have greatly facilitated both linkage analysis an d the can
didate gene approach. T he developm ent o f highly polym orphic DNA m arkers
known as m icrosatellites (W eber and May, 1989; Litt an d Luty, 1989; Weber, 1990)
has greatly sim plified genetic linkage studies. M icrosatellite m arkers are superior
to restriction fragm ent length polym orphism s (RFLPs) because they are m ore in
form ative and they can be assayed using the polym erase chain reaction (C hapter
1). T he second advance has been the developm ent o f m ethods for screening sin
gle-base sequence substitutions. These m ethods include d en atu rin g gradient gel
electrophoresis (DGGE) (Fischer an d Lerm an, 1980; Meyers e ta l., 1987; Sheffield
et al., 1989), single-strand conform ational gel analysis (SSCP) (O rita et al., 1989),
chem ical cleavage of m ism atch (C otton et al., 1988), and hetero d u p lex analysis
(Keen et al., 1991). As a group, they allow rapid screening o f a candidate gene for
m utations in m ultiple individuals. These techniques decrease the am o u n t o f labor
190 EDWIN M. STONE AND VAL C. SHEFFIELD
CONCLUSION
T he hum an genetic m ap is being refined to the centiM organ level with highly
polym orphic genetic m arkers and many genes are being characterized and
THE MOLECULAR GENETIC APPROACH T O MACULAR DEGENERATION 191
ACKNOWLEGEMENTS
REFERENCES
JO H N K. COWELL
INTRODUCTION
It appears that many hum an solid tum ours result from the inactivation o f critical
genes which are responsible for the norm al developm ent of the particular tissue
in question. Because these genes, th ro u g h their norm al function, ensure histio-
genesis an d prevent tum origenesis, they have been called tu m o u r suppressor
genes or recessive oncogenes. T he first such gene to be isolated was that
responsible for the developm ent of the ch ild re n s eye cancer, retinoblastom a.
T he study of this gene and the application o f this knowledge to the clinical m an
agem ent o f the disease has led, and continues to lead, the way in o u r u n d erstan d
ing o f m olecular events which result in tum our developm ent. T he details about
the isolation an d characterisation o f this gene is the subject o f this chapter.
RETINOBIASTOMA GENETICS
As the nam e implies, Rb is a tu m o u r of retinal cells and, with only rare excep
tions, affects children u n d e r the age o f 5 years. Individuals can present with Rb at
birth, dem onstrating that the tum ours have been growing since early fetal life.
This view' is sup p o rted by the histopathology o f the tum our which dem onstrates a
relatively undifferentiated, em bryonic-like organisation, im plying an arrest in
developm ent o f a retinal precursor cell. Thus, pools o f cells are frozen in a state
in which fu rth e r genetic changes can occur, giving rise to the full tum our p h en o
type. T he exact identity o f these p recursor cells, however, rem ains unknown.
Approxim ately 10% of patients will have a p rio r family history, the rest being ap
parently sporadic. Since the new m utation rate is relatively high (Vogel, 1979)
many o f these apparently sporadic cases will carry new germ line m utations. In
the familial form , the tu m o u r phenotype segregates as an autosom al dom inant
trait (Figure 1). This m eans th at inheritance o f a single m utant gene is apparently
sufficient to result in tum origenesis. In fact, pedigree analysis (Figure 2) shows
that, in 10% o f cases, individuals who inherit the m utant gene do no t develop a
tum our - so called incom plete p e n e tra n c e - so it is clearly only a predisposition
to tum origenesis that is in h erited an d o th er genetic events m ust happen. Thus, at
the cellular level, Rb gene m utations act in a recessive m anner. In fact, this must
be the case since no t all retinal cells in predisposed individuals develop into tu
m ours.
198 JO H N K. COWELL
* j O
C h ri l- jo
O r^BnO i in
i n f l
th at one o f the parents is a tissue m osaic (see Ribeiro et al., 1988 for review) car
rying the m utation in the germ line b u t n o t in th eir own retinal cells.
If tum ours are d etected early, they are usually m ore easily treated than those p re
senting later, although exactly w here the tu m o u r arises in the eye is im p o rtan t in
this regard. T reatm ent o f small tum ours usually involves cryosurgery, photocoag
ulation o r radiation therapy, whereas larger tum ours usually require enucleation.
Tum ours left to develop in the eye will eventually m etastasise, often down the optic
nerve, and prognosis in these cases is very poor indeed. Since early diagnosis of
fers a b etter prognosis, all at risk patients are screened regularly during the first
years o f life. In practice, this involves all relatives o f Rb patients since, because of
the possibility o f incom plete penetrance, tum our form ation is, for m ost patients,
the only unequivocal m eans o f identifying m u tan t gene carriers. Clearly a system
to identify those patients with germ line m utations would m ake the clinical m an
agem ent o f this disease m ore efficient (see below).
O ur u nderstanding o f the finer details of the genetics of Rb came with the clon
ing o f the predisposition gene, RBI. T h e circum stances leading to the actual clon
ing o f RBI resulted from painstaking analysis o f many patients over many years.
Thirty years ago Stallard an d colleagues (Stallard, 1962) showed that the associ
ation betw een Rb an d phenotypic abnorm alities such as m ental retardation, dis
tinctive dysm orphic features and, som etimes, abnorm al gonadal developm ent
200 JOHN K. COWELL
were due to the presence o f constitutional chrom osom e deletions in these individ
uals. T he deletions involved one o f the D-group o f chrom osom es (Lele et al.,
1963), later shown to be chrom osom e 13 (Yunis and Ramsay, 1978). In all cases,
region 13ql4 was involved (Figure 3), indicating the site o f the RBI gene. Since
the esterase-D gene (ESD) was also shown to be located in 13ql4 (Sparkes et al.,
1980), deletion carriers could be detected by a quantitative assay for this enzyme,
which form ed the basis for population studies (Cowell et al., 1986). D eletion car
riers, who have 50% o f norm al enzyme levels, constitute approxim ately 3% o f all
Rb patients (Cowell et al., 1989). Close genetic linkage between Rb and ESD was
re p o rte d by Sparkes et al. (1983), dem onstrating the unequivocal location o f the
h ereditary form o f the disease and, to date, there have been no re p o rted recom
bination events between the two loci (Cowell et al., 1987b). T he exact orientation
o f the two genes was established by the analysis of a p atien t with a constitutional
13ql4-q31chrom osom e deletion and norm al ESD levels (Cowell et al., 1987a). Us
ing somatic cell hybrids the ESD gene was shown to rem ain on the deletion chro
m osom e b u t the RBI gene did not. This placed the RBI gene distal to ESD
(M itchell an d Cowell, 1988), probably in 13ql4.3.
Sporadic tum ours from individuals heterozygous for polym orphic ESD alleles
(allowing each o f the parental chrom osom es to be distinguished) often lost one
allele (G odbout et al., 1983). This loss o f heterozygosity was also dem onstrated
using polym orphic DNA probes (Cavenee et al., 1983, Dryja e ta l., 1984). C hrom o
some analysis of these tum ours showed that two copies of 13 were still present. T he
in terp retatio n o f this observation was that an acquired m utation in retinal p recu r
sor cells was duplicated at some stage an d the norm al hom ologue was then lost. In
this way, the cell becom es hom ozygous for the initial loss o f function m utation in
RBI an d the cells do n o t have a functional protein. T he m echanism s by which this
loss o f heterozygosity (LOH) occurs were m ost frequently due to chrom osom e
non-disjunction and m itotic recom bination (Cavenee et al., 1983). Cavenee et al.,
(1985) showed that, in a tu m o u r from a p atien t with heredutary Rb, the chrom o
some retained in the tu m o u r was th at transm itted by the affected parent. It
appears that up to 70% of tum ours experience this LOH (Cavenee et al., 1983).
These kind o f analyses allowed the origin o f the parental m utation to be deter
m ined (Dryja et al., 1989). In sporadic cases there was no differential susceptibil
ity to som atic m utation between the hom ologous copies of the gene. However, for
new germ line m utations, the heritable m utation arose on the paternally derived
chrom osom e. These findings and those o f Zhu et al. (1989) argue against
genom ic im printing being im portant in Rb tum origenesis but point to new m uta
tional events arising predom inantly d u rin g sperm atogenesis. It has not, however,
been possible to attribute these to a paternal age effect (M atsunaga et al,, 1990).
9 *
8 % g 9
GOS 46 GOS 122
I a 5
GOS 49 GOS 140
S fc |
v
GOS 50
GOS 173
abnorm alities were found infrequently in tum our cells, although several interest
ing observations have com e ou t of these cytogenetic analyses (see Cowell and
Hogg, 1992, for review). T he m ost consistent finding was the presence o f (usually
two copies of) an isochrom osom e 6p (iso-6p) and trisomy for all or part of the
long arm of chrom osom e 1 (lq + ), in 45% an d 44% o f tum ours respectively.
C hrom osom e abnorm alities involving chrom osom e 13, usually resulting in
m onosom y 13, was only found in 20% o f cases. A bnorm alities involving lq are the
202 JO H N K. COWELL
m ost com m only seen in all tu m our cells. By contrast iso-6p is less frequently
observed, being restricted, largely, to Rb an d m alignant m elanom as (B echer et
al., 1983). It is possible, therefore, that duplication o f certain genes on the short
arm o f chrom osom e 6 are im p o rtan t in tu m o u r progression. However, since this
analysis is usually p erfo rm ed on advanced stage tum ours, it is difficult to deter
m ine w hether these changes are causal in tum origenesis or consequences of it.
C hrom osom e analysis o f in d ep en d e n t tu m o u r foci from a bilaterally affected
p atient showed that each had distinct abnorm alities, suggesting an in d ep en d e n t
origin for them (Squire et al., 1985). Tien et al. (1989) analysed a large, appar
ently unilateral, tum our and found cytogenetically distinct clones. This was inter
p reted to m ean that the tum ou r probably arose as the result of fusion of several
foci. This has im p o rtan t im plications for counselling since unilateral tum ours are
th o u g h t to be associated, predom inantly, with sporadic events. Multifocal
tum ours, however, even in only one eye, probably identifies th at patient as a
h ereditary case, especially if these tum ours have a relatively early age of onset.
W hether this analysis is justified for the relatively small re tu rn in term s of
im proved m anagem ent is questionable.
Following the random isolation of only 12 DNA probes from a flow-sorted chro
m osom e 13-specific DNA library (Lalande et al., 1984) one, H3-8, was shown to
be within the smallest of constitutional deletions in Rb patients. A djacent
sequences (Dryja et al., 1986) were shown to be within a gene which was soon iso
lated (Friend et al., 1986) and which detected structurally abnorm al mRNA in Rb
tum ours with varying frequencies (Friend et al., 1986; G oddard e ta l., 1988). Sim
ply isolating a gene from a particular p art o f the chrom osom e, however, is not, in
itself, conclusive evidence for the authenticity o f the gene. A djacent genes would
also be candidates although the identification of structural rearrangem ents o f
this gene in Rb tum ours strongly su pported its candidature. T he tissue distribu
tion o f expression o f RBI, however, was slightly surprising, being present in rela
tively high levels in all tissues exam ined (Friend et al., 1986). This was
u n expected since the hypothesis was that this gene controls im p o rtan t aspects of
the developing fetal retina. T he dem onstration of predisposing m utations involv
ing RBI provided m ore convincing evidence for its authenticity. In patients show
ing constitutional, predisposing reciprocal translocations the breakpoints on
chrom osom e 13 always in te rru p te d the RBI gene (Higgins et al., 1989; M itchell
an d Cowell, 1989). Thus, adjacent genes could n o t be involved.
A few cases have been re p o rted where the translocation p artn e r chrom osom e is
the X (Ejima et al., 1982; H ida et al., 1980; Nichols et al., 1980). Because random
X-inactivation occurs in females, w hen the derivative chrom osom e is inactivated in
retinal precursor cells the position o f the b reakpoint on 13 is no t im portant, since
the whole chrom osom e experiences genetic silencing, thereby constituting the
MOLECULAR GENETICS OF RETINOBLASTOMA 203
first hit. In two families in o u r series, with t( 1:13) (q22:ql4) and t (13:20) (q l4 :p l2 )
translocations, the p atien t with Rb in h erited the rearran g em en t from a p aren t
who was n o t affected (B. Gibbons, pers. com m ). A sim ilar situation was discussed
by Dryja et al. (1984) for 13ql4 deletions, in that deletion carriers often only have
unilateral, unifocal tum ours. In o u r survey (Cowell et al., 1989) half o f the 16 cases
re p o rted had unilateral tum ours. T h ere are also reports of deletion patients who
have never developed tum ours (Cowell et al., 1988; Fukushim a et al., 1987). T he
age o f onset of tum ours from patients with chrom osom e 13 abnorm alities also ap
pears to be later than those with germ inal m utations (Ejima et al., 1988). T he ex
planation for the low p en e tran ce in deletion patients is n o t clear although one
proposal is th at these deletions expose lethal m utations which are deleterious to
the rapidly growing tum our cells (Dryja et al., 1984). This does n o t explain the low
p en etran ce of the reciprocal translocation carriers, unless large deletions are as
sociated with the rearrangem ent. In o n e case o f a t (1:13) rearra n g em en t (Mitchell
and Cowell, 1989), although a deletion was associated with the translocation, it was
maximally 8 kb long and confined to the RBI gene. Keith et al. (1985) described
a p atien t with a 13ql4 deletion with a single tu m o u r in one eye and a retinom a in
the other, both o f which are considered to be 'm ild' form s o f the disease.
MUTATIONS IN RB TUMOURS
Only 20% o f tum ours showed structural abnorm alities of RBI an d very few p re
disposing m utations involve chrom osom e translocations. Clearly, the majority of
m utations were m ore subtle. T he n a tu re o f these m utations in tum ours has been
dem onstrated in a variety o f ways. D unn and colleagues (D unn et al., 1989) anal
ysed RNA from tu m o u r cells, although this is n o t always possible for reasons dis
cussed by Cowell an d Hogg (1992). N onetheless a variety o f d ifferent m utations
in tum ours and cell lines were rep o rted . T he RBI cDNA is 4.7 kb long (Friend
et al., 1986) and consists o f 27 exons clustered into three groups each separated
by two very large introns (Figure 4 ). T he 27 exons rep resen t the fragm ented cod
ing region of the gene as they ap p ear on the chrom osom e which are subse
quently jo in e d together in the final mRNA. In the absence o f mRNA, each
individual exon can be sequenced from DNA by first amplifying the exon and
flanking intron regions using the polym erase chain reaction (PCR). D irect
sequencing o f these PCR products would identify m utations by com parison with
the norm al sequence. This approach has been successful (Yandell et al., 1989)
and was im proved using the single strand conform ation polym orphism (SSCP)
technique to prescreen the PCR am plified exons before sequencing, which iden
tified those DNA m olecules m ost likely to carry m utations (Hogg et al., 1992).
In sum m mary, the available data shows th at there is no ap p a ren t hot-spot for
m utations within RBI. T he m ajority o f m utations are insertions, deletions or sin
gle base pair substitutions which result in the p roduction o f prem ature stop
codons. These m utations would be predicted to result in structurally grossly
204 JOHN K. COWELL
200 kb ------------------------------------------
36 kb 70 Kb
V V
H I I 114 II II I II I I M I I III 111
12 3 4 56 7 89 1011 12 13-17 18 19 20 21-23 24-26 27
abnorm al proteins, which is consistent with antibody analysis where Rb tum ours
mostly had n o detectable Rb protein (Horowitz et al., 1990). In approxim ately
10% of tum ours, the m utations affect the correct processing of the mRNA (splic
ing) by dam aging sequences essential for this process. Missense m utations, simply
substituting one am ino acid for another, appear to be less com m on. In o u r own
survey of hereditary Rb patients, one exam ple was found in exon 20, which was
associated with a low p e n e tra n c e phenotype (O nadim et al., 1992). It is tem pting
to speculate that the substitution of a single am ino acid only com prom ises the
function o f the protein and, unless the second m utation in the tu m o u r precursor
cell causes loss of RBI function, duplication of the weak m utation allows a suffi
ciently functional Rb protein to be produced, so preventing tum origenesis. This is
consistent with o u r observation in this particular family since many o f the m utant
gene carriers were eith er unaffected or had regressed tum ours (O nadim et al.,
1991). Sakai et al. (1991) also investigated low pen etran ce families and found m u
tations in recognition sequences for different transcription factors - the pro m o ter
-w h ic h lie at the beginning o f the gene and which controls the production of RBI
mRNA. Again the suggestion is that, as a result, a quantitative decrease in tran
scription occurs ra th e r than com plete inactivity. Sufficient pRB is produced, how
ever, and any phenotypic consequences are mild. W hether single am ino acid
changes will generally be found in patients with m ild phenotypes is still n o t clear.
T ranscription
Go G1 S G2 M
It has been known for some time that m u tan t RBI gene carriers also develop sec
o n d non-ocular tum ours; the conclusion always being that RBI also controls the
n o rm al developm ent of these tissues. Patients carrying constitutional RB gene
m utations are at significantly higher risk for the developm ent o f second, non-ocu
lar tum ours later in life (D raper et al., 1986) .These are usually osteosarcom as and
soft tissue sarcomas. Both o f these tum ours were shown to lose heterozygosity for
m arkers on chrom osom e 13 (Dryja et al., 1986; Friend et al,, 1987). T he same
classes of tum ours also show freq u en t structural an d transcriptional abnorm ali
ties o f RBI suggesting it plays a role in establishing the m alignant phenotype in
these cells. T he risk o f second tum ours is en h a n ced within the irrad iated tissues
following radiation treatm ent. This risk is greater for patients with germ line
m utations in RBI. This raises im p o rtan t issues ab o u t w'hether to treat these
patients with radiation although the risks m ust be offset with the benefits.
MOLECULAR GENETICS OF RETINOBLASTOMA 2 07
In addition to those tum ours known to be associated with RBI gene m utation
carriers, structural abnorm alities were also found in breast cancer (Lee et al.,
1988; T'Ang et al., 1988) and small cell lung carcinom a (H arbour et al., 1988) cells
at high frequency. A series o f o th er tum ours showing less freq u en t involvem ent
was presented by Horowitz et al. (1990). It is likely, however, that somatic RBI m u
tations in these o th er tissues only contribute to tu m o u r progression since, in many
cases, the frequency of tum ours with m utations is still relatively low.
O ne interesting tum our associated with hei'editary Rb is pineoblastom a. Be
cause the pineal is th o u g h t to be a vestigial p h o to rec ep to r organ, patients with ret
inal an d pineal tum ours have been described as having trilateral retinoblastom a
(Bader et al., 1982; Holladay et al., 1991). T he chances o f two such rare tum ours
occurring coincidentally is negligible an d suggests that the RBI gene also contrib
utes to the developm ent of pineoblastom a.
1-8Kb -1 -75 Kb
1-7Kb-
RISK ASSESSMENT
T here have been many estim ates of the relative risks for the developm ent o f Rb in
the children and siblings o f affected individuals. Many o f these calculations are
MOLECULAR GENETICS OF RETINOBIASTOMA 209
reviewed by Vogel (1979) bu t the figures do vary from study to study and alm ost
certainly reflect referral bias. In a review of the UK population o f Rb patients
involving over 900 cases over a 25 year period, D raper an d colleagues (D raper et
al., 1992) have re-evaluated the figures. Clearly, w here there is a family history the
chances o f inheriting the m u tan t gene is 50% but, because only 90% of gene car
riers actually develop a tum our, the actual risk is n ea rer 45%. It was n oted some
time ago (Cowell et al., 1986) that bilaterally affected m u tan t gene carriers gener
ally have bilaterally affected children and th at unilaterally affected gene carriers
have a greater chance o f having unilaterally affected children, o r som etim es unaf
fected gene carriers (O nadim et al., 1991). T he risk o f tum origenesis for the chil
d ren of unilaterally affected m u tan t gene carriers is therefore lower, estim ated by
D raper and colleagues at aro u n d 30%. In the UK series, where there is no family
history o f Rb the probability of a sibling being affected is 1.1% if the pro b an d is
bilaterally affected an d 0.6% if the p ro b a n d is unilaterally affected. T he differ
ence, however, is no t statistically significant. Finally, using the m ethod o f maxi
m um likelihood estim ation the probability of a unilateral case with no family
history being a gene carrier was calculated to be 1.7% giving th eir children a risk
o f approxim ately 0.8%.
to
' X *
I I
F ig u re 8 E x a m p le o f a fam ily p e d ig r e e sh o w in g m ild e x p re s s io n o f th e RB p h e n o ty p e a n d a
m is se n se m u ta tio n in e x o n 20. D N A s e q u e n c e analysis show s th e m u ta n t g e n e is p r e s e n t in
th e g r a n d p a r e n ts ( h a tc h e d sym bols) w h o b o th h a d re g re s s e d tu m o u r s as w ell as in th e u n a f
fe c te d in d iv id u a ls in d ic a te d by th e a rro w s. A ffe c te d in d iv id u a ls a re sh o w n w ith so lid sym bols.
In d iv id u a ls w ith u n ila te r a l R b a re r e p r e s e n t e d by h a lf-fille d sym bols, th o s e w ith b ila te ra l R b
by so lid sym bols.
REFERENCES
SUMMARY
In this chapter, the light sensitive visual pigm ents that underlie norm al and
defective color vision, an d the genes th at encode them are reviewed. New u n d er
standing o f these has developed quickly in the last few years since the techniques
o f m olecular biology have been b ro u g h t to bear on long p o n d ered problem s.
Decades before m olecular m ethods w ere available, a theory to explain the biolog
ical basis o f norm al and defective color vision was widely accepted. A favored ver
sion o f that theory held th at everyone with norm al color vision has, in com m on,
three types of cone photoreceptor, each containing a d ifferent type o f visual pig
m ent. T he com m on color defects were proposed to occur w hen one type o f cone
pigm ent was lost, or w hen the p h o to p ig m en t in one o f the cone types was
replaced by an abnorm al (or anom alous) pigm ent, d ifferent in absorption spec
trum from any o f the three stereotyped norm al pigm ents. However, over the
years, results have accum ulated th at are difficult to fit within the fram ework of
the classic view. Thus, an alternative theory is outlined. Its essentials are as fol
lows.
(1) H um ans can have many genes on the X-chrom osom e th at encode cone p h o
topigm ents (as many as ten are n o t uncom m on).
(2) Spectrally distinct subtypes o f pigm ents occur within each of the two classes of
X -encoded cone pigm ents. Both n orm al an d color defective observers draw
from the same pool o f pigm ents.
(3) People with norm al color vision all have at least one pigm ent from each of the
two X -encoded pigm ent classes, b u t they can have m ore than o n e subtype
from eith er class and, thus, can have m ore than three spectrally distinct cone
types.
(4) All of the com m on color vision deficiencies can be explained by a m utational
pathway in which genes that produce pigm ents req u ired for norm al color
vision are deleted by u n equal crossing over betw een norm al pigm ent gene ar
rays.
This is n o t offered as the only possible m odel, b u t h ere it serves as a fram ework
aro u n d which the facts o f color vision phenotypes an d genotypes can be organized
and re-evaluated.
218 JAY NEITZ AND MAUREEN NEITZ
INTRODUCTION
C olor vision capacities are characterized by the abilities to distinguish lights that
differ in th eir wavelength com positions. A lthough hum an color capacities are
extensive, there are many differences in wavelength com position to which we are
all quite blind. Deficiencies in o u r ability to tell spectrally different lights apart
are exploited, for exam ple, in color television th at employs m ixtures o f ju st three
color prim aries, red, green, and blue to synthesize a wide range o f colors. T he
wavelength com position o f the light from a banana seen on television is quite dif
feren t from a real banana, bu t we are nearly blind to the physical difference.
Inabilities to see the physical differences betw een lights th at differ in wavelength
co n ten t have proved convenient for classifying d ifferent form s o f color vision.
For exam ple, given a test light o f any wavelength com position, hum ans with nor
mal color vision can make an identical color m atch using ju s t three appropriately
chosen color prim aries, e.g., red, green, a n d blue - eith er by m ixing the right
am ounts o f the three prim aries, o r by m ixing two prim aries and adding the third
to the test light. This three-dim ensionality or trichrom acy is the hallm ark o f nor
mal hum an color vision. O th er form s of color vision (or color vision deficiency)
can be similarly classified by the nu m b er o f prim aries req u ired in color m atches.
An individual who is com pletely color-blind, classified in this way, is a m onochro-
mat. A m o n ochrom at can exactly m atch the appearance of any light, regardless
of its wavelength content, with a single fixed prim ary light by simply adjusting the
intensity o f the com parison prim ary. T he interm ediate category, dichom ats, need
only two prim aries. This form of color vision seems to be a standard am ong o th er
species (Jacobs, 1981). For exam ple, am ong m am m als, trichrom acy has been
fo und to occur only am ong prim ates. Many o th er fam iliar m am m als, e.g., dogs,
cats, an d squirrels, are dichrom ats. D ichrom acy is also fairly com m on am ong
hum ans. In Caucasian populations ab o u t 1 person ou t of every 100 is a dichro-
mat.
T he term s m onochrom acy, dichrom acy and trichrom acy strictly refer to the
n u m b er of prim ary colors req u ired in color m atches, however, these term s are of
ten (mis-) used to refer to the nu m b er of spectrally d ifferent cone pigm ents an in
dividual has, or is believed to have. W hile it is true that three pigm ents are the
m inim um required for trichrom atic color vision an d at least two are n eeded for
dichrom acy, there may be hazards in equating visual capacities with the nu m b er
of cone pigm ent types. It is possible, for exam ple, to im agine a system with dozens
of spectrally different receptors that still has m onochrom atic (color blind) vision,
since color vision requires th at the receptors be w ired in a way th at yields infor
m ation ab o u t spectral differences. Or, m ore to the p o in t in this review, the sim
plest (and favorite) theory of hum an color vision holds th at we have th ree types of
receptors each tu n ed to a d ifferent region o f the visible spectrum . Trichrom acy is
a fact o f norm al hum an color vision. No doubt, we have at least three types o f re
ceptors. But, we could have m ore than th ree and still be trichrom ats if the signals
from m ultiple receptors were channeled into three outputs at any stage in the
COLOR VISION DEFECTS 219
visual system. As will be seen, re cen t evidence suggests that many people may, in
deed, have m ore than three spectrally d ifferent cone pigm ents.
(1) Monochromacy
Am ong hum ans, com plete color blindness (m onochrom acy) is extrem ely rare,
affecting ab o u t 1 in 100,000 o f the general population. W ithin this group are
three types o f congenital m onochrom acy (reviewed by N athans et al., 1989;
220 JAY NEITZ AND MAUREEN NEITZ
(2) Dichromacy
T he m ost severe form s o f congenital color blindness that are com m only en co u n
tered, are the dichrom acies. As th ere are three classes o f hum an cone pigm ents,
th ere are three different types o f dichromacy. P rotanopes have a single m iddle-
and a single short-wave pigm ent b u t no long-wave pigm ents. D euteranopes have a
single long- an d a short-wave pigm ent bu t lack middle-wave pigm ent. T ritanopes
are missing short-wave receptors but have the o th e r two classes. T ritanopia is a
rare condition that is in h erited as an autosom al d om inant disorder. It is m uch
less com m on than the X-linked form s of color blindness bu t in some populations
the incidence is as high as 1 in 500 (van H eel et al., 1980). Recently, three differ
en t m utations have been identified within the gene encoding the short-wave pig
m en t in individuals with tritanopia, which segregated with the disorder and were
absent from general population controls (Weitz et al., 1992a; Weitz, W ent &
Nathans, 1992b). In each case, a non-conservative am ino acid substitution was
COLOR VISION DEFECTS 221
(3) Trichromacy
Less severe form s o f color vision deficiency are the anom alous trichrom acies.
A nom alous trichrom ats, like n orm al trichrom ats, require three prim aries in
color m atches. However, they generally have p o o re r color discrim ination ability
than norm al. T he term s for the anom alous trichrom acies: protanom aly, deutera-
nomaly, an d tritanom aly, parallel those for the dichrom atic types. D euteranom aly
is the m ost com m on form o f color vision deficiency affecting about 5% of males
in Caucasian populations. T he frequency o f protanom aly is about 1% o f males.
Tritanom aly is a rare condition.
T here is considerable variation in the ability to discrim inate colors within each
class of X-linked anom alous trichromacy. Some protanom als and deuteranom als
have re d /g re e n color vision that is nearly as p o o r as a dichrom at's. O thers have
m uch m ore m ild color deficiency; the color discrim ination ability of some ap
proaches norm al. P erceptual tests th at involve color m atching were useful for
broad classification o f color vision types. C olor m atching tests are also used in the
diagnosis o f color anom alies. A dram atic difference betw een the m ild anom alous
trichrom acies an d norm al can be seen in behavior in a color m atching task that is
referred to as the Rayleigh color m atch (after Lord Rayleigh, 1881, who intro
duced the use of this color m atch for diagnosing red-green color vision varia
tions). A person is given ju st two prim ary lights, a red and a green and is asked to
mix them in a p ro p o rtio n that will exactly m atch the appearance of a m onochro
matic yellow com parison light. Only two prim ary lights are req u ired in this color
m atch because the short-wave cones are very insensitive to the lights used in this
test. The observer needs only to satisfy the req u irem en t th at the m iddle- an d long
wave pigm en ts absorb photons at the same rate when stim ulated by the re d /g re e n
m ixture as they do when presented with the m onochrom atic yellow' light. Similar
to norm als, mildly anom alous trichrom ats can find a fairly narrow range o f re d /
green m ixtures th at identically m atch the yellow com parison. However, they
choose a ratio o f red to green light that is very different from norm al. Mildly pro-
tanom alous observers require considerably m ore red light and mildly deutera-
nom alous observers, considerably m ore green light in the m ixture than observers
with norm al color vision. For exam ple, mildly deuteranom alous observers typical
ly m atch the m onochrom atic yellow light with a re d /g re e n ratio that is 4 to 5 times
lower than norm al. People with m ore extrem e color anom alies, because o f their
p o o r color discrim ination ability, will accept a wide range o f re d /g re e n ratios as
indistinguishable from the yellow standard, often including the norm al re d /g re e n
ratio an d the ratio o f the corresponding m ild anomaly.
222 JAY NEITZ AND MAUREEN NEITZ
C olor m atches occur at the level of the pigm ents. In the color m atch described
above, the m iddle- and long-wave pigm ents each absorb some pro p o rtio n o f inci
d e n t photons from the m onochrom atic yellow light. T he m ixture ratio of red and
green light can be adjusted to produce the same p ro p o rtio n of quantal absorption
in these pigm ents as the yellow light does. T he im portant consequence o f the fact
th at the m atch occurs at the level o f pigm ent absorption is that once the lights are
set to m atch in their effects on the pigm ents, the two lights cannot be m ade distin
guishable by any trick of the nervous system, o r by changing eith er the ratio of
m iddle-to-long wave cones or the distribution o f pigm ents am ong the cone recep
tors. For exam ple, a pigm ent m atch m ade by a person who has long-wave pigm ent
in one set o f cones and middle-wave pigm ent in a separate set o f cones, would be
the same as a (hypothetical) person who has one cone type with long-wave pig
m ent and a second cone type containing a m ixture o f m iddle- and long-wave pig
m ents.
Individual differences in the density o f spectrally selective ocular filters, such as
the yellow pigm ents in the lens and m acula, do produce slight individual differ
ences in the ratio o f red-to-green light reaching the pigm ents, and these can influ
ence the color m atch. Also, differences in the effective optical density o f the visual
pigm ents within the cones can produce slight differences in the absorption spec
tra o f the receptors. O th er than the slight influences o f these factors, however, two
people can have different color m atches only if they have com plem ents o f pig
m ents th at differ in absorption spectra. T he fact th at anom alous trichrom ats re
quire extrem ely abnorm al ratios o f red-to-green light in color m atches indicates
that they have very different com plem ents o f pigm ent spectral sensitivities than
norm als.
T he visual pigm ents that u nderlie the two X-linked anom alous trichrom acies
have, at least to a first approxim ation, been characterized (e.g. DeM arco, Pokorny,
& Smith, 1992; Pokorny & Smith, 1977; P iantanida, 1976; Pokorny, Smith, & Katz,
1973). Protanom alous observers, like protanopes, have no long-wave pigm ents b u t
they m aintain two slightly d ifferent pigm ents th at peak in the m iddle wavelengths.
T hese two pigm ents are sufficiently different in their spectral properties to sup
p o rt some color discrim ination in the m iddle-to-long wavelengths. T he absence of
long-wave pigm ents in protanom alous observers confers a loss in sensitivity to very
long-wavelength (red) lights. In color m atching, protanom alous observers com
pensate for their insensitivity to red light by adding a m uch higher p ro p o rtio n of
the red prim ary to the m ixture. In contrast, deuteranom alous observers lack any
m easurable contribution to their vision from middle-wave pigm ents, b u t they
m aintain two types o f long-wave pigm ents. T he spectra o f the two long-wave pig
m ents expressed in deuteranom aly are similar bu t their difference supports some
degree of red-green color vision th at is variable across individuals.
Until recently it was believed that anom alous trichrom acy was caused by either
the replacem ent o f the gene th at encodes norm al long-wave pigm ent (in p rota
nomaly) o r the gene th at encodes norm al middle-wave pigm ent (in d eu teran o m
aly) with an abnorm al gene. T he abnorm al gene (previously believed to be allelic
COLOR VISION DEFECTS 223
with the norm al gene) was th o u g h t to produce an anom alous pigm ent with a spec
trum shifted from th at of norm als. Thus, for exam ple, deuteranom alous observers
were conceived as having a m u tan t (anom alous) middle-wave pigm ent an d a nor
mal long-wave pigm ent. Similarly, protanom alous observers were conceived as
having a norm al middle-wave pigm ent and a m u tan t (anom alous) long-wave pig
m ent. This is now th o u g h t to be in co rrect (N athans et al., 1986a). C u rre n t u n d er
standing suggests, to us, that a m uch m ore useful conception is that anom alous
trichrom ats, like th eir dichrom atic counterparts (deuteranopes an d protanopes),
are each missing all m em bers o f one class o f cone pigm ent. D euteranom alous ob
servers have no middle-wave pigm ents, however, unlike deuteranopes, they m ain
tain two slightly different long-wave pigm ent types. P rotanom alous observers have
no long-wave pigm ents b u t they m aintain two slightly d ifferent middle-wave pig
m ent types.
Dichromacy and anom alous trichrom acy are phenotypic color vision variants that
occur with relatively high frequency in the hum an population. People with these
color vision deficiencies have different com plem ents of cone pigm ents than nor
mal. In addition to the large color vision differences between norm al an d color
defective vision, m ost people with norm al color vision have experienced disagree
m ents with others ab o u t colors. Are som e o f these norm al individual differences
in color perception caused by individual differences in cone pigm ents? R ecent
evidence indicates that they are.
Individual differences in norm al color vision have been long recognized. How
ever, only recently has there been renew ed interest in these norm al variations and
their cause. Fifteen years ago, A lpern and colleagues m easured individual differ
ences in color defective observers th at they attrib u ted to variation in the cone pig
m ents (Alpern & Moeller, 1977; A lpern & Pugh, 1977; A lpern & Wake, 1977).
They concluded th at the long-wave pigm ents of different d eu teran o p es are vari
able in their spectral position as are the middle-wave pigm ents o f protanopes.
They reasoned th at if the dichrom acies are reduced form s of norm al vision, then
the m iddle- and long-wave pigm ents o f norm als m ust vary as well. They em pha
sized th at a finding o f individual differences in norm al pigm ents would dictate m a
jo r alterations in theories o f the basis o f color deficiency. However, the idea that
norm al pigm ents vary in spectral sensitivity has been controversial over the past
decade an d a h alf and the possibility o f variation in norm al pigm ents has contin
u ed to be ignored in the form ulation o f favored theories to explain color defects.
W hile all the controversies have yet to be settled, results have now accum ulated
to provide strong evidence th at the spectra o f norm al cone pigm ents do vary
across individuals. T he picture th at em erges is that am ong color norm als there
may be discrete form s o f both the long-wave pigm ents an d the middle-wave pig
ments. Most com m on are, perhaps, two m ajor alternative form s o f the long- and
two m ajor alternative form s o f the middle-wave pigm ent. T he idea o f discrete vari
224 JAY NEITZ AND MAUREEN NEITZ
ation in norm al hum an pigm ents was suggested earlier by W aaler (1967) from
analysis o f norm al color m atches, by Eisner & M acLeod (1981) who used psycho
physical m ethods to isolate responses from norm al m iddle- and long-wave pig
m ents and by Dartnall, et al. (1983), whose m icrospectrophotom etric study o f the
spectra from individual hum an ph o to recep to rs suggested two types o f m iddle- and
two types o f long-wave receptors am ong people with norm al color vision.
T he largest n u m b er o f recen t studies that provide evidence for variation in nor
mal pigm ents have exam ined norm al individual differences in color m atching.
T he Rayleigh color m atch (described earlier) where a m ixture o f red an d green
light is adjusted to m atch the appearance o f a m onochrom atic yellow light, has
long served to classify abnorm al vision. Since the re d /g re e n ratio set in the m atch
is directly related to the absorption spectra of the pigm ents, this test can be a very
sensitive indicator o f individual differences in norm al m iddle- and long-wave pig
m ents as well. If the cone pigm ents of norm al observers vary in spectral position,
the distribution of norm al color m atches will reflect that variability. R ecent studies
specifically designed to exam ine such norm al individual differences agree that
th ere is considerable variability in norm al color m atches, an d the range o f differ
ences is sim ilar across studies (Neitz & Jacobs, 1986; Eisner & Burns, 1987; Jo rd a n
& M ollon, 1988; W ebster & MacLeod, 1988; Lutze, Cox, Smith, & Pokorny, 1990;
Neitz & Jacobs, 1990; Lindsey, W inderickx, Sanocki, Teller, Deeb, & Motulsky,
1992; Shevell & He, 1992; W m derickx, Lindsey, Sanocki, Teller, Motulsky, & Deeb,
1992b; P iantanida & Gille, 1992). These differences in norm al color vision can be
striking. N orm al observers at one extrem e o f the distribution require a re d /g re e n
ratio that is m ore than twice as high as those at the o th er extrem e. This m eans that
a re d /g re e n m ixture that a norm al person from one e n d o f the distribution sees
as exactly m atching the m onochrom atic yellow is seen as a very conspicuous red-
o range m ism atch to a norm al person that falls at the o th er extrem e.
A greem ent about the issue o f discretely different pigm ents has been a d ifferent
m atter. If there are discrete form s of the n orm al m iddle- and long-wave pigm ents,
and if the steps in spectral peak are large enough, then different groups o f indi
viduals with distinct pigm ent com plem ents should be evident in the distribution
of norm al color m atches. Evidence for such m odality in the distribution o f color
m atches is claim ed by some studies (e.g. Neitz & Jacobs, 1986; Neitz & Jacobs,
1990; Eisner & Burns, 1987; Lindsey, et al., 1992; W inderickx, et al., 1992b;
P iantanida & Gille, 1992) bu t clear evidence o f m odality is lacking, o r at least, is
n o t as evident in o th er studies (e.g. Jo rd a n 8c M ollon, 1988; Lutze, et al., 1990;
Webster, 1992).
In the past year, the idea th at a small n u m b er o f discretely different types o f pig
m ents occur am ong observers with norm al color vision has gained considerable
im petus from studies that reveal a strong correlation between individual differ
ences in color vision and a polym orphism at a single am ino acid position in the
m iddle and long-wave pigm ents (Neitz, Neitz, & Jacobs, 1991c; Lindsey, et al.,
1992; W inderickx, et al., 1992b; Merbs & N athans, 1992a; Neitz, Neitz, &: Jacobs,
1993). These are discussed in detail in the section on spectral tuning, below.
COLOR VISION DEFECTS 225
N athans et al. (1986a; N athans, Thom as, & Hogness, 1986b) were the first to iso
late and characterize genes that encode cone visual pigm ents. They described the
goal o f their studies: to test the hypothesis th at hum an color vision is m ediated
by a family o f rhodopsin-like m olecules, the apoproteins of wLiich are encoded by
the corresponding m em bers o f a family o f g en e s... [and the] related hypothesis
that the com m on in h erited variations in hu m an color vision (color blindness)
are due to m utations in the m em bers of this gene family. A long history of
research on color vision and color blindness m ade clear predictions as to what
they would find. F undam ental to theories o f color vision is the idea that norm al
hum an vision is m ediated by three pigm ents, each presum ed to be en co d ed by a
single gene. C olor vision deficiencies that involve the short-wave pigm ent are
in h erited in an autosom al fashion whereas red-green color vision deficiencies are
X -chrom osom e linked. Thus, it was expected that two cone pigm ent genes, one
encoding a long-wave, and the o th er a middle-wave pigm ent w ould be found on
the X-chrom osom e, and a third, encoding the short-wave pigm ent, would be
found on an autosom e.
N athans et al. (1986b) isolated clones containing fragm ents o f at least th ree pig
m ent genes th at m ap to the X -chrom osom e from a genom ic DNA library of a m ale
( J. N athans) who has norm al color vision. They concluded th at the cloned frag
m ents, from this one person, co rresp o n d ed to one gene encoding the long-wave
pigm ent and two copies o f a gene encoding the middle-wave pigm ent. They also
isolated three X-linked pigm ent genes from a cDNA library m ade from several
dozen hum an eyes obtained at autopsy. They no ted th at these X-linked genes were
polym orphic. All the genes were slightly d ifferent in nucleotide sequence.
T he finding o f m ore than two X-linked cone pigm ent genes from one norm al
m ale was quite unexpected. Subsequently, S outhern hybridization analysis of the
X-linked pigm ent genes from a sam ple o f males with norm al color vision suggest
ed that the presence o f e x tra pigm ent genes was no t uncom m on, an d th at the
n u m b er o f pigm ent genes was variable across individuals. N athans an d colleagues
concluded th at everyone had a single copy of the long-wave gene, b u t individuals
could have eith er one, two, or three copies o f the middle-wave gene. Two copies
o f the middle-wave gene were proposed to be the m ost com m on arrangem ent.
In a second study (N athans, et al., 1986a), the genes of 25 subjects with congen
ital color defects were exam ined using S outhern hybridization analysis. It was long
believed that X-linked color vision deficiencies are caused by alterations in genes
that encode m iddle- and long-wave pigm ents and, indeed, in every case, the gene
arrangem ents o f the color blind subjects, as in ferred from S outhern analysis, were
different from any observed in the N athans et al. (1986b) sam ple o f norm als. T he
results an d conclusions can be sum m arized as follows.
1) D euteranopia. Six o f nine d eu teran o p es exam ined h ad a single long-wave
gene an d no middle-wave genes. T he three o th er d eu teran o p es h ad m ultiple
226 JAY NEITZ AND MAUREEN NEITZ
pressed in the green cones to g eth er with the norm al green pigm ent gene or
genes. This arran g em en t th erefo re confers a G R+ [mild] or G R+ [extrem e
deuteranom alous] phenotype. This cannot, however, explain the deu tera
nom alous phenotypes. As described earlier, the X-linked pigm ents that u n d e r
lie deuteranom aly are sim ilar in spectrum . Close enough, in fact, th at they can
b oth be classed as long-wave pigm ents. Mixing both long- an d middle-wave
pigm ents in the same middle-wave cones would n o t cause subjects to set the
abnorm al Rayleigh m atches th at characterize deuteranom aly. As discussed
earlier, this is because color m atches occur at the level of the pigm ents and not
the cones. Individuals who expressed both norm al m iddle- and deu teran o m a
lous pigm ents in the same middle-wave cones would set norm al or n ear nor
mal pigm ent m atches. From a study th at investigated this issue in m ore detail
(Neitz, Neitz, & Jacobs, 1991b) it was concluded th at deuteranom alous sub
jects do n o t express any detectable am o u n t o f middle-wave pigm ent even
though they often appear, by S outhern analysis, to have m ultiple norm al mid-
dle-wave genes.
With the exception o f an adequate explanation for the behavior o f deu teran o m
alous observers, the in terp retatio n s o f N athans an d colleagues seem to offer sim
ple explanations o f the genetic basis for red-green color blindness. O ne greatly
simplifying aspect o f their theory is th at some o f the color-blind genotypes are ex
plained as arising from unequal b u t hom ologous recom bination of norm al gene
arrangem ents. This can be simply diagram m ed as in the exam ple o f Figure 1.
T he im portance o f the work of N athans an d colleagues c an n o t be underestim at
ed. They established the prim ary structures of the pigm ent genes an d their chro
m osom al locations. They suggested th at the X-linked pigm ent genes are in a
tandem array, and dem onstrated that m ost people have m ore pigm ent genes than
previously believed. They also d em onstrated differences betw een norm al gene ar
rangem ents an d those o f color blind observers and illustrated how some o f the
gene arran g em en t m ight com e about by hom ologous recom bination.
N athans et al. (1986a,b) came to som e new conclusions about the genetic m ech
anisms o f color vision: 1) m any X-chrom osom es o f norm al observers were pro
posed to have an extra copy (or two) o f the middle-wave gene, 2) the abnorm al
pigm ents o f anom alous trichrom ats were proposed to result from recom bination
between the long- and middle-wave genes, an d 3) many deuteranom alous observ
ers were proposed to have both n orm al m iddle- and long-wave genes in addition
to m iddle- /long-wave fusion genes. W ith these exceptions, th eir results were in
terp reted as generally confirm ing conventional theories. Botstein (1986), fo r ex
am ple, in terp re ted the m olecular genetic results as, at last, confirm ing the Young-
H elm holtz theory of color vision in which the existence o f three types of light ab
sorbing particles (one for each prim ary color) are proposed to explain why nor
mal hum an vision is trichrom atic (Young, 1802). F urtherm ore, inheritance of
m u tan t pigm ents with abnorm al spectra, different from any expressed in norm al
observers, was in terp re ted as the cause o f anom alous trichromacy.
228 JAY NEITZ AND MAUREEN NEITZ
Normals
Deuteranope
Now, seven years later, the body of experim ental results th at relate to the m olec
ular biology o f color vision has grown. It may be time to reconsider the theories
p u t forth in 1986. A new theory may b etter explain the experim ental results - a
theory in wrhich all the ideas in the preceding paragraph w ould be held as substan
tially incorrect. As will be elucidated in subsequent sections, the evidence suggests
th at it is likely that people with norm al color vision have, on average, m ore X-
linked pigm ent genes than suggested by N athans and colleagues. F u rth erm o re,
m any observers wdth norm al color vision may have m ultiple long- as well as m ulti
ple middle-wave genes. T he facts are consistent with an alternative theory in which
the fusion genes n eith er produce abnorm al pigm ents, n o r do they cause color de
fects. T hat is, fusion genes an d the variant pigm ents they produce may be found
an d expressed in individuals with norm al color vision. Finally, results from m olec
ular biology may refute, ra th e r than confirm , the Young-Helmholtz theory as the
explanation for hum an trichromacy.
COLOR VISION DEFECTS 229
T he discoveries that the m iddle- an d long-wave pigm ent genes are highly hom ol
ogous, lie closely spaced in a tandem array, and are variable in n u m b er suggested
a m utational pathway to gene arrangem ents that confer color defects that is sim
ple and com pelling - the arran g e m e n t o f the pigm ent genes is one that would
prom ote a high frequency o f u n equal but hom ologous recom bination, a m echa
nism that could increm ent and d ecrem en t gene nu m b er an d produce fusion
genes, all seemingly, ju st as req u ired to explain the gene arrangem ents that
underlie color defects. This idea is illustrated in Figure 1; an intergenic exchange
between two norm al arrays transfers a middle-wave gene from one chrom osom e
to the other, giving rise to one chrom osom e with only a single long- and no m id
dle-wave pigm ent genes. T he o th er chrom osom e that results from this crossover
has one long- an d two middle-wave genes. In a stroke, both the occurrence of
d euteranopes with only a long-wave gene an d norm al trichrom ats with extra mid-
dle-wave genes are explained.
Difficulties arise, however, when one tries to explain the gene arrangem ents of
o th er color defective observers by sim ilar one-step m utational pathways. For exam
ple, several o f the genotypes in ferred for deuteranom alous subjects cannot result
from ju st one crossover betw een norm al arrays (see Figure 2). Fusion genes can
result from crossovers between norm al gene arrays bu t no m ore than one fusion
gene can be produced in each crossover. Yet, frequently, deuteranom alous sub
jects seem to have m ultiple fusion genes. A crossover betw een two norm al arrays
is illustrated at the top o f Figure 2. T he pro d u ct of the crossover that is assem bled
with a 5 m iddle- 3 long-wave fusion gene also has the com plete long-wave gene
from one parental chrom osom e an d the com plete middle-wave gene from the
other. To get two fusion genes by crossing over, an arran g e m e n t th at already has
one fusion gene (Figure 2) m ust u n d erg o a second recom bination. T he same type
of recom bination m ust occur three separate times for three fusion genes. T he
probability o f a recom bination occurring twice is expected to be the square of the
probability o f one occurrence ancl the likelihood of accum ulating three fusion
genes by this m echanism is expected to be equal to the cube o f the probability of
a single occurrence. T he frequency of deuteranom alous phenotypes should equal
the sum of the frequencies o f each of the different genotypes. Satisfying these re
quirem ents yields that, in the population, 95.2% o f deuteranom alous subjects
should have a single fusion gene, 4.5% should have two fusion genes, an d only
0.2% should have three fusion genes.
Table 1 shows com putations o f the n u m b er o f fusion genes for each o f the eight
d euteranom alous subjects exam ined by N athans et al. (1986a). T he calculations
suggest th at two subjects may have two fusion genes an d two m ore may have three
fusion genes each. This is wildly d ifferent from what would be expected if the in
heritance o f a single fusion gene was sufficient to cause deuteranom aly. If 95% of
d euteranom alous individuals in the population have only a single fusion gene,
then the probability of drawing four subjects (out of eight) with m ultiple fusion
230 JAY NEITZ AND MAUREEN NEITZ
N orm als
D euteranom alous?
D euteranom alous?
D euteranom alous?
Key:
Long-Wave gene i N Middle-wave Gene Fusion Gene
genes an d two of those with three fusion genes is less than one in a m illion. Earlier,
it was n o ted th at a m ysterious feature o f deuteranom aly is the presence o f seem
ingly norm al middle-wave genes. Now we have an added curiosity. D euteranom a
lous subjects seem to have m ore fusion genes than would be expected from a
m odel in which the inheritance of a fusion gene causes deuteranom aly.
COLOR VISION DEFECTS 231
Table 1. The number of fusion genes in deuleranomalous observers are calculated from
the results of Nathans et al. (1986a). The total number of X-linked pigment genes in each
subject was determined from his Ag/Ar fragment ratio. The ratio of band intensities was
translated to a gene fragment ratio using the intensity ratios suggested to correspond to a
1:1 gene ratio (Ar:Ag = 0.97; Dr:Dg = 1.24). Gene number was taken to equal Ag/Ar + 1; these
values were rounded to the nearest whole number. The number of genes that contain frag
ment Dr was calculated using the equation: Genes with Dr = Total number of X-linked pig
ment genes / (Dg/D r +1 ). These were rounded to the nearest whole number. The number
of fusion genes equals 1 less than the number of genes with fragment 1). because one gene
with Dr is the normal long-wave gene. These calculations assume that all subjects have ont}
one copy of fragment A,.. If some subjects have more than one copy of A,, the number of
fusion genes would be higher.
34 5 0.27 3
35 3 0 2
36 6 0.57 3
37 6 1.61 1
38 5 0.84 2
39 3 0,32 1
40 3 0.45 1
41 4 1.32 1
N athans et al. (1986a) exam ined the genes of twenty-five color deficient males.
We have organized the gene arran g em en ts th at can be inferred from their results
into 10 d ifferent categories. These are illustrated in the left-hand colum n of Fig
ure 3. T he po p u lar idea is that color defective arrangem ents can be explained by
simple, one-step recom binations betw een norm al arrays that result in gene dele
tions or produce a fusion gene. A rran g em en t num ber 1 of Figure 3, can be pro
d uced by a crossover in which bo th products o f the recom bination fit perfectly
with this idea (as was shown in F u re 1); one pro d u ct confers d eu teran o p ia and
the o th er has two middle-wave genes ju st as fo u n d to occur am ong norm als. The
o th er nine color-blind gene arran g em en ts are m ore difficult to fit with this
scheme. T he p ro tan o p e arrangem ents, like num bers 2 and 3 an d the protanom a-
lous arrangem ents, like num ber 4 o f Figure 3 can arise by a sim ple crossover. How
ever, two distinct gene arrangem ents arise from every crossover. W hen a protan
arran g em en t is produced, an o th er array with an intact norm al long-wave gene
from one parent, all the norm al middle-wave genes from the o th er parent, and a
5 m iddle- 3long fusion gene is also produced. T he 5 m iddle- 3 long fusion gene
is expected to encode a pigm ent with a long-wave spectrum . P rotanope gene ar
rangem ents are missing sequences th at encode a long-wave pigm ent, b u t in their
production, an o th er chrom osom e effectively gains the long-wave gene (as a fusion
gene) th at the p ro tan o p e lost.
232 JAY NEITZ AND MAUREEN NEITZ
10
F ig u re 3 A n e w th e o r y o f th e p h o to p ig m e n t g e n e a r r a n g e m e n ts th a t u n d e r lie n o r m a l c o lo r
v isio n allow s a v e ry s im p le e x p la n a tio n f o r h o w c o lo r d e fe c ts a rise. T h e in f e r r e d g e n e a r r a n g e
m e n ts o f th e 25 c o lo r d e fe c tiv e o b s e rv e rs s tu d ie d by N a th a n s e t a l. (1 9 8 6 a ) c a n b e c la sse d in to
10 c a te g o rie s sh o w n in th e le ft-h a n d c o lu m n (a b b re v ia tio n s : D, d e u te r a n o p e ; P p r o ta n o p e ;
PA, p ro ta n o m a lo u s ; D A d e u te r a n o m a lo u s ) . I n s o m e cases, th e re s u lts fro m S o u th e r n analysis
in d ic a te th e p r e s e n c e o f a n in ta c t g e n e w h o se e x p re s s io n is n o t e v id e n t in th e su b je c ts' b e h a v
ior. F o r e x a m p le , th e c o lo r v isio n o f d e u te r a n o m a lo u s o b s e rv e rs is th a t p r e d ic te d b y th e e x
p r e s s io n o f tw o slig h tly d if f e r e n t long-w ave p ig m e n ts b u t n o m id d le-w av e p ig m e n ts , h ow ever,
d e u te r a n o m a lo u s su b je c ts o fte n h a v e all th e g e n e fra g m e n ts c o r r e s p o n d in g to in ta c t m id d le -
w ave g e n e s . A lig h tn in g b o l t sym bol d ra w n t h r o u g h a g e n e in d ic a te s lac k o f e x p re s s io n o r
f u n c tio n . T h e g e n e a r r a n g e m e n ts o f c o lo r d e fe c tiv e su b je c ts a r e o fte n c o m p lic a te d , h a v in g
m a n y g e n e s , a p p a r e n tly u n e x p r e s s e d g e n e s , a n d m u ltip le fu s io n g e n e s . T h e s e s u g g e st a p re v i
o u sly u n r e a liz e d c o m p le x ity in th e n o r m a l g e n e a r ra n g e m e n ts fr o m w h ic h th e y a ro s e . T h e
r ig h t- h a n d c o lu m n show s h y p o th e tic a l g e n e a r r a n g e m e n ts th a t c o u ld c o n f e r n o r m a l c o lo r
visio n . E a c h p a ir o f n o r m a l g e n e a r ra n g e m e n ts c o u ld u n d e r g o a sin g le c ro sso v e r to y ie ld th e
c o lo r-d e fe c tiv e g e n e a r r a n g e m e n t sh o w n d ire c tly to its left. In th is n e w th e o ry , n o r m a l o b s e rv
e rs a re p r o p o s e d to c a rr y d e fe c tiv e g e n e s s o m e tim e s b u t in e a c h case th e y h a v e a t le a s t o n e
f u n c tio n a l m id d le - a n d o n e f u n c tio n a l long-w ave p ig m e n t g e n e . S o m e n o r m a l o b s e rv e rs a r e
sh o w n to h a v e f u s io n g e n e s . T h e e x p re s s io n o f th e s e g e n e s in n o r m a l o b s e rv e rs is p r o p o s e d
to c o n tr ib u te to th e o b s e rv e d v a ria tio n in n o r m a l c o lo r v isio n b u t d o e s n o t in te r f e r e w ith n o r
m a l c o lo r vision. P re v io u s th e o rie s r e q u ir e d p r o ta n o m a ly a n d d e u te r a n o m a ly to a ris e by d if
f e r e n t m u ta tio n a l p athw ays, o fte n w ith m a n y ste p s. In c o n tra s t, th e c ro sso v e rs sh o w n h e r e all
give rise to c o lo r d e fe c ts by th e sa m e o n e -s te p m e c h a n is m . T h e y p r o d u c e g e n e a r r a n g e m e n ts
t h a t lack e it h e r f u n c tio n a l m id d le -w av e g e n e s ( d e u ta n d e fe c ts ) o r long-w ave g e n e s ( p r o ta n d e
fe cts).
COLOR VISION DEFECTS 233
It seems that either one o f two alternatives m ust be true with regard to the gene
arrangem ents that are pro d u ced as co u n terp arts to the protan gene arrays. O ne is
th at the inheritance o f a 5 m iddle- 3 long-wave fusion gene usually prevents the
expression o f the otherw ise norm al middle-wave genes and, thus, d eutan gene ar
rangem ents are pro d u ced as the com plem ent to protan ones. This would explain
why deuteranom alous subjects, like those illustrated by arran g em en t num ber 5 of
I- ' lire 3, can often have n orm al middle-wave genes. T he alternative is that the
gene arrays th at are pro d u ced in the same crossovers that give rise to protan d e
fects can confer norm al color vision. This would allow the possibility that norm al
observers can have, a n d may express fusion genes. T he question o f w hether or not
color norm al observers can have a n d express fusion genes is central to u n d er
standing the m olecular genetics o f color vision.
The rem aining five categories o f gene arrangem ents illustrated in Figure 3 are
all bew ildering because of their com plexity relative to the sim ple gene arran g e
m ents that have been proposed to u n d erlie norm al color vision. N one of them can
be explained by a sim ple single crossover between two of the proposed com m on
norm al gene arrays.
N um ber 6, a protanom alous gene arrangem ent, apparently has at least six
genes. Protanom alous arrangem ents are proposed to arise from deletion of the
one norm al long-wave gene. To result from a single crossover, an arran g em en t like
n u m b er 6 would have to arise from a norm al arran g em en t with at least seven
genes. T he suggested counts of the n u m b e r o f pigm ent genes in norm als would
indicate that the o ccurrence of norm als with seven genes is exceptionally rare.
Such a p rotanom alous arran g e m e n t ca n n o t have arisen by a single crossover from
any of the proposed com m on norm al gene arrays.
N um ber 7, a pro tan o p e, also has m any m ore genes (perhaps seven) than are
proposed for color norm al observers. In addition, he appears, by S outhern analy
sis, to have a norm al long-wave gene. A likely explanation o f his protanopia, is that
his long-wave gene has a m utation th at ren d ers the pigm ent p ro d u ced non-func
tional, perhaps similar to the m utations fo u n d to occur in blue-cone m o n o ch ro
m ats (N athans et al., 1989) (as are discussed earlier). In Figure 3, genes which do
n o t appear to contribute to the phenotype have a lighting b o lt symbol drawn
through them . A deleterious m utation may explain the loss o f long-wave pigm ent
function. But, the extrem ely high gene num bers fo u n d in p ro tan subjects relative
to those proposed for norm als are n o t w hat would be predicted from a single
crossover betw een com m on norm al arrays.
Some deu teran o p es (num ber 8) have only two genes, a norm al long-wave gene
and a 5 m iddle- 3long-wave fusion gene. Middle-wave genes cannot be deleted in
the same crossover th at p ro d u ced the fusion gene. N athans an d colleagues no ted
that eith er two in d ep en d e n t crossovers o r a gene conversion are req u ired to ex
plain these arrangem ents.
N um ber 9, a deuteranom alous arran g e m e n t with m ultiple fusion genes, and no
middle-wave genes can only be explained by some com bination of crossovers, or
234 JAY NEITZ AND MAUREEN NEITZ
A new concept o f norm al genes makes abnorm al gene arrangem ents easily
explainable
G enetic m echanism s that give rise to color defects could be quite simple, if: 1)
people with norm al color vision can often have m ultiple long-wave genes, 2)
color norm al people often have fusion genes, an d 3) the average nu m b er o f pig
m en t genes p er X -chrom osom e o f color norm al observers is substantially larger
than previously im agined. T he c u rre n t view o f norm al gene arrangem ents comes
from the analysis o f pigm ent gene fragm ents by S outhern hybridization
(Nathans, et al., 1986b; Vollrath, N athans, & Davis, 1988; D rum m ond-B org,
Deeb, & Motulsky, 1989; Deeb et al., 1992). Art exam ination o f those results lends
insight into w hether o r n o t they may have been m isinterpreted. For exam ple, is it
likely that a norm al array with m ultiple long-wave genes an d with fusion genes
m ight have been in terp re ted as having only a single long-wave gene, and no
fusion genes?
Figure 4 shows the restriction m aps for m iddle- and long-wave genes. Four re
striction fragm ents, labeled A, B, C, an d D have been used in S outhern analysis to
distinguish m iddle- from long-wave genes. T he A fragm ent from the long-wave
gene (A, s is larger than the A fragm ent from the middle-wave gene (Ag) because
of an insert (of about 1.9 kb, in the first intron) about one th ird o f the way into
the gene. This same insert accounts for the size difference betw een the C g and C r
fragm ents. T he fragm ent, B r , is sm aller than B^ because it is produced by cleav
age at a Bam HI site that occurs in un iq u e sequences flanking the 5 end o f the
array. These unique sequences are n o t part of the tandem ly rep eated sequences
236 | \Y M l IV \M> MAI RI 1- \ Ni l : /
B B B Middle-wave
(B o tto m ) B e c a u se th e in s e r t th a t d is tin g u is h e s fr a g m e n ts A r a n d C r f ro m A g a n d C ,, o c c u rs
w ith in th e g e n e , a c ro sso v e r a n y w h e re in a b o u t th e first th ir d o f th e g e n e w ill d u p lic a te th e
long-w'ave g e n e in c lu d in g fra g m e n ts A ,., C r . a n d D r . T h e d u p lic a te d long-w ave g e n e w o u ld
h a v e f r a g m e n t B . It m ay also h a v e e x o n 1 d e r iv e d fro m a m id d le-w av e g e n e b u t th a t e x o n is
id e n tic a l b e tw e e n m id d le - a n d long-w ave g e n e s . T h u s , th e d u p lic a te d long-w ave g e n e a n d th e
long-w'ave g e n e fro m w h ic h it was d e riv e d will h a v e id e n tic a l c o d in g s e q u e n c e s .
COLOR VISION DEFECTS 237
A g / A r =2 B g / B r =8 C g / C r =2 Dg / D r =2
o f long-wave genes within groups that are classified by A g / A r ratio. This is exactly
what is found. For exam ple, N athans et al. (1986a) found that for people with a
1:1 A g / A r ratio ( m ean A g / A r = 0.97) the o th er fragm ents had the following-
m eans and standard deviations: Bg /B r , 2.00 0.49;C g /C r , 0.88 0.18; Dg / D p
1.24 0.30J u st as predicted if some people have m ultiple long-wave genes and,
thus, their higher Bg /B r ratios are included in the sam ple, the m ean Bg / B r ratio
is high, m ore than twice the A o r C ratios. As predicted by the idea that the sam ple
is inhom ogeneous in gene num ber, the B ratio is highly variable across subjects.
T he B g /B r ratio has a standard deviation 2.7 times greater than the C g / C r ratio.
If everyone had a single long-wave gene, B g/ B r would be expected to be always
equal to A g/ A r and C g / C r both in m agnitude an d in degree o f variability. Nei
th e r o f these are b o rn e out by experim ental results.
Two specific exam ples can serve to illustrate, in detail, how nicely a new inter
p retation o f norm al gene arrangem ents can explain color defective genotypes and
can explain results from S outhern analysis o f norm al subjects as well. C onsider two
cases from N athans et al. (1986a), subject #27, a protanope, and subject #40, a
d euteranom alous observer. Figure 6 shows gene arrangem ents that could yield the
fragm ent stoichiom etries observed for each subject. Both are problem s for earlier
theories because the arran g em en ts can n o t com e ab o u t by any simple pathway
COLOR VISION DEFECTS 239
from norm al arrangem ents. Also shown for each subject are theoretical norm al ar
rangem ents that could crossover ju st once to yield the color defective genotype.
These differ from the norm al genotypes proposed previously in th at they have
m ore genes (as many as eight) an d they all have one o r m ore 5 m iddle- 3 long-
wave fusion genes. C ould such arran g em en ts really be typical o f color norm al ob
servers? D rum m ond-B org et al. (1989) exam ined the pigm ent genes of 134 Cau
casian males using S outhern analysis. Subjects were classified according to their
A g /A r ratios. T he results for the m ales who were considered to have norm al gene
arrangem ents with nom inal A g / A r ratios o f 3:1 (about 20% of th eir subjects) are
illustrated in the lower half o f Figure 6. T he observed m ean for each of the A, B,
an d D fragm ent ratios is shown together with bars that extend two standard devi
ations on each side o f the m ean. T he C ratio is om itted because the inform ation
it yields about the genes is re d u n d a n t with the A ratio. O verlaid are the fragm ent
ratios that co rresp o n d to the two hypothetical norm al arrangem ents illustrated
(in the u p p e r half of Figure 6) to give rise to the p ro tan o p e (squares) an d the two
proposed to give rise to the deuteranom al (circles) (m iddle panel, Figure 6). Even
though the arrangem ents are very different from those previously th o u g h t to u n
derlie norm al color vision, the stoichiom etries are consistent with those consid
ered norm al. This suggests th at the norm al gene arrays exam ined previously
probably included such arrangem ents - e.g., with large num bers of genes, m ulti
ple long-wave genes and fusion genes. It would seem that norm al arrangem ents
similar to those illustrated in Figure 6 were in terp reted as having only a single
long-wave gene an d no fusion genes because the interp retatio n em phasized the re
sults for the A fragm ents.
Each recom bination illustrated in Figure 6 simply deletes genes req u ired for
norm al color vision b u t the process alters the fragm ent stoichiom etries substan
tially, such th at those o f the color defective observers fall outside n orm al limits.
This is illustrated in the bottom panel o f Figure 6. T he p ro tan o p e inherits only
one long-wave gene (a non-functional one). Even though his X -chrom osom e has
the same n u m b er o f pigm ent genes (seven) as one o f the norm al arrangem ents
from which it is proposed to arise, the reduction to a single long-wave gene in
creases his A g /A r an d D g /D r ratios to the high value considered rare for norm als.
In the case of the prod u ctio n of the deuteranom alous arrangem ent, previous in
terpretations would be oblivious o f the fact th at the two norm al arrays shown as
giving rise to the deuteranom alous genotype have fusion genes, b u t the crossover
im parts a deuteranom alous gene arran g em en t in which the presence of fusion
genes becom es obvious because o f the extrem ely low Dg/ D r ratio (even though no
additional fusion genes were p ro d u c e d ).
Pigm ent gene fragm ent stoichiom etries o f color-deficient observers alm ost al
ways differ from those of typical norm al observers. However, there are exceptions;
Deeb et al., (1992) found four o f 129 norm al observers in which the presence o f
fusion genes was obvious from the fragm ent stoichiom etries. It was previously be
lieved th at the abnorm al stoichiom etries were the result of fusion genes present
in color defects but nearly always absent in norm al subjects (Nathans, et al.,
240 JAY NEITZ AND MAUREEN NEITZ
Ll_
OL
A g/A r B g /B r Dg/ D r
Figure 6 Two specific examples o f how a new theory o f norm al gene arrangem ents can ac
count for results from color defective and color n o rm al subjects. T he results from S outhern
analysis for two subjects exam ined by N ath a n se t al. (1986a) are as follows: subject #27 (a pro
tanope), A g /A r = 6.08, B g / B r = 5.40, C g / C r = 3.77, D g /D r = 5.87; su b ject#40 (deuteranom
alous), A g /A r = 2.31, B g /B r = 5.36, C g/ C r = 1.42, Dg/ D r = 0.56. Those fragm ent
stoichiom etries suggest the gene arrangem ents shown (m arked #27 and #40). T he o rd er of
the genes was arbitrarily chosen. Above each color-defective gene array, a pair of hypothetical
arrays proposed to confer norm al color vision is shown. T he pairs of norm al gene clusters
could each undergo a single crossover to yield the observed color defective genotypes. Each
norm al array illustrated has m ore pigm ent genes than previously proposed for norm als and
has m ultiple genes that encode long-wave pigm ents. T hree o f the norm al arrays have defective
genes in addition to both norm al m iddle- and long-wave genes. Plotted in the lower h alf o f the
figure are the ranges of fragm ent stoichiom etries (bars, drawn as the m ean2 SD) d eterm in ed
for genes, considered to confer norm al color vision, from a sample of males that had nom inal
A g /A r fragm ent ratios of 3:1 (from D rum m ond-B org, e t al., 1989). Superim posed on those
ranges are the fragm ent ratios for the four hypothetical norm al gene arrangem ents shown in
the u p p er half o f the figure (circles, parents to the deuteranom alous gene arrangem ent;
squares, parents to the protanope gene arran g em en t). Even though the hypothetical norm al
gene arrangem ents are m uch different than those previously proposed for norm al subjects
they yield fragm ent stoichiom etries that are consistent with those previously considered nor
mal. Fragm ent stoichiom etries for the two color defective gene arrangem ents are plotted in
the bottom -m ost panel (circles, deuteranom alous; squares, pro tan o p e). They fall outside the
norm al limits.
CO LO R VISION DEFECTS 241
D efective genes
We suggest th at defective genes are com m on am ong color defective and color
norm al populations. By defective we m ean th at individual genes have altered cod
ing or regulatory sequences, as m ight com e about by point m utations o r dele
tions, th at prevent expression or function of expressed product. Such defects
m ight be expected to be especially com m on in the X-linked pigm ent genes
because often people have genes to spare (perhaps, som etim es as m any as seven
or eight m ore than are req u ired to sup p o rt trichrom atic color vision). Thus,
defects m ight be acquired with little consequence to vision. We propose that d eu
teranom alous individuals with middle-wave genes may be people who in h erit all
their middle-wave genes as defective genes. They could arise from norm al gene
2 42 JAY NEITZ AND MAUREEN NEITZ
arrangem ents with defective genes by u n fo rtu n ate recom binations (as illustrated
in Figure 6) th at leave no functional middle-wave genes. R ecent evidence sug
gests that defects in individual genes are a com m on cause of o th er visual defects.
It is, thus, plausible that defective genes underlie deuteranom aly as well. T hree
d ifferent p oint m utations within the gene encoding the short-wave pigm ent have
been fo u n d to be associated with tritanopia (Weitz et al., 1992a; Weitz, Went, &
N athans, 1992b). P oint m utations within X-linked pigm ent genes have also been
found associated with some cases o f blue cone m onochrom acy (N athans, et al.,
1989). An exam ple o f defective genes in red-green color blindness has also been
found. W inderickx et al. (1992c) exam ined the middle-wave pigm ent genes of a
d eu tan observer who had an apparently norm al gene array, i.e., by S outhern
analysis he had long- and middle-wave genes an d no fusion genes. From these
results he would be expected to have norm al color vision. Analysis of his m iddle-
wave genes revealed sequences encoding the same cysteine to arginine substitu
tion observed in blue cone m onochrom ats. These m utations probably explain
the lack of middle-wave pigm ent function in this observer. T he same alteration
was detected in one middle-wave gene o f a mildly deuteranom alous observer. It
was also detected in a middle-wave gene o f one out o f 52 norm al subjects dem o n
strating that defective genes can occur in observers with norm al color vision.
T h e possibility th at defective cone pigm ent genes may be fairly com m on in ob
servers with norm al color vision may have im p o rtan t clinical im plications in light
o f the discovery th at p h o topigm ent gene defects cause retinal degeneration. At
least 32 m utations in the rhodopsin gene have been found associated with autoso
mal d o m in an t retinitis pigm entosa (e.g., Dryja et al., 1990; Sung et al., 1991a;
Sung et al., 1991b). C one pigm ent gene defects may similarly cause cone-specific
degenerations. Some blue cone m onochrom ats show retinal degeneration
(N athans, et al., 1989). Cone degeneration has also been observed in one family
w here functional middle-wave genes were present but there was a deletion in the
long-wave gene (Reichel, Bruce, Sandberg, & Berson, 1989). A possibility that
seems w orth considering is that while in m ost cases the inheritance o f a defective
cone pigm ent gene in an array o f otherw ise norm al genes may have no serious
consequences for vision in young eyes, som e cone pigm ent gene defects may cause
late-onset retinal (m acular) degenerations.
A nother re cen t study relates to the issue o f defective pigm ent genes and their
expression. T he expression o f photopigm ent genes was investigated in an exam i
nation o f cone pigm ent mRNA transcripts from hum an retinas (W inderickx, Bat-
tisti, Motulsky, & Deeb, 1992a). A com m on silent polym orphism (A vs. C at the
th ird position o f codon 283) occurs in exon 5 o f the middle-wave gene. G enom ic
DNA derived from blood samples and cDNAs from retinas were exam ined from 13
males. Both the C and the A versions of the middle-wave genes were detected in
the genom ic DNA from 10 of the males. Middle-wave cDNAs were detected in sam
ples from all eyes, however, middle-wave cDNAs with C at the third position of
codon 283 were no t detected in the samples from any o f those 10 males. T he ex
pression o f the C allele was detected in only one subject, a male who had no A
COLOR VISION DEFECTS 243
alleles o f the gene. O ne possible explanation for this result is th at the C serves to
m ark middle-wave genes th at have a n o th er m utation that interferes with their ex
pression. If this is true, middle-wave genes with such defects occur at a high fre
quency in the norm al population, as we propose. An alternative explanation
(p referred by W inderickx, et al., 1992a) is that only the first two genes are ex
pressed from the pigm ent g ene arrays of all observers. T here is good evidence for
a locus-control region (LCR) about 4 kb upstream of the transcription initiation
site (N athans, et al., 1989; W ang et al., 1992). An interaction betw een the LCR and
a pigm ent gene is proposed to be re q u ired to switch on transcription. W inderickx
et al. (1992a) suggest th at the LCR allows transcription o f only the two genes clos
est to the LCR; m ore dow nstream genes are supposed no t to interact with the LCR
an d are, thus, n o t transcribed. If so, it is curious that in individuals with m ultiple
middle-wave genes, the expression o f only the A allele was observed. C onsidering
the high frequency o f recom bination betw een opsin genes, one m ight expect that
eith er allele could occur in the second position, and across individuals both alleles
would be found to be expressed.
T here is p re ced e n t from o th er systems for the idea th at m ore proxim ally located
genes have a com petitive advantage to interact with the LCR. It is im aginable that
the probability o f a particular pigm ent gene being expressed in a cone is p ro p o r
tional to its proxim ity to the LCR, i.e., the m ost 5 gene would be expressed in the
highest n u m b er o f cones, the second gene expressed in fewer cones than the first
and so on, u ntil the m ost distal genes o f large arrays are expressed in very few
cones. Wre have suggested th at the n u m b er o f genes p er array m ight often be larg
er than has been suggested from previous interpretations o f results from S outhern
analysis. It could be th at the A alleles o f the middle-wave genes usually o u t n u m b er
the C alleles in individual arrays an d the C alleles may tend to occur at m ore distal
positions relative to the LCR, where they are expressed at low levels. This m ight
explain why W inderickx et al. (1992a) detected the expression o f only the A ver
sion in their subjects who carry both alleles. Such a proposal is attractive, and it
does n o t conflict with a theory in which m ore than two X-linked pigm ent genes
can be expressed in an individual eye. Even the expression o f a gene at very low
levels is expected to have im p o rtan t consequences for vision in some cases. T here
are about 5 m illion cones in each eye. If a gene were expressed at a level o f only
5%, it m ight n o t be detected in experim ents like those o f W inderickx et al.
(1992a) bu t a person would still have a quarter-m illion cones with th at expressed
pigm ent. It is virtually certain that a p erson would no t suffer extrem e deu teran o m
aly if he h ad that n u m b er o f norm al middle-wave cones. Extrem ely biased cone ra
tios still seem to support norm al color vision. For exam ple, Pokorny, Smith &
Baron (1988) re p o rte d the case of a fem ale heterozygous carrier o f a protan color
defect who, they concluded, has an extrem ely small n u m b er o f long-wave cones;
the subject has norm al color vision. T he possibility th at levels o f expression d e
pen d on gene position (W inderickx, et al., 1992a; Deeb, et al., 1992) is interesting
an d extrem ely im portant, b u t it needs to be tested fu rth e r before the consequenc
es for genotype-phenotype relationships in color vision are understood.
244 JAY NEITZ AND MAUREEN NEITZ
Red-green color vision depends on having at least two pigm ents with different
spectral sensitivities in the m iddle-to-long wavelengths. U nderstanding the
genetic basis of norm al color vision and the cause o f color defects d epends criti
cally on knowing which am ino acid differences betw een the cone pigm ent opsins
are responsible for giving the m iddle-to-long wave pigm ents different spectra. A
com parative study o f the genes th at encode eight pigm ents with various spectra,
two from hum ans, an d three each from two species o f South A m erican monkey,
indicated that am ino acid substitutions at ju st three am ino acid positions could
account for the spectral difference betw een hum an long- and middle-wave pig
m ents (Neitz, et al., 1991c). T he positions o f these substitutions in the opsin are
illustrated in Figure 7. Two am ino acid differences betw een long- an d m iddle-
wave pigm ents, at positions 277 and 285 are encoded by nucleotides in exon 5 of
the pigm ent genes. A substitution o f Tyr for Phe at position 277 produces a spec
tral shift o f ab o u t 10 nm, and a substitution o f T h r for Ala at 285 produces a shift
o f about 15 nm. Together, these two substitutions shift pigm ents from ones that
can be categorized as middle-wave to those th at can be categorized as long-wave,
confirm ing an earlier conclusion that substitutions in exon 5 are principally
responsible for the difference between m iddle and long-wave pigm ent classes
(Neitz, Neitz, & Jacobs, 1989). Thus, if all com m on hum an X-linked pigm ents are
categorized into two classes, m iddle- o r long-wave sensitive, any gene th at has
exon 5 from a long-wave gene will encode a long-wave pigm ent. Any gene that has
exon 5 derived from a middle-wave gene will encode a middle-wave pigm ent. A
third am ino acid substitution, of Ser for Ala at position 180, encoded by a nucle
otide substitution in exon 3 o f the genes is responsible for a sm aller spectral shift
of ab o u t 4-6 nm. This is one o f the substitutions that appears to be responsible for
spectral differences within the classes o f m iddle- and long-wave pigm ents. Sub-
types o f long-wave pigm ents and sub-types o f middle-wave pigm ents th at have dif
ferences at position 180 could arise from point m utations o r from crossovers
betw een the m iddle- and long-wave genes occuring dow nstream o f exon 3 bu t
upstream o f exon 5.
T he finding that substitutions at the three positions, 180, 277, and 285 are re
sponsible fo r spectral differences am ong the pigm ents has been confirm ed (C han,
Lee, 8c Sakmar, 1992; Ibbotson et a l , 1992; Merbs & N athans, 1992a; Merbs &
N athans, 1992b; Williams et al., 1992). However, som e studies suggested th at these
th ree are n o t the only substitutions involved in tuning the spectra o f m iddle to
long-wave pigm ents. Specifically, several results (Ibbotson, et al., 1992; Williams,
et al., 1992; W inderickx, et al., 1992b) h in ted that o n e o r both of two closely sep
arated substitutions, at positions 230 an d 233 encoded in exon 4 m ight also be in
volved. Recently, Merbs and N athans (1992b) have m easured the absorption
spectra of hybrid pigm ents expressed in vitro. Pigm ents en co d ed by genes with
long-wave exon 4 were found to have spectra that differ by ab o u t 2 -4 nm from
those en co d ed by genes with middle-wave exon 4. Further, they found that substi-
COLOR VISION DEFECTS 245
Figure 7 Amino-acid substitutions at three sites can account for most of the differences in
spectral sensitivity among middle-to-long wavelength sensitive visual pigments. The pigment
molecules consist of seven membrane-spanning helices. The filled square in the seventh he
lix indicates the lysine residue that binds the chromophore, 11-cis-retinal. Filled circles indi
cate the positions of three amino acid residues that participate in controlling the pigment
spectrum. The identities of the two residues in helix six determine whether a pigment will
fall into the middle- or the long-wave class. Both residues are encoded in exon 5 of the gene.
Substitutions at the site in helix four (position 180) produce a smaller spectral shift that ac
counts for spectral differences between pigment subtypes within each of the middle- and
long-wave classes. In addition to the three residues indicated, substitutions at other sites en
coded by exons 2 and 4 of the genes are associated with small spectral shifts that are likely to
contribute to spectral variation within the middle- and long-wave classes.
tutions in exon 2 were also associated with small shifts in the spectral peak o f the
long-wave pigm ents.
T he small spectral shifts p roduced by nucleotide substitutions in exons 2, 3 and
4 may seem hardly significant, bu t together they may occasionally be responsible
for im p o rtan t differences betw een subtypes within each of the classes o f m iddle-
and long-wave pigm ents. For exam ple, we would class as long-wave pigm ents, all
pigm ents pro d u ced by genes with a long-wave exon 5, however, a gene with m id
246 JAY NEITZ AND MAUREEN NEITZ
wave genes. Psychophysical results (e.g., W ebster & MacLeod, 1988; Neitz & Ja
cobs, 1990) suggest th at individual differences in both m iddle- an d long-wave
genes underlie norm al color vision variation. A lthough the exact frequencies still
n eed to be clarified, it appears that there are two subtypes o f m iddle- and two sub-
types of long-wave pigm ents with respect to the identity o f the am ino acid, Ser or
Ala, at position 180 in the population with norm al color vision.
25
20
JdL
I 15
LU
3
O
10
i i i i i i________i i i i j
As discussed earlier, one o f the m ost engaging questions raised by the finding
that m ost people have extra X-linked pigm ent genes is; w hat are the extra genes
doing? Are they expressed; and if so is each gene expressed in its own subset of
cones? T he investigations o f polym orphism in norm al color vision lend insight
into these questions. O ne idea is th at only the first two genes in the array are ex
pressed (Deeb, et al., 1992; W inderickx, et al., 1992a). If the favored view of nor
248 JAY NEITZ AND MAUREEN NEITZ
mal gene arrangem ents was true and alm ost everyone with norm al color vision has
only a single long-wave gene, the first in the array, then the second gene could al
ways be a middle-wave gene in norm als. If only the first two genes were expressed,
then everyone with a norm al gene arran g em en t would express one long- an d one
middle-wave gene. This would fit well with the traditional view of norm al color vi
sion in which everyone is supposed to express only three different cone pigm ents.
F u rtherm ore, this m echanism could account for the fact that deuteranom alous
observers do n o t express the norm al middle-wave genes they are often believed to
carry. T he idea is that, in deuteranom aly, the addition o f a 5m iddle- 3long-wave
fusion gene in the second position of the array displaces the norm al middle-wave
genes to m ore 3 positions w here they are no t expressed (Deeb, et al., 1992; W in
derickx, et al., 1992a). Conversely, however, we have proposed here that many col
or norm al subjects have m ultiple long-wave genes. In Figure 3, the extra long-wave
genes are illustrated as occuring in the second position of the array to explain how
crossovers between norm al gene arrangem ents can produce color defects. This
would seem to preclude the possibility that only the first two genes are expressed.
N orm al individual differences lend insight into this issue as follows. We find that
the norm al color m atches o f m ost males fall into one o f two m ain groups as shown
in Figure 8 (Neitz & Jacobs, 1986; Neitz & Jacobs, 1990). However, there is a small
g roup of males, circled in Figure 8, who require less red light in the Rayleigh mix
ture than those in eith er m ain group. T he differences in color vision betw een in
dividuals in the two m ain groups correlate w'ell with am ino acid differences at
position 180 (Neitz, et al., 1993; W inderickx, et al., 1992b). We find that the m ore
extrem e color m atches are also predicted by the p ro p o rtio n o f X-linked genes with
Ala vs. Ser at position 180 (Neitz, et al., 1993). It has b een suggested (M erbs &
N athans, 1992a; Merbs & N athans, 1992b; W inderickx, et al., 1992b) that the two
large m ain groups of observers differ because individuals in the large g ro u p who
req u ire less red light in the m ixture, express a long-wave gene with S e ri80 and
middle-wave genes with A la i80 while those in the o th er main group have Ala at
position 180 o f both m id d le-an d long-wave pigm ents. Since the m ost extrem e col
or m atches also correlate with the pro p o rtio n o f genes encoding A la i80 vs.
S e ri80, in o rd e r to be consistent with the idea that only two genes are expressed,
people who fall into the extrem e group (circled in Figure 8) m ust have Ser at po
sition 180 o f both their expressed m iddle- and long-wave genes. W inderickx et al.
(1992b) suggest that although about 60% o f the long-wave genes encode Ser at po
sition 180, only a small percentage of the middle-wave genes have S eri 80. They
used nucleotide differences in exon 2 to identify m iddle- from long-wave genes. It
is unlikely th at those changes would reliably distinguish m iddle- from long-wave
genes, but nonetheless, it does appear, from th eir experim ent, that Ala is m uch
m ore com m on than Ser at position 180 am ong the middle-wave pigm ent genes.
From this, u n d e r the hypothesis that everyone has a single long-wave gene and
only the first two genes are expressed, the com m on gene arrangem ents that
should give rise to the extrem e color m atches (circled in Figure 8) can be predict
ed. They are illustrated in ] gure 9, the first two genes m ust have Ser at 180 but
COLOR VISION DEFECTS 249
the other, unexpressed genes are likely to have Ala. It can be seen that if people
are allowed to have unexpressed genes with A la i80, the pro p o rtio n of genes that
encode Ala vs. Ser at 180 should vary widely for this extrem e phenotype d ep e n d
ing on how many unexpressed middle-wave genes a person has. Indeed, there
should be very little co rresp o n d en ce betw een genotype and phenotype for this
group. To the contrary the correlation is very high. O u r experim ent suggests that
all these extrem e subjects have num erous genes (perhaps, betw een 4 an d 10) of
which the vast p ro p o rtio n have Ser at position 180. T he chance o f obtaining this
result if only the first two genes were expressed is vanishingly small. Conversely,
however, if all the functional genes are expressed, then the only way a person
could fall into this extrem e group is if the prep o n d eran ce o f his genes have
S erl80, ju st as is observed.
If m any o r all the functional pigm ent genes are expressed, then because o f the
polym orphism in the norm al pigm ents it seems likely th at many people would in
dividually have and express genes for both subtypes of m iddle- a n d /o r bo th sub-
types o f long-wave pigm ents. T hat is, contrary to the long favored view th at only
three pigm ent types u n d erlie norm al color vision, many people could have four
or five spectrally d ifferent pigm ents. This is consistent with the distribution of col
or m atches am ong color norm al males (Neitz, et al., 1993). A nother recen t result
suggests that the expression of additional pigm ents is also evident in the behavior
of individual males. A fundam ental property o f color vision based on three cone
types is that color m atches m ade with the un ad ap ted eye will persist after selective
color adaptation (Brindley, 1970; Jam eson & H urvich, 1972). To the contrary, in
an experim ent designed to test the idea that norm al males have m ore than three
spectrally d ifferent cone types, it was fo u n d that some norm al males change their
color m atches after adaptation to re d light, as predicted if they have m ore than two
different types o f long-wave cones (Neitz, Neitz, & Jacobs, 1991a).
1.0
CD 0.8
0
0.6
0.4
0.2
0.0
Color Match (R / R + G)
Proportion of
Genes with
Aia1SO
F ig u re 9 Individual differences at a single am ino acid position (180) of the m iddle- and long
wave pigm ents can account for individual differences in norm al red-green color vision. A G /
T polym orphism at nucleotide position 1032 o f the genes produces a S er/A la 180 polymor
phism am ong the X -encoded pigm ents. Filled circles plot the pro p o rtio n of red light in a mix
ture o f red and green light that each subject req u ired to m atch the appearance of a
m onochrom atic standard light versus the relative p ro p o rtio n of X -chrom osome pigm ent
genes that have G at position 1032. T he six subjects with the smallest (R/R+G) settings in the
color m atch would fall into the extrem e group th a t is circled in Figure 8. Each has a low pro
portion o f genes with G at position 1032 am ong his total X-chrom osome pigm ent genes, i.e.,
they all have a p reponderance o f genes encoding Ser 180. C om pare this result with the pre
diction from the hypothesis that males with norm al color vision each express only the two 5-
m ost genes in their array, one long- and one middle-wave gene. If only two o f the genes were
expressed, to explain their color matches, these extrem e subjects m ust have S erl8 0 for both
the expressed m iddle- and long-wave pigments, i.e., they m ust express the m ore long wave
length sensitive subtype of each pigm ent. Because o f a prepo n d eran ce o f genes th at encode
Alai 80 in the population, the unexpressed genes are expected to m ost often specify Alai 80.
The m ost com m on gene arrangem ents predicted for these extrem e subjects are illustrated at
the bottom o f the figure. This distribution p red icted by the hypothesis that only two genes are
expressed, is superim posed in histogram form , on the observed results in the u p p e r graph.
T he descrepency between the prediction an d the observed results suggests that most or all of
the functional X -chrom osome genes are expressed and contribute to the observed behavior.
COLOR VISION DEFECTS 251
m en t spectra. A lthough they recognize position 180 as a polym orphic site in the
norm al pigm ents, long-wave genes th at include the active substitutions in exons 2
or 4 of the middle-wave genes would have to be fusion genes. If norm al observers
do n o t express fusion genes, then only a subset of the pigm ents that occur in
anom alous observers would co rresp o n d to n orm al pigm ent variants, others, p ro
duced by genes with fusions betw een exons 2 an d 5 would be exclusively expressed
in anom alous trichrom ats. To the contrary, here, it is suggested that fusion genes
are characteristic of norm al gene arrangem ents. We fu rth e r suggest that norm al
observers can express their fusion genes along with their o th er m iddle- and long
wave pigm ent genes. T he expression o f fusion genes in norm al subjects merely
contributes to the variability in n orm al color vision. Thus, norm al observers can
have and express any o f the pigm ents that occur in anom alous trichrom acy and
vice versa. In the traditional sense, anom alous pigm ents have been conceived as
pigm ents th at differ in spectrum from those that ever occur in norm al eyes. If the
picture o f the pigm ents and genes o f n orm al color vision presented here is the cor
rect one, then anom alous pigm ents do n o t exist. As originally envisioned by Alp
ern an d colleagues, anom alous trichrom ats draw from exactly the same set o f long-
and middle-wave pigm ents as do n orm al trichrom ats. Thus, there are no pigm ents
that are unique to color anom aly - i.e., no anom alous pigm ents. T he difference
between norm al an d anom alous trichrom acy is that for X-linked pigm ents, n o r
mal subjects draw a set th at includes o n e or m ore m em bers from each o f the m id
dle- and long-wave classes; deuteranom alous observers have functional pigm ents
from only the long-wave class; protanom alous observers have pigm ents from only
the middle-wave class.
In summary, the traditional theory held that the replacem ent o f a norm al pig
m en t with an anom alous one caused color anomaly. This thinking has carried over
in m ore recen t explanations o f anomaly, i.e., th at the fusion genes cause anom a
lous trichromacy. We hold that the fusion genes do not, in any way, cause color
anomaly. Q uite the opposite, the norm al variant pigm ents, including those p ro
duced by fusion genes, serve the advantageous role, of protecting som e observers
who loose the genes for (or function of) one class o f pigm ents, from becom ing
dichrom ats. They can in h erit anom alous trichrom acy instead. T he advantage of
anom alous trichrom acy com pared to dichrom acy may be quite significant for in
dividuals who in h erit the m ildest form s o f anomaly. This advantage may explain
why polym orphism s within the m iddle- and long-wave pigm ent classes have been
m aintained in the norm al hum an population.
O u r view o f the relationship betw een genes, cone pigm ents, an d color vision is
a simple one (illustrated in Figure 10). T he cone pigm ents are encoded by a gene
for the short-wave pigm ent on chrom osom e 7 and an array of genes on the X-chro
m osom e. T he n u m b er o f X -chrom osom e pigm ent genes is variable, with a range
across individuals, o f one to ten (or m o re ). Genes for middle-wave pigm ents are
m ore num erous than genes for long-wave pigm ents, b u t people with m ultiple
long-wave genes are n o t unusual. T he genes that encode middle-wave and long
wave pigm ents are highly polym orphic. An enorm ous variety o f pigm ent genes
252 JAY NEITZ AND MAUREEN NEITZ
SUM M ARY A T H E O R Y O F T H E C O N E S , C O N E P IG M E N T S A N D
C O N E P IG M E N T G E N ES O F H U M A N C O L O R V IS IO N
Normal
Genes
Pigments
liSi~
KPr
Color Defects
r
Key: Middle-wave subtypes: Q Q Long-wave subtypes: tj:
Figure 10 Sum m ary of a new theory of the genes, pigm ents, an d cone types that underlie nor
mal and defective hum an color vision. Among observers with norm al color vision th ere is an
enorm ous variety of different pigm ent gene arrangem ents. Some are relatively simple as illus-
COLOR VISION DEFECTS 258
trated by the exam ple in the u p p er left. Many are m ore com plicated and the gene com ple
m ents can include fusion genes an d genes with defects, as illustrated by the m ore right-hand
u pper gene arrays. Most or all of the functional genes are expressed. T he pigm ents produced
by the X -chrom osome genes fall into two classes, middle- an d long-wave, b ut each of these
classes is com posed of subtypes. Perhaps, two major svibtypes of middle-wave and two m ajor
subtypes of long-wave pigm ent are m ost com m on. Exam ples o f absorption spectra of the ex
pressed pigm ents are shown directly below each exam ple gene array. Each includes a short
wave-pigment (encoded by a separate gene on chrom osom e 7) an d at least one pigm ent from
each of the middle- and long-wave classes. However, norm al individual males can express m ul
tiple m iddle a n d /o r m ultiple long-w'ave pigm ent subtypes. Below the spectral curves, small
patches o f the cone mosaic that would result from each gene arrangem ent, are diagram m ed.
Each cone is illustrated as a circle. D ifferences in fill a n d outline illustrate cones with spectrally
different pigm ents (short-wave cones, solid black; middle-wave cone types, dark outline; long
wave cone types; no outline; subtypes have different fills). Each gene is expressed in its own
subpopulation of cone, thus, individuals can have as many as four or five spectrally different
cone types. T he situation in the color defects is simple. Each o f the four illustrated is reduced
in functional genes, in pigments, and in cone types from the norm al arran g em en t shown
above it. All have a short-wave pigm ent. T he p ro tan o p e and deu teran o p e are each red u ced to
a single type of X -encoded pigm ent. T he protanom alous observer is red u ced to only norm al
middle-wave subtypes, the deuteranom alous observer to only norm al long-wave subtypes.
254 JAY NEITZ AND MAUREEN NEITZ
plained by the loss o f norm al pigm ent genes. All such losses can occur by one-step
recom binations in which norm al genes are deleted from gene com plem ents that
u n derlie norm al color vision. Anom alous trichrom ats with red-green deficiencies
m aintain two or m ore o f eith er the middle-wave subtypes or the long-wave pig
m ent subtypes that are expressed in norm al color vision. D ichrom ats with red-
g reen defects have X-linked genes that pro d u ce only a single type o f cone pig
m ent.
We offer this m odel as an alternative to conventional theories o f the basis of nor
m al an d defective color vision. It has been useful for us in providing a fram ework
for organizing and re-exam ining what is known about the biological m echanism s
that u nderlie norm al an d defective color vision. We believe it is the sim plest m odel
th at can explain the facts as we un d erstan d them . This m odel will, no doubt, be
controversial. If it stim ulates fu rth e r research directed toward un d erstan d in g the
intriguing mysteries of color vision, it will have served its purpose.
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COLOR VISION DEFECTS 257
1MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU,
UK and
2 Department of Clinical Ophthalmology, Moorfields Eye Hospital, City Road, London EC1V
2PD, UK
SUMMARY
X-linked retinitis pigm entosa (xlRP) is one o f the m ost severe form s o f this group
of retinal dystrophies. It has been fo u n d to be genetically heterogeneous, with
two loci (RP2 an d RP3) on the short arm o f the X-chrom osom e. T he RP3 gene
has been localised by genetic linkage analysis an d m ore finely m apped by analysis
of deletion patients. T he RP2 locus is less well defined by linkage analysis alone
and the lack o f associated cytogenetic abnorm alities is likely to m ake the identifi
cation o f this gene m ore problem atic. H eterogeneity is a source of difficulty in
genetic counselling an d gene m apping studies, since many families cannot be
unam biguously assigned to one or the o th er locus.
INTRODUCTION
tion o f pigm ent laden cells into the retina. P igm ent often adheres to the narrow ed
retinal vessels giving a characteristic bone-spicule appearance, but it may also be
diffusely scattered. T he retinal blood vessels, particularly the arterioles, gradually
constrict as the disease progresses (Heckenlively, 1988).
RP may be in h erited as an autosom al dom inant, autosom al recessive o r X-linked
recessive trait. In general, the d ifferent m odes of inheritance cannot be distin
guished clinically, although carriers o f the X-linked disorder may show a charac
teristic fundus appearance (see below). T he m ajority o f cases with bilateral,
symmetrical disease probably have a genetic basis (see Jay, 1982). However, 40-50%
o f patients have no family history o f RP (sim plex RP) (Fishman, 1978; Jay, 1982;
Bundey and Crews, 1984b; Kaplan et al., 1990).
X-LINKED RP
X-linked RP (xlRP) is one of the m ost severe form s o f RP. In the first decade of
life, affected males com m only have night blindness, an d early visual field loss may
be ap p a ren t in the child tripping over things or appearing clumsy. If th ere is a
family history of RP, then the significance o f these symptoms may be recognised
and a diagnosis m ade as early as age four years (Heckenlively, 1988). In the teens,
d eg eneration of the RPE becom es apparent, visual field loss is m ore m arked and
visual acuity may deteriorate. T he p atien t starts to experience problem s in bright
light as well as in the dark. In the third decade, the loss in visual acuity progresses
and visual fields may be red u ced to 10-15. Secondary cataracts may develop. By
the age of forty, visual acuity is often re d u ced to counting fingers o r the p ercep
tion o f light and searching nystagmus may develop.
RP patients tend to be myopic and those with high myopia have the ad d ed prob
lem o f myopic degeneration. This is particularly tru e o f X-linked RP. Sieving and
Fishm an (1978) found th at 75% o f all RP patients (268 eyes) and 90% o f xlRP pa
tients (41 eyes) were myopic, com pared to 12% o f the general population. xlRP
patients had, on average, h ig h er m yopia (m ean 5.51D, SD 4.04D) than those
w'ith non-X-linked RP (m ean T.20D, SD 2.79D). T here was a suggestion th at the
X-linked group m ight be divided into two subgroups with low o r high m yopia re
spectively, bu t the sample size was small. RP patients were also found to have a
m uch hig h er frequency o f astigmatism, with 74% having cylindrical errors o f
>0.5D com pared to 19% o f the general population. T here was no significant dif
ference betw een the X-linked an d non-xlRP groups with regard to astigm atic cor
rection.
In fem ales who are heterozygous for xlRP, random inactivation of one o r o th er
o f the two X chrom osom es in each cell d u rin g early em bryogenesis (Lyon, 1961)
causes the phenotype to range from asym ptom atic to severely affected, d ep en d in g
on the p ro p o rtio n o f retinal cells with an active disease-bearing X-chrom osom e
(Bird, 1975; Fishm an et al., 1986). Many carriers over the age o f 20-30 years have
one or m ore of the following features: p erip h eral retinal d egeneration with pig
X-LINKED RETINITIS PIGMENTOSA 261
m ent deposition, ERG abnorm alities o r a tapetal reflex in the m acular region. T he
latter is a golden m etallic sheen which is seen in the fundus o f some, bu t n o t all,
carriers (Fishman et al., 1986) an d occasionally also in young hemizygous males
(Heckenlively, 1988; van Osch et al., 1990). However, its detection can be subjec
tive an d the reflex dim inishes with age, so it is not always a helpful indicator o f car
rier status (Bird, 1975). C arriers may have clinical signs at any age but tend not to
becom e sym ptomatic until m iddle to late life (Bird, 1975). Severely affected carri
ers may be n ight blind, have reduced visual field and som etim es loss of visual acu
ity. Like affected males, myopia and astigmatism are com m on in heterozygous
females an d carriers may show myopic degeneration of the fundus (Fishman et al.,
1986).
Fishm an et al. (1986) exam ined 33 obligate carriers (i.e. females with an affect
ed father, son, or both) from 22 families, ranging from 13 to 83 years o f age. T he
carrier state was detectable by the presence o f pigm ent a n d /o r an abnorm al ERG
in 28 patients (85% ), although 3 o f these (aged 28-45) showed only myopic
changes of the fundus and the abnorm al ERG may have been due to high myopia
rath er than RR A fourth patient (aged 31) who showed myopic degeneration as
the only fundus abnorm ality refused ERG. T he rem aining four patients showed a
b orderline norm al phenotype. T hree had norm al ERG and the only fundus abnor
mality was a tapetal reflex. O ne o f these patients was only 18 years old, bu t the o th
er two were bo th aged 49. T he rem aining patient h ad a com pletely norm al fundus
appearance and was only abnorm al in one o f five ERG tests, at age 16 years. Details
o f this study are sum m arised in Table 1.
Table 1 - Phenotypes of 33 obligate xlRP carriers (summarised from Fishman et al. (1986))
PREVALENCE
xlRP was first localised by B hattacharya et al. (1984), who re p o rted linkage of
xlRP in five kindreds to DXS7, a m arker localised in X p ll.4 - p ll.3 (Figure 1).
However, ensuing reports were in conflict over the location of xlRP relative to
DXS7. Friedrich et al. (1985) and W right et al. (1987) found that xlRP was m ost
likely to be proxim al to DXS7, while others favoured a m ore distal location
betw een DXS7 and DXS84, tightly linked to OTC (Nussbaum et al., 1985; D enton
et al., 1988; M usarella et al., 1988, 1989b). This distal localisation was su pported
by two patients who had deletions of Xp21 associated with RP (Francke et al.,
1985; de Saint-Basile et al., 1988). W right et al. (1987) suggested that xlRP is
genetically heterogeneous and C hen et al. (1989) found evidence o f genetic het
erogeneity in nine xlRP families. M eitinger et al. (1989) re p o rted linkage o f xlRP
to DXS255, a probe 15-20cM proxim al to DXS7. F our o f their five families
showed no recom binants betw een DXS255 and xlRP in a total of 17 meioses, but
th e o th er family contained two such recom binants in 12 meioses. O ne o f these
also recom bined with DXS7, placing xlRP distal to DXS7 (Figure 1) in th at fam
ily. Several authors suggested th at xlRP with tapetal reflex in one o r m ore carriers
was a distinct genetic form m apping to the distal location (Nussbaum et al., 1985;
D enton et al., 1988; Curtis and Blank, 1989; M usarella et al., 1989b), although
one ou t o f 14 carrier females in a family with the proxim al locus has been
re p o rted to show a tapetal reflex (Friedrich et al., 1985).
M ultipoint linkage analysis o f 62 families with xlRP gave overw helm ing evidence
for the existence o f two xlRP loci, with odds o f 6.4 X 109:1 (O tt et al., 1990). O ne
locus, term ed RP2, was m apped to X pl 1.3-pl 1.2. T he m ost likely location o f RP2
was 2cM proxim al to DXS14, bu t the confidence limits were wide, extending to
3cM proxim al to DXS7. T he second locus, RP3, was m apped to X p2 1 .1 -p ll.4 at a
position IcM distal to OTC (Figure 1). T h ere was also evidence that the propor-
X-LINKED RETINITIS PIGMENTOSA 263
DXS85
DXS41
DXS28
r DXS164
DMD
DXS206
0XS84
CYBB ____
RP3
L OTC
- DXS228
DXS7
MAOA
SYN/ARAF1
__TRP2
DXS426
l CSNB
TiMP
L 0ATL1
DXS225
DXS146
DXS14
DXS1
21.1
21.2
21.31
21.32
21.33
22.1
22.2
22.3
26.1
26.2
26.3
27.1
27.2
27.3
don o f RP2 and RP3 families varied between investigators, explaining the previ
ously discrepant results from d ifferent groups.
M usarella et al. (1988) had previously proposed a n o th er xlRP locus located be
tween DXS28 and the DMD locus. O tt et al. (1990) found some evidence for a
th ird locus, located in the same region, with odds o f 293:1, while M usarella et al.
(1990) found weaker evidence from an analysis o f 20 xlRP families. However, pa
tients deleted for this region have n o t b een re p o rted to have RP (Clarke et al.,
1986; Francke et al., 1987; reviewed by W right, 1990), which argues against an
xlRP gene in this location.
T he following sections sum m arise o u r c u rre n t knowledge with regard to the lo
calisations o f RP3 and RP2.
RP3
T he region containing RP3 has been defined both by linkage analysis and by
identification o f patients with deletions o f the X-chrom osom e. Linkage analysis
indicates that RP3 is proxim al to DXS84 an d distal to OTC (Nussbaum et al.,
1985; D enton et al., 1988; M usarella et al., 1988 and 1989b), a genetic distance of
ab o u t 6cM. This localisation is su pported by two patients that were identified with
RP an d an associated deletion. Patient BB (Francke et al., 1985), a m ale with a vis
ible deletion in Xp21, suffered from D uchenne m uscular dystrophy (DMD),
chronic granulom atous disease (CCD), M cLeod phenotype, RP and m ental retar
dation. A nother male, SB, was re p o rted with CGD, RP and M cLeod phenotype
(de Saint-Basile et al,, 1988). No visible cytogenetic abnorm ality was detectable in
SB, b u t both the CGD locus (CYBB) an d DXS140 were deleted from his DNA.
T h ere was evidence of X-linked transm ission, as his m o th er had dim inished neu
trophil cytochrom e b content, weak expression o f Kell antigens an d some bilat
eral retinal pigm entation, indicating that she is a carrier o f all three traits.
O th er patients with deletions overlapping those of BB and SB bu t who do n o t
have RP have been reported. P atient NF, a nine year old male, was re p o rted with
DMD and CGD (B aehner et al., 1986), while p atien t OM, a twelve year old male,
h ad CGD an d M cLeod phenotype b u t n o t RP (Bertelson et al., 1988). Both pa
tients had deletions th at included an d ex tended distally from CYBB, indicating
th at RP3 is proxim al to CYBB (Figure 2 ). T hus physical and genetic linkage data
indicate th at the region containing the RP3 gene is flanked distally by DXS84 and
CYBB, an d proxim ally by OTC an d the proxim al BB deletion breakpoint.
As a first step towards isolating the RP3 gene, the sequences at the proxim al BB
deletion b reakpoint have been cloned (B runs et al., 1988; M usarella et al., 1991)
an d used as a m arker for long-range physical m apping and chrom osom e walking.
A detailed physical m ap spanning the region assum ed to contain the M cLeod lo
cus (XK) an d RP3 has been constructed (M usarella et al., 1991; Ho et al., 1992)
and five CpG islands have been identified th at could m ark candidate genes for XK
an d RP3. O ne o f these islands is located 50 kb distal to the BB proxim al breakpoint
X-LINKED RETINITIS PIGMENTOSA 265
and an adjacent DNA fragm ent showed cross-species hom ology (McDowell et al.,
1990). This conserved fragm ent was used to screen retinal and retinal pigm ent ep
ithelial cDNA libraries. A 2.1 kb cDNA clone was identified. It is expressed in re t
ina an d brain, and shows hom ology to the m ouse t com plex sterility locus
(McDowell et al., 1990). However, extensive exam ination of the gene in RP3 pa
tients has failed to identify any m utation that would indicate that this is the RP3
gene (R o u x e ta l., 1992).
LOCUS BB SB NF OM S/H
BB SB NF OM S/H
DXS164 - NT - NT +
DXS142 - NT - NT +
DXS84 - + - + +
DXS141 - NT - + -
CGD cDNA - - - - +
DXS140 - - - - +
OTC + + NT NT NT
DXS7 + + NT NT NT
Figure 2 D eletio n analysis a n d RP3. S ch em atic d iag ram show ing She e x te n t o l d e le tio n s in 5
patien ts, BB (F ran ck e e t a l., 1985; K unkel e t a l., 1985), SB (d e Saint-Basile e t a l,, 1988), NF
(R oyer-Pokora e t a l., 1986), O M a n d S /H (B ertelso n e t a l., 1988) ( S /H a re first cousins with
M cL eod p h e n o ty p e a n d have id en tical d e le tio n s). In terv als I to VIII c o n ta in th e follow ing lo
ci: D u c h e n n e m u scu lar d y stro p h y (DMD) (I), DXS84 (II), DXS141 (III), M cL eod p h e n o ty p e
(XK) (IV), c h ro n ic g ra n u lo m a to u s disease (CG D) (V), DXS140 (VI), RP3 (VII) a n d O T C a n d
DXS7 (V III). NF an d O M d o n o t have RP, in d ic atin g th a t th e RP3 g e n e is p ro x im al to CGD,
while d e le tio n s in O M a n d S /H d e fin e th e sm allest re g io n o f overlap th a t m u st co n ta in th e
M cL eod locus, distal to CGD, T h e tab le shows th e p re se n c e o r ab sen ce o f various m ark ers in
DNA fro m th ese p atie n ts, su m m arised fro m th e re fe re n c e s d e ta ile d above. N T = n o t
tested.
T he RP3 gene may be associated with one of the o th er CpG islands, b u t there
could be o th er candidate genes betw een CYBB an d the BB breakpoint th at do n o t
have CpG islands. It is also possible th at p art of the RP3 gene is situated proxim al
to the BB b reakpoint an d that BB was only deleted for the term inal p art of it. Al
ternatively, the deletion in th at p atien t may have caused a position effect, such that
a change in the chrom atin structure influenced the expression o f the RP3 gene,
even though it lay outside the deletion. However, the p atien t BB suffered from a
very severe form o f RP, suggesting th at a com plete loss of gene function was m ore
likely than red u ced expression d u e to a chrom osom al position effect.
266 M. A. ALDRED ET AL.
RP2
T he RP2 locus has been m apped by linkage analysis alone, since no associated
cytogenetic abnorm alities have been rep o rted . As m entioned above, O tt et al.
(1990) localised RP2 to a broad region extending from DXS7 to the centrom ere.
M ore re cen t data have refined the localisation o f RP2 to a position proxim al to
DXS7 an d distal to DXS255 (Friedrich e t al., 1992; A ldred et al., subm itted).
However, this still represents a region o f 13-18cM (M ahtani et al., 1991). Cole
m an et al. (1990) suggested that RP2 is localised distal to DXS426 on the basis of
a recom binant in one family. However, th at family has since been shown also to
be linked to m arkers in the RP3 region (L. Hardwick, personal com m unication)
and therefore is n o t unam biguously RP2-type.
O ne of the m ain problem s with the genetic m apping o f RP2 is the difficulty in
identifying a hom ogeneous subset o f families to study. Bayesian probabilities of
RP2 versus RP3 can be calculated for individual families (W right et al., 1991; van
D orp et al., 1992), b u t the p osterior probabilities are very sensitive to the assum ed
locations o f these loci, which in the case o f RP2 is n o t accurately known.
T he m ost robust m ethod o f classifying families is to inspect the haplotypes and
identify recom binant meioses. T hose families that have recom bination events lo
calising the gene relative to OTC a n d /o r DXS7 can be unam biguously classified,
as illustrated in Figure 3. However, only a very small n u m b er o f families can be
classified in this m anner. A recen t m ulti-point analysis o f seven such RP2 families
th at includes both new and previously published data indicates th at the m ost likely
localisation for RP2 is proxim al to DXS7 a n d distal to DXS255, with the m axim um
lod score at the TIMP locus (Aldred et al., su b m itted ). This com pares well with the
data from a heterogeneity analysis of 37 predom inantly British xlRP families, in
which the m ost likely location for RP2 was 3.5 cM distal to TIMP (Teague et al.,
1994).
T here is still some scope for refining the localisation o f RP2 fu rth e r by linkage
analysis, bu t given the classification difficulties outlined above, the resolution of
the genetic localisation is unlikely to be b etter than 5 cM. F u rth er m apping studies
will increasingly rely on physical m apping techniques, such as the developm ent of
a YAC contig spanning the region. C andidate genes could be identified by search
ing for CpG islands or for sequences with cross-species homology. However, de
tailed physical m apping of a 5 M egabase (Mb) region is a m ajor undertaking. An
alternative approach is to identify expressed sequences from the region of inter
est. H um an-specific cDNAs could be cloned from a YAC intro d u ced into a ro d e n t
cell line (Eliceiri et al., 1991), o r alternatively from a h um an-rodent som atic cell
hybrid by subtractive hybridisation or by cDNA synthesis from unprocessed h eter
ogeneous nuclear RNA (Sive an d St Jo h n , 1988; Liu et al., 1989; C orbo et al.,
1990).
X-LINKED RETINITIS PIGMENTOSA 267
I ff I S13
! 2
i 1
I 1
1
! 2
I II $ i* $ j m
L j
i
2 ' 1 1
2! 1 2
1 i 1 2
2 1 | 2
3 i 1 3
2 ' 1 2
I I1! 1 1
IV
Markers:
DXS164
1
2 DXS84 1
2 DXS7 2
2 TIMP 1
3 DXS255 i 4
2 DXS14
DXYS1X ii
F igure 3 D istin g u ish in g b etw een RP2 a n d RP3 types o f xlRP by h a p lo ty p e analysis. Sections o f
two xlRP fam ilies illu strate th e use o f h a p lo ty p e analysis to d e te rm in e w hich locus is seg reg at
ing. In (a), u n a ffe c te d m ale filjj.h a s th e affe c te d h ap lo ty p e a t DXS7 a n d m o re distal m ark ers,
in d ic atin g th a t this is an RP2 family. T h is is su p p o rte d by a n o th e r re c o m b in a n t in individual
III 13, a fem ale with p h e n o ty p ic ev id en ce o f th e c a rrie r state. A g ra p h o f th e m u ltip o in t linkage
d ata fro m this fam ily co n firm s a b ro a d p e a k in th e RP2 re g io n (W right et a l, 1991).
1
II
Markers:
0
DXS84 2
CYBB 2
OTC 1
DXS7 2
OATH 2
DXS255 3
III
(O (?)
2 6 |7
IV 1I
2 2 2
2 - 2
1 - 1
2 1 2
2 - 1
3 3 2
F igure 3 (b) shows th e reverse situ atio n , in w hich affected m ale IVg is re c o m b in a n t w ith DXS7
a n d DXS255, in d ica tin g th a t th e disease locus is distal to DXS7, co n sisten t with a n RP3 local
isation. It sh o u ld be n o te d th a t th e m ajority o f fam ilies (8 6 % in this la b o rato ry ) d o n o t show
re c o m b in a n ts th a t allow such u n a m b ig u o u s classification. W hile p ro b ab ilities can be calcu lat
ed, assig n m en t difficulties re m a in o n e o f th e m ajo r p ro b le m s in resea rc h a n d co u n se llin g in
xlRP.
Since it is difficult to distinguish the two xlRP loci, both clinically an d genetically,
the answer to this interesting and im p o rtan t question rem ains som ewhat elusive.
In the m ulticentre analysis of O tt et al. (1990), 75% o f families were RP3-type
with confidence limits extending from 45% to 90%. However, the relative p ro p o r
tions varied between different investigators. T he highest p roportion o f RP2 was
found in families contributed from this laboratory, whereas M usarella et al. (1988
and 1990) found no evidence o f the RP2 locus am ongst 20 families. This raised
the possibility that RP2 m ight be m ore com m on in Britain than elsewhere.
270 M. A. ALDRED ET AL.
To investigate this further, published families that included linkage data for two
or m ore m arkers between DXS84 an d DXS255 inclusive were reviewed. W here
possible, they were tentatively classified as RP2 o r RP3-type on the basis o f haplo-
types and lod scores. As detailed above, families showing recom bination with OTC
a n d /o r DXS7 can be unam biguously classified. In addition, for this study, families
th at showed one o r m ore recom binants with DXS84 but were linked to the RP2
region (TIMP-DXS255) were classified as possible RP2 families an d vice versa.
T hose families showing no recom bination, or recom bination in both regions, and
those with recom binants that only involved m ore proxim al or distal m arkers were
n o t classified. Families which were n eith er RP2 n o r RP3 (four from this laboratory
an d one re p o rted by M usarella et al. (1988 and 1990)) were excluded.
T he results (Table 2) show that overall, 50 o f the 83 families (60%) can be clas
sified. C om paring Britain with the com bined data from the rest o f E urope and
Scandinavia, the proportions o f classified families are very similar, as are the rela
tive proportions of RP2 and RP3 am ongst the classified families. Thus there is no
evidence th at RP2 is m ore com m on in Britain than the rest o f Europe on the basis
o f this approxim ate classification.
T he com bined British an d E uropean data suggest that 38% of the 34 classified
families are RP2-type. This is alm ost twice the pro p o rtio n o f RP2 found in Austra
lia (22%) and nearly three times that in A m erica (14% ). RP2 may therefore be
m ore com m on in Europe than elsewhere, bu t the num ber of classified families
from Am erica and Australia is small (n = 16), so it is n o t clear w hether this re p re
sents a true difference.
D eterm ining the relative proportions o f the two genes and w hether there is vari
ation betw een countries is an im portant issue with im plications for genetic coun
selling. Overall, 32% of families in this study were RP2-type, agreeing with Teague
et al. (1994) who found 30% RP2 in a heterogeneity analysis o f 37 predom inantly
British families. However, given the difficulties in categorising families and the
small sam ple sizes, the question o f ethnic differences may n o t be resolved until
one of the genes is cloned.
Nationality Total no. Total no. No. RP2 No. RP3 Reference
classified unclassified families families
It has been suggested th at th ere are closely linked genes for CSNB and myopia
on the X-chrom osom e an d that hyperopic XL-CSNB patients result from crossing-
over between these genes (K houri et al., 1988). However, although X-linked myo
pia has been docu m en ted (Bartsocas and Kastrantas, 1981), a linkage study of a
family with two cousins affected by XL-CSNB, one myopic an d one hyperopic,
showed no evidence o f exchange of flanking m arkers (Dry et al., 1993). In both
CSNB and RP, m yopia may be a pleiotropic effect o f the m utation, perhaps influ
272 M. A. ALDRED ET AL.
encing the growth o f the eye pre- a n d /o r post-natally. Since ocular refraction is a
m ultifactorial trait, the variation in refraction observed in RP and CSNB could be
explained by m odulation o f the disease phenotype by the genetic background. Al
ternatively, m yopia m ight be secondary to the defective vision. It has been shown
th at neonatal lid fusion in m onkeys causes the eye to enlarge and becom e myopic
(Wiesel and Raviola, 1977). It is therefore possible that the early visual defect in
CSNB o r RP causes myopia. This m ight explain why Miyake et al. (1986) found
high m yopia to be associated with a total lack o f rod function in CSNB, while those
with some residual function ten d ed to have sm aller refractive errors, and why pa
tients with non-X-linked RP have lower m yopia that those with the m ore severe X-
linked disease (Sieving and Fishm an, 1978). However, the question o f allelic rela
tionships between these disorders will n o t be answered definitively until the genes
are isolated.
CONCLUSION
ACKNOWLEDGEMENTS
We are grateful to the British Retinitis Pigm entosa Society, the N ational Retinitis
Pigm entosa Foundation an d G eorge G und Foundation for financial support.
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11. CONGENITAL STATIONARY NIGHT BLINDNESS:
A CRITICAL REVIEW FOR MOLECULAR APPROACHES
SUMMARY
INTRODUCTION
some work has been done on the autosom al d o m in an t CSNB (Yijian et al., 1991),
most o f the linkage studies have been co n ducted on the X-linked form (Volker-
D ieben & W ent, 1975; M usarella et al., 1989; B ech-Hansen et al., 1990; Schwartz &
Rosenberg, 1991; A ldred et al., 1992; Bech-H ansen, M oore & Pearce, 1992;
M usarella et al., 1992). T he results suggest a single gene may be responsible for
several clinical entities on the X-chrom osom e.
(Kandori, 1959; Kandori, Setogawa & Tam ai, 1966; K andori et al., 1972) should be
considered.
CLINICAL MANIFESTATIONS
m ost freq u en t sign is nystagmus (jerky eye m ovem ents), patients are often ini
tially diagnosed as having ocular albinism o r congenital nystagmus. It may be dif
ficult to differentiate these conditions from CSNB w ithout p ro p e r electrodiag
nostic evaluation.
T he phenotypic distinction o f each p attern o f in h eritan ce is not strict and over
laps do occur. However, both visual acuity and refraction b ear some relationship
to inheritance. Visual function varies from norm al acuity (20/20) to legal blind
ness (20/200). In autosom al d om inant CSNB, im paired night vision is often the
m ain defect (C uniers, 1838). Visual acuity is usually within the norm al range (2 0 /
2 0 -2 0 /4 0 ) with a m ild refractive e rro r ( 0.50 D - +0.50 D) if any. Myopia is rarely
seen. In the autosom al recessive phenotype, vision is usually m oderately im paired
(about 2 0 /8 0 o r b etter), an d the myopia tends to be m ild to m oderate.
In males affected with X-linked CSNB, visual loss can show intra- and inter-famil-
ial variability (Pearce, Reedyk & C oupland, 1990). In these cases vision is usually
abnorm al, ranging from 2 0 /3 0 to 20/200. Visual acuity o f 2 0 /2 0 appears to be
rare in this subgroup. A lthough refractive e rro r is usually o f high myopia, it may
range from m ild hyperopia (+0.50) to severe myopia (up to 20 D) (D onders,
1855; C arroll & Haig, 1952; Francois, Verriest & De Rouck, 1965; Carr, 1974; Krill,
1977; Haim , 1985; K houri et al., 1988; Fishm an, 1990; Noble, C arr & Siegel, 1990;
Pearce, Reedyk & C oupland, 1990).
Nystagmus is a com m on finding in patients born with subnorm al vision (less
than 20/60) (Krill, 1977). In m ost cases o f CSNB the nystagm us is horizontal and
may be fine o r coarse. Fine rotary nystagmus has also been described (M erin et al.,
1970). Strabism us is often present, bu t th ere is no predom inance o f esotropia or
ex otropia (N ettleship, 1912; M erin et al., 1970; Carr, 1974; Krill, 1977; M erin,
1991). A nisom etropia (a difference in refractive e rro r greater than 1.5 D between
eyes) an d astigm atism are usually m ild (1.0-2.0 D). Amblyopia, which is n o t spe
cific, may also be seen (M erin et al., 1970; Krill, 1977; K houri et al., 1988; Pearce,
Reedyk & C oupland, 1990).
Findings o f the fundus exam ination are generally norm al. However, w hen myo
pia is m ore than 4.0 D, the retin a may show characteristic changes o f myopia, such
as a tigroid fundus and thinning o f the retina, but blood vessel caliber is always
n o rm al and no pigm ent clum ping is seen. Cases have been re p o rted o f the optic
disc being tilted, pale or dysplastic (Mintz H ittner, Borda & Justice, 1981; H ecken
lively, M artin & R osenbaum , 1983). C arr (1966b) also describes an occasional in
crease in granularity of the macula, which can alter the norm al foveal reflex.
Barricks et al. (1977) described paradoxical pupillary responses, in which the
pupil constricts in the dark, in three boys with CSNB of unspecified inheritance
(Barricks, Flynn & Kushner, 1977). Since this p h en o m en o n is no t seen in older
affected males (> 55 y.o.), it is considered a useful diagnostic finding in young chil
d ren w hen present (Khouri et al., 1988).
Vitreous fluorophotom etry, a useful tool in the evaluation o f the blood-retinal
barrier, was p erfo rm ed on three types o f n ight blindness: O guchis disease, X-
A CRITICAL REVIEW FOR MOLECULAR APPROACHES 281
linked CSNB an d fundus albipunctatus. All eyes showed norm al values, strongly
suggesting th at the blood-retinal b a rrie r is intact (Miyake et al., 1983).
C olour vision is usually unaffected in CSNB, bu t occasionally a m ild tritan defect
can be seen (C arroll & Haig, 1952; A rm ington & Schwab, 1954; Franceschetti, Ba
bel & Francois, 1963; Miyake e ta l., 1986). R ed-green deficiency has been re p o rt
ed, regardless o f the p attern o f in h eritan ce (M erin et al., 1970; Ponte, Lodato &
Lauricella, 1974; Miyake et al., 1986), an d is n o t felt to be related specifically to
CSNB bu t ra th e r to an u n related red-green colour gene defect located at Xq 28
(McKusick, 1990a).
Visual fields are usually norm al w hen tested u n d e r lighted condition (photo-
pic). However, if patients are tested in dim illum ination, visual field constriction
may be no ted (Carr, 1974; Krill, 1977; Miyake et al., 1986).
Physical anom alies are usually n o t re p o rted in patients affected with CSNB;
however, M aeder (1946) re p o rted a family of autosom al d om inant CSNB in four
generations w here p lan tar syndactyly (fusion o f two toes) was a regular finding.
U nfortunately, no long-term follow-up of this family is available.
Basics o f Electroretinography
and rods, as does a single red flash on a dark-adapted retina. Pure cone responses
are o btained with either a red or white light flickering at a frequency o f 30 cycles
p er second (H z), o r from a light-adapted retin a following a single flash o f yellow-
re d o r blue-green stimulus (Fishman, 1985).
T he three main com ponents of the ERG (Einthoven & Jolly, 1908; Jim enez-
Sierra, O gden 8c Van Boemel, 1989) are the a-wave, b-wave an d c-wave (Figure 1).
T h e a-wave, the initial cornea-negative com ponent, is g enerated by the p h o to re
cep to r layer. It is followed by a cornea-positive b-wave that arises from the in n er
nuclear layer (bipolar, M uller and am acrine cells). T he c-wave, p roduced by the
retinal pigm ent epithelial (RPE) cells, is a cornea-positive response th at is n o t rou
tinely used clinically (Fishman, 1985; Jim enez-Sierra, O gden & Van Boemel,
1989). T he ganglion cell layer does no t contribute to the ERG.
T he m ost freq u en t m easurem ents taken with the ERG are the a- and b-wave am
plitudes and the b-wave im plicit time (tim e from flash to peak o f b-wave). In
CSNB, the b-wave is m ost severely affected, an d in some form s of the condition, its
am plitude can be red u ced so drastically that it becom es negative. Several o th er
co m ponents o f the ERG are clinically useful. For exam ple, the am plitudes o f the
oscillatory potentials (OPs) (O gden, 1973), wavelets on the ascending limb o f the
b-wave, can be affected by diseases that are due to neurotransm ission defects in
the vicinity o f the in n er plexiform layer (Lachapelle, Little & Polom eno, 1983;
Miyake, Yagasaki & H origuchi, 1987c).
A CRITICAL REVIEW FOR MOLECULAR APPROACHES 283
eno, 1983). A lthough this phenotype is m ost frequently seen with X-linked
in h eritance, it has also been re p o rted in autosom al d om inant an d autosom al re
cessive pedigrees (Noble, C arr & Siegel, 1990; Yijian et al., 1991).
Patients with incom plete CSNB exhibit m easurable, albeit reduced, scotopic b-
wave am plitudes, decreased night vision sensitivity an d less severe m yopia than
those with the com plete form (m ean 2SD; 0.8 10.8 D) (Heckenlively, M artin
& R osenbaum , 1983; Miyake et al., 1987a; Miyake, Yagasaki & H orogushi, 1987b).
Visual acuity may be norm al o r n ear norm al. Oscillatory potentials are m ore easily
reco rded than in com plete CSNB (Lachapelle, Little & Polom eno, 1983; Miyake,
Yagasaki & H origuchi, 1987c; Miyake & Kawase, 1984). This phenotype has also
been described in the three M endelian patterns of inheritance. Initially, the in
com plete CSNB was th ought to be a variant o f the com plete form , distinguished
mainly by the severity of functional disturbance. However, Miyake et al. (1986) fur-
A CRITICAL REVIEW FOR MOLECULAR APPROACHES 285
Complete Incomplete
th er characterized both subgroups an d provided evidence that the com plete and
the incom plete types o f CSNB are different clinical entities. O thers have debated
w hether these types are phenotypically an d genotypically distinct (Khouri et al.,
1988; Pearce, Reedyk & C oupland, 1990). In considering these questions, we be
lieve it is im portant to recognize th at the ERG test stim ulus used by Miyake et al.
(1986) is unique. T h eir use of a rectangular ra th e r than a flash-evoked stimulus
resulted in d ifferent ERG patterns, m aking com parison with o th er groups diffi
cult. This may contribute to th eir distinction betw een com plete and incom plete
CSNB. As we note later, linkage studies o f Miyake's incom plete families do no t
support separation into two disorders.
1989). O f these, special atten tio n will be given to Aland Island eye disease (AIED)
an d D uchenne m uscular dystrophy (DMD) in a later section.
1) Stationary
1.1 complete congenital stationary night blindness
1.2 incomplete congenital stationary night blindness
l.SO guchi's disease
2) Retinal dystrophies
2.1 Early retinitis pigmentosa
2.2 Infantile Refsums disease
2.3 Goldman-Favre vitreoretinopathy
2.4 Juvenile retinoschisis
2.5 Progressive myopia
3) Vascular disorders
3.1 Ischemic central vein occlusion
3.2 Ischemic central artery occlusion
4) Retinal toxicity
4.1 Quinine
4.2 Vincristine
5) Paraneoplastic melanoma
6) Familial optic nerve atrophy
7) Duchenne muscular dystrophy
8) Aland Island eve disease
Modified from Weleber, RG; Pillers, D -A; Powell, B R, H anna, CE, Magenis, R E, Buist, N R M (Wele
ber, 1989)
Most carriers o f X-linked CSNB are asym ptom atic. However, because o f random
inactivation o f the X chrom osom e (lyonisation), they may show signs and symp
toms o f the disease. Some may show evidence o f nystagmus a n d /o r ERG changes
(Pearce, Reedyk & C oupland, 1990), b u t the majority of these fem ale carriers are
n o t identified by routine ocular exam ination (Miyake, Yagasaki & H orogushi,
1987b; Bech-Hansen e ta l., 1990). Subtle changes have been d o cum ented by ERG
testing. Miyake an d Kawase (1984) re co rd ed statistically sm aller oscillatory p o ten
tials (OPs) and am plitudes in carriers o f X-linked CSNB than in non-carrier
women. T he im plicit tim e o f the OPs, a n d the am plitudes o f the a- and b-waves,
were within norm al limits. Young et al. (1989) established that the OPs are opti
mally reco rd ed in the X-linked carriers when the dark-adapted eye is stim ulated
by a blue flash.
288 ELISE HEON AND MARIA A. MUSARELLA
Recently, R uttum , Lewandowski and B atem an (1992) re p o rted the clinical find
ings an d the results of ERG testing and dark adaptom etry in four sym ptomatic car
rier fem ales from a five-generation family with X-linked CSNB. The scotopic ERGs
showed a negative b-wave consistent with the S chubert-B ornschein type of CSNB.
T he p o o r rod-m ediated dark adaptations found in some sym ptomatic carriers are
th o u g h t to be a consequence o f lyonization.
an d AIED (W eleber et al., 1989). T he ERG responses o f this child were sim ilar to
those for AlED an d identical to those re p o rte d for incom plete CSNB, as described
earlier. It was suggested that AlED an d incom plete CSNB m ight be the same dis
ease, thus initially localizing this form o f CSNB to Xp21 (W aardenburg, 1970;
W eleber et al., 1989). F u rth er m olecular studies o f this patient localized the dele
tion to the subbands Xp21.3-p21.2, betw een DXS67 an d DMD (Miyake, Yagasaki
& H orogushi, 1987b; Pillers et al., 1989a, 1989b, 1990a, 1990b; W eleber et al.,
1989) (Figure 4). In 1991, linkage studies were re p o rted on two families with AlED
(Alitalo et al., 1991; Schwartz & Rosenberg, 1991). Both studies positioned AIED
on the proxim al p a rt o f the short arm o f the X chrom osom e. A lthough these re
sults did n o t support the work o f Pillers et al., th eir data suggested th at AIED and
com plete CSNB may be allelic and th at the ocular findings in the p atien t de
scribed by W eleber were probably p a rt o f the syndrom e resulting from the dele
tion at Xp21, ra th e r than AIED. To clarify the m atter, the ocular disorder of this
p atien t was nam ed O regon eye disease, and the origin o f the negative ERG of
O regonJR w as suggested to lie in the Xp21.3-p21.2 region, the location of the glyc
erol kinase deficiency and DMD genes.
Since then, the gene for DMD has been isolated and its gene product, dystro
phin, has been identified. A ntibodies to dystrophin were used to d eterm in e
w hether it was expressed in the retina, and dystrophin was identified in the o uter
plexiform layer, the putative site for CSNB (Bulm an et al., 1992). In o u r experi
290 ELISE HEON AND MARIA A. MUSARELLA
ence, m ost young DMD patients tested by ERG were found to have a significantly
red u ced b-wave am plitude, suggesting th at dystrophin may play a role in norm al
retinal neurotransm ission (M usarella et al., unpublished d a ta ). F u rth er studies to
d eterm in e the role o f dystrophin in the retina may contribute to a b etter u n d er
standing o f the X-linked form s o f CSNB.
Because dark adaptom etry m easures the ability o f the p hotoreceptors to adapt
over time in the dark, it is mainly indicated for patients suffering from nyctalopia.
In this test the patient is initially dark-adapted for 2 m inutes, then light-adapted
for 10 m inutes to bleach the photoreceptors. T he background light is then extin
guished an d the p atien t is asked to identify the test target, which has adjustable
intensities ( Jim enez-Sierra, O gden & Van Boemel, 1989; Fishm an, 1990). The
ability to dark-adapt is then m easured over a 30-m inute period with the dark
adaptom eter. A norm al curve has a b ipartite shape; the first 10-minute segm ent
rep resents the cones adaptation, an d the rest o f the curves rep resen t the rods'
adaptation (Figure 5).
o "complete" CSNB
. J _____________ i____________ I __ I | i
5 10 15 20 25 30
Francois, 1963; Carr, 1974; Krill, 1977). However, there appears to be a subgroup
of retinitis pigm entosa (RP), a form of n ig h t blindness, m arked by extrem ely slow
progression. This forme fruste of retinitis pigm entosa should n o t be confused with
CSNB (M aeder, 1946; Babel, 1963; Franceschetti, Babel & Francois, 1963; Auer
bach, Godel 8c Rowe, 1969). Because m any tapetoretinal degenerations may show
n o rm al retinal findings, usually in the early stages, it is essential to rep eat retinal
exam inations periodically. If progression is docum ented, the diagnosis o f CSNB
m ust be re-evaluated an d a tapetoretinal degeneration considered (France
schetti, Babel & Francois, 1963). T he ERG is useful in distinguishing these disor
ders. However, cases o f patients with initial findings consistent with CSNB (Zorn,
1920; A m m ann, 1946; Frangois, 1961; A uerbach, Godel & Rowe, 1969), including
the ERG characteristics, have been rep o rted , bu t in these cases the condition
developed very slowly, like a progressive retinitis pigm entosa. Sieving et al. (1992)
re p o rted a family that fell betw een the definitions for CSNB an d RP, since they
d em onstrated ERG criteria for CSNB and a progressive course consistent with RP.
In m ost family m em bers, this autosom al d o m in an t pedigree has no detectable
rod vision, norm al cone function and n orm al G oldm ann visual fields. O lder
m em bers have bone-spicules, a sign usually seen in retinitis pigm entosa. Since all
family m em bers tested had norm al (or n ear norm al) cone function, they are
believed to have a congenital night blindness, probably progressive (a type o f rod
dystrophy) ra th e r than retinitis pigm entosa (which would imply a progressive
rod-cone dysfunction). M olecular studies o f this family will be discussed later.
HISTOPATHOLOGY
PATHOGENESIS
ANIMAL MODELS
Two anim al m odels have been used in the study o f CSNB. In the first, Witzel et al.
(1978) studied electroretinographically nyctalopic A ppaloosa horses. U nder pho
topic and scotopic conditions, ERG changes were sim ilar to those in hum ans with
the S chubert-B ornschein type o f CSNB. T he photopic abnorm alities consisted of
red u ced b-wave and slower-than-norm al im plicit time. T he dark-adapted ERGs
showed a sim ple negative potential, the b-wave being non-recordable. F urther
m ore, the presence o f a norm al a-wave in the dark-adapted eye suggested that the
rod photoreceptors were responding norm ally an d that the rod-RPE relationship
was n o t disturbed. Absence of the scotopic b-wave localizes the functional defect
to the o u ter plexiform layer a n d /o r in n e r nuclear layers. Histological and ultra-
structural studies showed no abnorm ality.
T he second anim al m odel studied was the pearl m u tan t m ouse (Balkema,
M angini Sc Pinto, 1983; Pinto et al., 1985). T he m ouse is known to have no gross
m alform ations, decreased sensitivity in dark-adapted conditions, an abnorm al
ERG a-wave, absence of p h o to rec ep to r degeneration, an d norm al rhodopsin con
centration. T he pearl is an autosom al recessive m utation in the m ouse that has
been m apped to m ouse chrom osom e 13. T he m ouse is characterized by hypopig
m entation o f the RPE an d reduced ipsilateral retinofugal projections, as observed
in albinos. This phenotypic characterization o f the pearl m ouse raises questions
ab o u t w hether it should be considered an anim al m odel o f CSNB.
CONCLUSION
A lthough th ere are distinct clinical entities re ferred to as CSNB, litde is known
about the m olecular defects o r even the defective neurotransm itters that are
responsible for any o f their forms. T he localization of putative genes for the auto
somal form s o f CSNB rem ains unknow n. T he X-linked type o f CSNB is located at
X p l 1.3 in a region n ea r m onoam ine oxidase A an d B, synapsin an d synapto-
phorin. However, linkage studies d o n e with these m arkers have excluded these
neurotransm itters as putative sites for this form o f CSNB. At this point, little else
rem ains at this location as possible candidate genes for X-linked CSNB. T he iden
tification o f dystrophin in the o u ter nuclear layer o f norm al retinas may indi
rectly provide clues to this enigm atic g ro u p of disorders.
ACKNOWLEDGEMENTS
This work was sup p o rted by the M acLaughlin F oundation (E. H.) an d the R P
F oundation o f C anada (MAM). This ch a p te r was p rep ared with the assistance of
Medical Publications, T he H ospital for Sick C hildren, Toronto.
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A CRITICAL REVIEW FOR MOLECULAR APPROACHES 301
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12. CHOROIDEREMIA
SUMMARY
CLINICAL ASPECTS
C horoiderem ia was first described by M authner in 1872 who coined the nam e of
this disease because o f presu m ed congenital absence o f the choroid. Progressive
degeneration o f the choroid, retinal pigm ent epithelium (RPE) and retin a was
established only m uch later (Bedell, 1937; Schutzbach, 1938; Friedm an, 1940;
304 FRANS P. M. CREMERS AND HANS-HILGER ROPERS
Several males have been described who show cytogenetically detectable deletions
o f (parts of) the Xq21 ban d which are associated with com plex phenotypes
(Tabor et al., 1983; R osenberg et al., 1986,1987; Schwartz et al., 1986; H odgson
et al., 1987; Nussbaum et al., 1987; Wells et al., 1991; R eardon et al., 1992). These
phenotypes alm ost invariably include CHM, congenital m ixed deafness, m ental
retard atio n (MR), and in a few cases cleft lip and palate (CLP), obesity, u rin ary
tract abnorm alities, hypertelorism , m acrocephaly, an d myopia. In o rd e r to posi
tion the CHM locus m ore accurately, these deletions were characterized in great
detail em ploying S outhern analysis (Schwartz et al., 1986, 1988; H odgson et al.,
1987; C rem ers et al., 1988, 1989a; M erry et al., 1990; Yang et al., 1990; Bach et al.,
1992). Based on these results, the Xq21 region was subdivided into a n u m b er of
intervals. As depicted in Figure 1, the sm allest region of deletion overlap th o u g h t
to contain the CHM gene encom passes interval 3 which is defined by the loci
DXS233 (pJL68), ZNF6 (CMPX1), DXS165 (p lb D 5 ), and DXS95 (pXG7c). The
CHOROIDEREMIA 305
LG L 7 .6 3 3 .1 D20 DM XL62 NP X q
2906
lo cus p ro be 25,6 3.5 MBU SD XL45 RvD
DXS 7 2 p X 65H 7
DXS169 pX104f
DXS 2 6 PHU16
DXS 2 3 2 pJL.68
DXS12 1 p784
DXS2 33 p JL 8
ZN F6 CMPX1
DXS1 65 p 1bD5
DXS 9 5 pXG7c
DXS1 10 p 72 2
pXG8b
DXYS1 PDP34
DXYS5 p 4 7 b /p 3 1
DXS2 14 pPA20
DXYS12 pSt2 5.2
DXS 1 1 2 p753
DXS 3 p 19.2
DXS 7 3 P X20R 42
DXS 9 6 pXG3b
DXS 1 18 p776
pF8
pF 1
onlY discrepancy in this assignm ent is the absence of clinical signs o f CHM in
patient RvD, who, at the age of 15, does n o t show the characteristic features of
CHM despite the fact th at interval 3 is com pletely absent (J. J. P. van de Kamp
and E. M. Bleeker-W agemakers, personal com m unication). All o th er patients
show CHM or fundus abnorm alities reflecting an earlier stage o f CHM because of
their young age.
L G L2905
3.5
25 .6
7.6
C3
C759
2082
LGL1134
LGL1101
33.1
t(X;13) . / -
Figure 2 Deletion map of the CHM locus at Xq21.2. Deleted segments are given as horizontal
bars. The location of the X-chromosomal breakpoint in a female with an X;13 translocation
and choroideremia is shown as a broken bar at the bottom. The exact physical distances be
tween the anonymous probes is not known, except for pJ7.6A/plbD5 and pJ15/pJll/pJ60
which have been linked by genome walking (Cremers et al., 1989b, 1990c).
CHOROIDEREMIA 307
Em ploying FIGE analysis o f control DNA, the DNA probes dem arcating the criti
cal region for CHM, i.e. p lb D 5 an d pJ60, were shown to hybridize to the same
625kb Sfil fragm ent indicating th at at least part o f the CHM gene was located on
this fragm ent (Van de Pol et al., 1990). High m olecular w eight DNA from a cell
line that contained only the hu m an X chrom osom e in a ro d e n t background was
digested with Sfil an d subjected to FIGE. DNA was extracted from agarose slices
containing the 625 kb Sfil fragm ent, digested with EcoRI an d subcloned into the
lambdaZAP vector (Van de Pol et al., 1990). From this library, 7 clones were iso
lated which derived from the correct 625 kb Sfil fragm ent. Two o f these probes,
i.e. pZ l72 and p Z ll, are depicted in Figure 2. pZ172 was located centrom eric to
plbD5 an d p Z ll was positioned telom eric to pJ60.
As indicated above, the centrom eric b reakpoint o f the deletion in p atien t 7.6 was
located proxim al to p lb D 5 . C onventional S outhern analysis using a centrom eric
ju n ctio n probe showed th at the deletion junction was situated on a 10.5 kb EcoRI
fragm ent (C rem ers et al., 1990a). This fragm ent was gel-purified from genom ic
DNA o f p atien t 7.6 an d cloned into a lam bda vector. Using the centrom eric ju n c
tion clone, several clones were isolated, one of which was analysed in m ore detail.
T he deletion ju n c tio n was located on a 0.9 kb H indi II fragm ent which in norm al
genom ic DNA detected the expected 7.5 kb EcoRI fragm ent (pJ7.6A) and a 8.5
kb EcoRI fragm ent (pj7.6B) spanning the distal deletion breakpoint. pJ7.6B is
located distal to the ju m p clo n es an d to p Z ll (Figure 2), an d is located on a dif
feren t Sfil fragm ent (C rem ers et al., 1990a).
Em ploying the new probes, six additional deletions were fo u n d in patients with
CHM (Figure 2) which enabled us to locate p a rt o f the CHM gene to a 50 kb re
gion flanked by p j l l an d p Z ll. Recently, two balanced translocations were found
in females showing choroiderem ia (Kaplan et al., 1989; Siu et al., 1990). In both
translocations, the breakpoints on the X -chrom osom e were situated in band
Xq21.2, while the autosom al breakpoints were at 7 p l4 and 13pl2, respectively.
A part from CHM, both females showed prim ary am enorrhoea. T he X;13 translo
cation breakpoint could be positioned betw een pJ60 an d p j l l , in the critical re
gion for the CHM gene (Figure 2; C rem ers et al., 1989b). M erry et al. (1990)
isolated a ju m p in g clone in the vicinity of p jl 1 which also detected the X;13 trans
location breakpoint. S outhern blotting has indicated th at the X;7 translocation
breakpoint is located m ore distally (C. Philippe et al., unpublished data).
Em ploying two ju m p clo n es from the critical region for CHM, i.e. p j l l an d pJ60
(Figure 2), hum an genom ic DNA phage clones were isolated, which together
308 FRANS P. M. CREMERS AND HANS-HILGER ROPERS
sp an ned a region of approxim ately 45 kb. Single and low copy probes were subse
quently tested for evolutionary conservation by hybridization to S o u th ern blots
containing DNAs from a variety of vertebrates. Using one o f the probes that
detected hom ologous sequences in e.g. m ouse and chicken, eight overlapping
cDNAs were isolated from a adult hum an retinal cDNA library (C rem ers et al.,
1990c). R escreening of the adult retina, a fetal retina, and a fetal brain cDNA
library with DNA probes from the 5 and 3 ends o f the consensus cDNA resulted
in th e isolation o f a nu m b er o f d ifferent clones, together spanning 4.7 kb o f the
cDNA including a poly(A) tail indicative o f the 3 end. An overview of the most
relevant CHM cDNA clones is given in Figure 3; the sequence o f the ORF is given
in Figure 4. T he consensus cDNA contains an ORF o f 1257 bps encoding a
polypeptide o f 419 am ino acids. At the 5 end, no in-frame stopcodons have been
fo u n d indicating that the gene m ight be longer.
M erry et al. (1992) independently isolated a CHM cDNA clone from an adult
h u m an retin a library which contains a sequence similar to the above cDNA, T120,
except for the presence o f a 190 bp inversion flanked by short inverted repeats at
the 5 end. T he co rrect o rientation of the 5 sequence could be deduced from the
sequences o f the closely related m ouse CHM an d hum an CHML cDNAs (C rem ers
e ta l., 1992).
T8-
T2-
T 45 -(A)n
T110
T120 -
EcoRI
fr a g m e n ts 7 .5 4 .5 1 2 .5 2 .6 4 .0 1.0 7 .0
(kb)
C T G C T G T A T T C T C G AG G A T T A C T A A T T G A T C T T C T A A T C A A A T C T A A T G T T A G T C G A T A T G C AG A G T T T A A A A A T A T T A C C A G G A T T C T T 90
L L Y S R G L L I D L L I K S N V S R Y A E F K N I T R I L 30
><
GCATTTCGAGAAG GA CG A G TGG A A CA G GTTCC G TG TTCC AG A G CA G ATG TCTTTA ATA G CA A A CA AC TTAC TATG G TAG A A A AG CG A A TG 180
A F R E G R V E Q V P C S R A D V F N S K Q L T M V E K R M 60
><
C T A A T G A A A T TT C T T ACA TTTTG TA TGG A A TA TG A G A A A TA TC C TG A TG AA TA TAA A G G ATA TG AA G A GA TCA C ATTTTA TG A A TATTTA 270
L M K F L T F C M E Y E K Y P D E Y K G Y E E I T F Y E Y L 90
A A G A CT C A A A A A TT A A .CC C C C A A C C T C C A A T A T A T T G T C A T G C A T T C A A T T G C A A T G A C A T C A G A G A C A G C C A G C A G C A C C A T A G A T G G T 360
K T Q K L T P N L Q Y I V M H S I A M T S E T A S S T I D G 120
CTC A A A G CTA CCA A A AA CTTTCTTCACTGTCTTG G G CG G TATG G CA A CA CTCCATTTTTG TTTCCTTTA TA TG GCCA A GG A G AA CTCCCC 450
L K A T K N F L H C L G R Y G N T P F L F P L Y G Q G E L P 150
><
CAGTGTTTCTG CAG G A TG TG TG CTG TG TTTG GTG G A ATTTA TTG TCTTCG CCA TTCA GTA CA G TG CCTTG TAG TG G ACA AA G A ATCCA G A 540
Q C F C R M C A V F G G I Y C L R H S V Q C L V V D K E S R 180
A A A T G T A A A G C A A TTATA G A TC A GTTTG GTC AG A G A ATA A TCTCTG A GC A TTTCC TCG TGG A G G AC AG TTA CTTTCCTG A G AA C ATG TG C 630
K C K A I I D Q F G Q R I I S E H F L . V E D S Y F P E N M C 210
>< ><
T C A C G T G TG C A A T A C A G G C A G A T C T C C A G G G C A G T G C T G A T T A C A G A T A G A T C T G T C C T A A A A A C A G A T T C A G A T C A A C A G A T T T C C A T T 720
S R V Q Y R Q I S R A V L I T D R S V L K T D S D Q Q I S I 240
><
T T G A C A G T G CC AG CA G A G GA A CC AG G A AC TTTTG CTG TTCG G GTC ATTG A G TTA TG TTC TTCA AC G ATG A CA TG CA TGA A A GG C AC CTA T 810
L T V P A E E P G T F A V R V I E L C S S T M T C M K G T Y 270
TTG GTTCA TTTG A C TTG C A C A TC TTC TA A A A C A G C A A G A G A A G A TTTA G A A TC A G TTG TG C A G A A A TTG TTTG TTC C A TA TA C TG A A A TG 900
L V H L T C T S S K T A R E D L E S V V Q K L F V P Y T E M 300
><
G A G A T A G AA A A TG A A CA A G T A G A A AA G CC AA G A A T T C T G T G G G C T C T T T A C T T C A A T A T G A G A G A T T C G T C A G A CA T C A G C A G G A G C T G T 990
E I E N E Q V E K P R I L W A L Y F N M R D S S D I S R S C 330
><
TA TAA TG ATTTA CC ATC C A AC G TTTA TG TC TGC TC TG GC C C AG A TTGTG G TTTA G G A AA TG ATA A TG C A GTC AA A C A G G C TG AA A C A C TT 1080
Y N D L P S N V Y V C S G P D C G L G N D N A V K Q A E T L 360
TT C C A G G A A A T C T G C C C CA A TG AA G A TTTC TGTC CCC CTCC AC CA A ATC CTG AA G A CA TTATC CTTGA TG G AG A CA G TTTA C AG CC A GA G 1170
F Q E I C P N E D F C P P P P N P E D I I L D G D S L Q P E 390
G C T T C A G A A T C C A G T G C C A T A C C A G A G G C T A A C T C G G A G A C T T T C A A G G A A AG C A C A A A C C T T G G A A A C C T A G AGG A G T C C T C T G A A T A A 1260
A S E S S A I P E A N S E T F K E S T N L G N L E E S S E * 419
kb
- - 12.5
- 7.5
b
- 4.5
T1E 0.5
Figure 5 S o u th e rn blo t analysis o f EcoRI d ig ested DNAs fro m several p a tie n ts with classical
CHM . T h e cDNA p ro b e T 1E0.5 en co m p asses ex o n s A3, B l / 2 , B3, a n d B4 w hich a re lo cated
o n E coR I fra g m e n ts o f 7.5 kb, 4.5 kb, 12.5 kb, a n d 2.6 kb, respectively (F ro m C re m e r s e t a l.,
1990c, by p erm issio n o f Nature).
N o rth e rn blot analysis o f RNA from several hum an cell lines and tissues using
CHM cDNA clones revealed th at the CHM gene is no t only expressed in ocular
tissues or ocular derived cell lines, bu t also in various cells o f non-ocular origin,
e.g. HeLa, lym phoblast cells and fibroblasts (Figure 6, C rem ers et al., 1990c;
M erry et al., 1992). Yet clinical symptoms are confined to the eye. This finding is
n o t u n p reced en ted , however. Gyrate atrophy (GA), a choroidal disease with clini
cal similarity to choroiderem ia, is also caused by a deficiency o f an ubiquitously
expressed protein, the enzyme o rn ith in e am inotransferase (Valle and Simell,
1989). A part from ocular symptoms resulting from the typical sharply dem ar
cated, circular areas o f chorioretinal degeneration, m ost patients with GA are
CHOROIDEREMIA 311
asym ptom atic. Proxim al m uscle weakness which is seen in less than 10% of the
patients is the only extraocular m anifestation o f this disease (Valle an d Simell,
1989).
4"
> X*
T1 EN0.6
4 M > I
pAct 1
F ig u re 6 N o rth e rn b lo t analysis o f RNA fro m several h u m a n cell lines a n d tissues usin g cDNA
clo n e T 1 . a. B lot c o n ta in in g RNA fro m H eL a, two re tin a l cell lines (H E R RC2 a n d H E R X C 2 ),
an EBV -im m ortalized ly m p h o b lasto id cell lin e (L C L1154), as well as h u m a n c h o ro id /re tin a l
p ig m e n t e p ith e liu m a n d re tin a sc re e n e d w ith clo n e T lE N 0 .6 .b . H y b rid izatio n o f a h a m ste r
actin cD NA c lo n e (pA ct-1). T h e p o sitio n s o f th e 18S a n d 28S rib o so m al RNA b a n d s a re in d i
cate d (F ro m C re m ers e t a l., 1990c, by p e rm issio n o f Nature).
312 FRANS P. M. CREMERS AND HANS-HII.GER ROPERS
MUTATION SPECTRUM
N o rthern Analysis
SSCP Analysis
(ATG) TAA
CHM cDNA
CHM exons
2084 CC G
100 bp
1.2 -------- del(A)
I-------- 1
2.1 ----------- G T
17 .1 ------------- - C A
C O P -C -------------- C- A
C O P -D
3'ss A G
C O P -E /2 0 8 6 - - del(TGTT)
C O P -B --------------------------------------- del(TT)
10.1 3'ss G -* A
Em ploying a cDNA clone from the 5 e n d o f the CHM gene (T8; Figure 3), a
cDNA corresponding to the m urine CHM gene, mCHM, was isolated. T he
mCHM cDNA sequence is m ore com plete than the consensus hum an CHM
cDNA sequence; it contains 173 additional am ino acids at its 5 end. Overall,
mCHM an d hCHM show 88% nucleotide an d am ino acid sequence identity, illus
trating th eir high degree o f evolutionary conservation. W hen conservative am ino
acid replacem ents are disregarded, mCHM displays 97% similarity to hCHM
(C rem ers et al., 1992; Figure 8).
hC H M L M A D N L P T E F D W IIG T G L P E S IL A A A C S R S G Q R V L H ID S R S Y Y G G N W A S F S F S G L L S W L K E Y Q Q N N D IG E E S T W W Q D L IH E T E E A IT L R 90
mCHM M E Q - L - N ---------L - S 16
h CHML KKD E T IQ H T E A F S Y A S Q D M E D N V E E IG A L Q K N P S L G V S N T FT E V L D S A L P E E S Q L S Y F N S D E M PA K H T Q K SD T E IS L E V T D V E E SV E K E K 1 8 0
mCHM S K --------- V - V - C ----------- LHKD-------A ---------------- A S V M -A Q A A A E A -E A A -A T E A A E A A E A -E A A C L P T A -E S T R S C -L P A - Q S Q 1 0 4
hCHML, YCGDKTCMHT V SD K D G D K D E SK ST V E D K A D E P IR N R IT Y S Q IV K E G R R F N ID L V S K L L Y S Q G L L 1 D L L IK S D V S R Y V 2 5 7
mCHM C M -P E S S PQ V N D AE - G E K E TQ S - A K S E Q S S E I L P K - Q - N T E T -K K V --------------1 ------------------------------------- R ----------------------- N---------A 1 9 4
hCHM ------R --------------- N------A 21
hC H M L E SS C T T ID G L N A T K N F L Q C L G R F G N T P F L F P L Y G Q G E 1 PQ G F C R M C A V F G G IY CLR H K V Q CF W D K E SG R C K A 11D H F G Q R IN A K Y F I V E 4 3 7
HiCHM - T T S S - V -------K------ K ---------------- Y ---------------------------------L C -------------------------------------- S -------L --------------R K ---------V - Q ----------- I S - H - V I - 3 7 4
hCHM - T A S S ----------- K--------------H --------- Y ---------------------------------L C --------------------------------------S -------L --------------R K --------------Q ----------- I S E H - L 2 0 1
h CHML K L F T P Y T E T E IN E E E L T K P R L L W A L Y F N M R D S S G IS R S S Y N G LPSN V Y V C SG PD C G LG N EH A V K Q A E T L F Q E IF P T E E F C P P P P N P ED 1 1 6 1 7
mCHM -------------------I - A E N - Q V E -------1 ---------------------------- D ------ D C D-------------------------------N -------DN Q-------I V K - C - N - D -------A ------------------ 5 5 4
hC H M -------V ---------M E N -Q V E -------1 ---------------------------- D ---------C D---------------------------------------- DN---------------------------- C - N - D ---------------------------- 3 8 1
hC H M L F D G D D K Q P E A P G T N N W M A K L E SS E E SK N L E S PE K H L Q N 6 5 6
mCHM L -------S S Q - V S E S S V I P E T N S - T P K T V - G D S - E P S E 592
hCH M L -------S L --------- S E S S A I P E - N S - T F K T G N L - E S S E 419
chrom osom es. These studies showed th at the hCHM L gene resides on chrom o
some 1. In addition, hCHM L subclones were hybridized to S outhern blots with
DNAs from three somatic cell hybrids carrying defined segm ents o f chrom osom e
I. In this way, hCHM L could be assigned to lq42-qter (C rem ers et al., 1992;
F.P.M.C., unpublished data). Recently, the gene for an o th er form o f retina degen
eration, U sher syndrom e type 2 (U SH 2), has been localized in approxim ately the
same chrom osom al interval by linkage analysis (K im berling et al., 1990; Lewis
et al., 1990). U sher syndrom e consists o f retinitis pigm entosa with hearing loss and
is in h erited as an autosom al recessive condition (C hapter x x ). T he chrom osom al
region containing the USH2 locus a n d hCHM L is estim ated to com prise no m ore
than 2% o f the hum an genom e. This co-localization, together with the clinical
similarities betw een CHM an d USH2, renders hCHM L a prom ising candidate
gene for this disorder. M ore accurate physical m apping o f hCHM L and closely
linked flanking m arkers o f USH2, as well as hCHM L segregation studies in USH2
families, are n ee d ed to substantiate this hypothesis. Final p ro o f for the identity of
hCHM L a n d USH2 may com e from the identification o f m utations in the hCHML
gene in patients with the USH2 phenotype. It is o f note, however, that so far,
screening o f 10 patients with USH2 has failed to detect gross rearrangem ents or
point m utations in the hCHM L gene (T. J. R. van de Pol et al., u n p ublished d a ta ).
sequence hom ology may indicate that these proteins have sim ilar binding do
m ains for this small G TP-binding protein.
Assuming that CHM and Rab GGT are identical, there are several rem aining
questions related to the pathogenesis o f choroiderem ia, as follows.
1. Both CHM (Rab GGT) and Rab3A are expressed in a variety o f tissues, includ
ing brain. Why then, is the defect lim ited to the choroid and retina?
2. W hat is the role of the CHML gene product? Does it geranylgeranylate the
same or a n o th er class o f small GTP binding proteins? In view o f its high de
gree of similarity with CHM, is it possible that CHML expression m odulates
the CHM phenotype? Is there a CHML hom ologue in mice and rats?
3. Rab3A is tho u g h t to regulate vesicle tran sp o rt in brain synapses (Fischer von
M ollard et al., 1990; Mizoguchi et al., 1990). C ould Rab3A or a related gera-
nylgeranylated GTP binding protein be involved in m em brane turnover in
ro d o u ter segments? In accordance with this hypothesis, the fundus ab n o rm al
ities observed in early stages o f the disease suggest that lesions first occur in or
n ea r the retinal pigm ent epithelium (W aardenburg, 1942; Pam eijer et al.,
1960; M cCulloch, 1969; H am m erstein et al., 1979; H am m erstein and Bohm,
1985).
F uture studies will concentrate on the identification of Rab G G T/C H M gera-
nylgeranylated small GTP binding proteins in the retina, RPE, an d choroid. The
subcellular expression o f these m olecules as well as the CHM gene p ro d u ct will be
studied by im m unohistochem ical techniques. Also, a b etter u n d erstan d in g of the
pathological processes in choroiderem ia may be achieved by the construction of
a m ouse m odel via insertional inactivation o f the m ouse CHM gene. Eventually,
com plete un d erstan d in g of the pathological processes underlying choroiderem ia
may provide a basis for rational therapy.
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13. NORRIE DISEASE
CLINICAL FEATURES
N orrie disease (NDP) was d elineated by W arburg (W arburg, 1961; 1966) who
subsequently fo u n d th at patients with this disorder had been m entioned by the
Danish ophthalm ologist to the Royal Institute for the Blind, G ordon N orrie
(N orrie, 1927). As N orrie was the first to recognise the genetic origin o f the dis
order, W arburg decided to nam e the disease after him . T he condition has been
observed in all ethnic groups. Since the disease is X-linked it is m anifest in boys
only. Two affected fem ales have b een observed (I. Craig, personal com m unica
tion). It is probably the m ost com m on disorder with bilateral retrolental vascular
masses.
Figure 2 E lo n g ate d ciliary p rocesses (arro w ), w hich can be seen b e h in d a cle a r len s (L ).
NORRIE DISEASE 323
Histology
Sometim es the retrolental masses are m istaken for retinoblastom a and the eye is
enucleated. Histological exam ination shows that the detached retina contains
m alform ative rosettes, m any large retinal vessels, haem orrhage, gliosis, and optic
atrophy. Hyperplasia of the retinal pigm ent epithelium was also observed
(W arburg, 1966; A ndersen and W arburg, 1961; L iberfarb et al., 1985; Blodi and
H unter, 1969). In ultram icroscopical sections (Liberfarb et al., 1985) the in n er
and o u ter neuroblastic cells are undifferentiated.
Systemic signs
T he patients are usually healthy an d have a norm al life span. Many are very intel
ligent; in m ost cases procreation is norm al. D uring their lifetime, about one third
of the patients develop progressive neurosensory hearing im pairm ent. This may
begin in childhood o r as late as in the 6th decade. O ne q u arter o f those affected
show dem entia or autistic signs which may develop at any time between the age of
2 and 60 years. In som e cases the psychiatric im pairm ent may be a com plication
o f the com bined visual and auditory com m unicative disorder, while in others the
blind m en an d boys show autistic signs even when hearing is still norm al. In a few
324 A. A. B. BERGEN' ET AL
families, the affected blind males had systemic m alform ations, seizures, and
severe m ental retardation (Bleeker-W agemakers et al., 1988; De la C hapelle et al.,
1985; D onnai et al., 1988; Zhu et al., 1989). Microcephaly, large ears, p ro m in en t
nasal bridge, and thin u p p er lips have been described (D onnai e ta l., 1988). O ne
p atien t (Zhu et al., 1989) was autistic and self abusive, he had growth failure,
abnorm al sexual m aturation, hypotension, paroxysmal irregularity o f respiration,
flushing, atonic seizures, m yotonic contractions and hyper-reflexia w ithout spas
tic paresis. H e was unable to talk and walk. A D utch p atien t (Bleeker-W agem ak-
ers et al., 1988) had cutaneous hyperkeratosis and microcephaly, cryptorchidism
an d penis hypoplasia. Brain stem audiography showed sensory hearing loss. A
Danish patient recently re p o rted was mildly m entally re ta rd ed an d lived in an
o p en institution. His only extraocular m alform ations were m icropenis and cryp
torchidism . He was childish and had very little confidence in the people aro u n d
him o r in himself. In these atypical patients, deletions or o th er rearrangem ents
involving the NDP locus were d em onstrated by m olecular genetic exam ination
(B erger et al., 1992a; C hen et al., 1992b). It is conceivable th at th eir com plex
p henotype is due to the involvem ent o f o th e r genes which have n o t yet been
identified. An X /autosom al translocation was also found in some o f the N orrie
p atients (O hba and Yamashita, 1986; M cM ahan e ta l., 1991).
C arrier phenotype
In general, fem ale carriers are com pletely healthy, O phthalm ological an d audio-
logical exam inations are usually norm al (W arburg, 1966; 1975; Parving and
Schwartz, 1991) and both carriers of deletions and o f o th er m utations have had
children. Two fem ale carriers m anifesting signs o f the disease have been
observed. For one of these cases evidence has been fo u n d supporting a m odest
inactivation bias (70:30) in favour of expression of the defective allele (I. Craig,
personal com m unication).
Pathogenesis
U ltrastructural studies (Enyedi et al., 1991) showed that the in n er and o u ter neu-
roblastic layers of the retina were u n differentiated. This may explain the lack of
coaption o f the retinal pigm ent epithelium and the n euroretina, leading to con
genital detachm ent. Most o f the clinical signs are secondary, nam ely the persis
tence o f the prim ary vitreous, the elongation o f the ciliary processes, the cataract
an d the shrinkage with resulting corneal opacities. T he progressive hearing
im pairm ent an d the freq u en t autistic signs may be in terp re ted as a progressive
neurological disorder o f unknow n character.
D ifferential diagnosis
Diagnosis is sim ple if there are o th er affected males in the family. W hen only a
single person is affected, the diagnosis can only be tentatively established. The
NORRIE DISEASE 325
differential diagnosis depends on the time when the patient is first observed. In
the neonate, it includes retinoblastom a, retinopathy of prem aturity, persistent
hyperplastic prim ary vitreous (PHPV), oculo-palato-cerebral dwarfism (Frydm an
et al., 1985), the osteoporosis-pseudogliom a syndrom e (Saraux an d Frezal, 1967),
X-linked prim ary retinal dysplasia (G odel and G oodm an, 1981) which is probably
identical with X-linked familial exudative vitreo-retinopathy, trisomy 13, and
incontinentia pigm enti. In this respect, it is of particular interest to note th at the
locus for X-linked exudative vitreo-retinopathy is localized to the same Xp region
as NDP, and, recently, a m utation in the NDP gene has been found in affected
m em bers o f an X-linked exudative vitreo-retinopathy family (C hen et al., in
p ress).
T he patients with NDP are usually b o rn at term and n eed no oxygen, which
excludes retinopathy o f prem aturity. T h eir disorder is bilateral while PHPV is
unilateral, except in the autosom al recessive oculo-palato-cerebral dwarfism char
acterised by m icrocephaly, hypotonia, bilateral leukocornia, an d cleft lip-palate.
X-linked retinal dysplasia and X-linked falciform folds m anifest as a falciform
retinal fold, and carriers of X-linked retinal dysplasia may show p eripheral folds;
in NDP the affecteds are m ale - n o t fem ales as in incontinentia pigm enti. In chil
dren, the differential diagnosis com prises infections, X-linked congenital cataract,
X-linked m icrophthalm os and autosom al d om inant familial exudative vitreoretin-
opathy (FEVR). C ataract an d small eyes are n o t congenital in NDP, and FEVR is
rare in children, in addition to which one of the relatives may show the retinal
vascular anom alies o f FEVR (von Nouhuys, 1991). In adults, the differential diag
nosis com prises xerophthalm ia an d m icrophthalm os o r phthisis due to a different
aetiology.
Clearly, m olecular genetic exam ination will soon facilitate the differential diag
nosis.
M ore than 300 m ale patients have b een described worldwide, (W arburg, 1961;
1966; 1975; Blodi et al., 1969; N ance et al., 1969; Lomickova an d Raska, 1969; Mor-
eira-F ilho an d N eustein, 1979; B leeker-W agem akers 1981; Jo h n sto n et al., 1982;
Bleeker-W agem akers et al., 1985; L iberfarb et al., 1985; Phillips et al., 1986; Hill
et al., 1987; Kivlin et al., 1987; Esakowitz et al., 1988; Gal et al., 1988; H arenda de
Silva et al., 1988; Curtis et al., 1989; N adol et al., 1990). P enetrance was com plete,
b u t expression was variable except for congenital blindness which is always
present. T he incidence is no t known. Phillips (Phillips et al., 1986) calculated a
m utation rate o f 3.9 x 10'6 in the Scottish population, but ascertainm ent was p ro b
ably n o t com plete.
326 A. A. B. BERGEN ET AL.
Linkage
As early as 1965, W arburg em ployed the classical X chrom osom al Xg blood group
m arker in an attem p t to establish linkage with NDP (W arburg, 1965). No linkage
could be found between NDP and the Xg blood group system. T he same results
were re p o rted within a Brazilian N egro sibship (M oreira-Filho a n d Neustein,
1979). A loose a n d unreliable linkage was fo u n d betw een NDP an d the glucose-6-
pho sphate dehydrogenase m arker in an A m erican N egro family (Nance et al.,
1969). In D utch and Danish families, Gal et al. (1985a) n o ted close linkage o f
NDP with an anonym ous RFLP m arker, L I .28 (DXS7) (Bakker et al., 1983) from
the X p ll.4 - p ll.3 region. After this rep o rt, extensive linkage studies were per
fo rm ed on N orrie families from all over the w orld by several research groups.
T h e results from these studies clearly assigned NDP to the proxim al Xp n ear
o th er regionally assigned eye disease loci (I *ure 4). T he accum ulated LOD
scores are sum m arized in Table 1. A recom bination between NDP and DXS7 was
re p o rted in only one family (Ngo et al., 1988). S ubsequent analysis o f the same
family with an RFLP detected by a hum an o rn ith in e-8 -am in o tran sferase (OAT1)
cDNA probe (Ngo et al., 1989, L afreniere et al., 1991b), a polym orphic CA-repeat
at the 5'-end o f the MAOB gene (Sims et al., 1992) and the DXS426 CA-repeat
(Lindsay et al., 1992) suggested the genetic order: Xpter-(DXS7,MAOB)-NDP-
(DXS426,OATL)-Xcen (Figure 5).
Cytogenetics
T he first physical confirm ation for the localization o f NDP in b an d X p l 1.4 on the
sh o rt arm o f the X chrom osom e came from a pap er by O h b a and Yamashita in
which they described a fem ale infant NDP p atien t carrying a balanced X;10 trans
location (O hba an d Yamashita, 1986). T he expression o f the m utated gene in
this fem ale could be explained by the fact th at in an X-autosome translocation
th e translocated X chrom osom e is preferentially activated (T herm an et al.,
1974). M ore recently, a second X-chrom osom al ab erratio n associated with NDP
NORRIE DISEASE 321
Xp2i .1 Xpter
RP3
[
ND X p 1 1.4
CSNBX
RP2 X p 1 1.3
WAS
IP
Cen
was published in which a familial pericentric inversion (X) (p ll.4 -q 2 2 ) was found
in males affected with NDP. Fem ale carriers o f this inversion showed no clinical
symptoms o f NDP (M cM ahan e ta l., 1991).
Im p o rtan t progress towards the m apping an d isolation of the NDP gene came
from the cloning and ord erin g o f genes and anonym ous DNA probes to defined
X -chrom osom al intervals. O ne o f the first m arkers, L I.28 (DXS7) (Bakker et al.,
1983), was assigned to the proxim al Xp by linkage analysis in D uchenne M uscular
Dystrophy families (W ieacker et al., 1984). Subsequently, a large n u m b er o f m ark
ers and genes have been localized to the proxim al Xp region using linkage analy
sis, deletion m apping, in situ hybridization an d by m apping probes in hum an
ham ster cell hybrids containing parts o f the hum an X chrom osom e (Lafreniere
e ta l., 1991; Davies et al., 1991) (Figure 4).
328 A. A. B. BEROEV F.T Al
L I.2 8 MAOB
ploA6 pX59 MAOA de l 2 cpXr31 8
tel cen
----- 1 J ----------------- 1
-------
-^ 1
Deletion studies
In 1985, two research groups sim ultaneously re p o rted physical evidence for close
proxim ity of the NDP gene to the DXS7 locus (Gal et al., 1985b; 1986; de la
C hapelle et al., 1985). In each case the DXS7 locus was found to be deleted, in a
D utch an d in a Finnish m ale with atypical features of NDP respectively. Co-dele
tion o f the N orrie gene to g eth er with the DXS7 locus could explain the NDP p h e
notype of these patients. T he physical length o f the deletions was n o t known, but
as no changes could be found in high resolution chrom osom e banding experi
m ents, the deletion in the D utch patient could n o t be m uch larger than about 3
m illion bp (Gal e ta l., 1986).
NORRIE DISEASE 329
After the discovery of the first subm icroscopic X p-deletions associated with NDP,
inform ation on the relative gene m arker o rd e r in the proxim ity of NDP came
from yet an o th er source: long range restriction analysis.
D iergaarde etal., (1989) assigned the probes plA a6 (DXS228), pX59 (DXS77),
L I.28 (DXS7) and M AOA/B to a region o f 1500 kb, as they all hybridized to an
identical BssHII restriction fragm ent o f this size. In the same study, the distal
b reakpoint of the D utch deletion (Gal et al., 1985b) was m apped within a 400 kb
Sail fragm ent shared by pX59 (DXS77) an d L I.28 (DXS7), but which could not
be identified with M AOA/B. F u rth erm o re, with different com binations o f single
and double digests using a set of restriction enzymes, the physical o rd e r DXS228-
DXS77-DXS7-(MAOA/B, NDP) was suggested (D iergaarde et al., 1989). Refine
m en t and extension o f this m ap cam e from physical m apping experim ents using
YACs corresponding to the L I.28 a n d MAOA/MAOB loci (C hen et al., 1992a,
Sims et al., 1992), and from genom ic pulsed-field gel electrophoresis (PFG) stud
ies with a novel probe, cpXr318 (DX S742), which is localized ju st proxim al to the
N orrie disease locus (Bergen et al., 1993). The resulting o rd e r o f m arkers and
physical distances betw een them were in general agreem ent with the data ob
tained from deletion studies (Figure 5).
330 A. A. B. BERGEN ET AL.
Xcen Xpter
G8------------------------------------------------------------ -V -
M8---------
Al0-*~ ---- --- - ------------------------
kbp j 3.7 12.5| 9.5 J 5.4 j2jlj 10 J 4 (1.3J
F ig u re 6 T h e ex o n -in tro n -stru ctu re o f th e N o rrie disease g e n e as p u b lish e d elsew here (B erg
e r e t al., 1992c; M ein d l e t al., 1992). T h e co sm id clo n es u sed fo r se q u e n c e analysis are given
by th e co d es M 8 , G8 a n d A10. EcoRI re stric tio n sites a re in d ic a te d by arrow s a n d fra g m e n t
sizes in kb. O p e n arrow s in d ic a te x o n c o n ta in in g g en o m ic frag m en ts. T h e p ro te in co d in g
p a rt o f th e e x o n s is given as h a tc h e d boxes, th e p ro m o te r re g io n as d o tte d , a n d th e 3' a n d 5'
flan k in g re g io n s as o p e n boxes. P ositions o f th e TATA box, th e start a n d sto p c o d o n as well
as th e p o ly ad en y latio n signal c o rre sp o n d to seq u e n c e s p u b lish e d e a rlie r (B e re e r e t al., 1992a;
M ein d l e ta l., 1992).
B E EB EBBEB B B E EBE
U III I
exon 1
1 ... S &=
Cen Tel
13545
8838
3883
6446 a H M H I
12316
GTTAGTATTT
Figure 7 (b)
lated. The ATG start codon lies within exon 2, and the stop codon as well as the
polyadenylation signal are located in exon 3 (Figures 6 an d 7). T he open reading
fram e of 399 bp encoding a p rotein of 133 am ino acids, is com pletely contained
within exons 2 an d 3. W ithin the first intron, a canonical TATAAAT sequence was
identified 185 bp 5' to the start o f the second exon. This structural organisation
would allow an alternative start of transcription, suggesting that expression o f the
NDP een e may be reeulated by the activity of two in d ep en d e n t p ro m o to r regions
(C hen e ta l., 1993).
C onvincing evidence for the involvem ent of the isolated candidate gene in the
etiology o f N orrie disease has com e from th e detection o f point m utations in
patients. Berger et al., (1992b) described the identification o f small sequence
alterations in 10 out of 12 non-deletion patients with classical NDP. F urtherm ore,
m utation analysis established the diagnosis in 2 out o f 5 patients, in whom the
clinical diagnosis o f NDP was suspected. Sim ilar results were re p o rted by M eindl
et al., (1992) and C hen et al., (1993). Most o f the m utations described so far give
NORRIE DISEASE 333
An extensive DNA an d p rotein sequence com parison betw een the hum an NDP
gene and related genes from the NBRF and Swiss protein database revealed sig
nificant hom ology with C -term inal cysteine-rich dom ains shared by a family of
mucin-like proteins an d von W illebrand factor (M eindl et al., 1992; C hen et al.,
1993). Many m ucins are secreted proteins and some are integral m em brane p ro
teins an ch o red at a m ucin-like, glycosylated dom ain. No glycosylation sites are
identified in the NDP gene product. However, an N -term inus hydrophobic region
of the NDP p ro d u ct contains features o f a signal sequence containing a basic
region, a hydrophobic "core" an d a m ore polar region (C hen et al., 1993). A weak
hom ology between this N -term inal dom ain and the rat-horsefall protein supports
the secretory status of the NDP gene p ro d u c t (M eindl et al., 1992). Prediction of
signal sequence cleavage sites based on the m eth o d o f G. von H eijne (1986)
defined a signal peptide o f 24 am ino acid residues with a 0.744 probability
(B erger et al., unpublished data). T he same region also exhibits hom ology with
two im m ediate early genes; the p h o rb o l ester-repressive an d v-src-inducible Cyr61
gene identified in m urine fibroblast (M eindl et al., 1992; C hen et al., 1993).
334 A. A. B. BERGEN ET AL.
Based on the observed hom ologies, it can be postulated th at the NDP gene is
involved in a pathway that regulates neural cell differentiation and proliferation.
Also, because o f the potential o f cysteine rich regions to form disulphide bonds,
it can be postulated th at the function o f the protein may concern interaction with
o th er growth factors im p o rtan t in neurological developm ent.
O utlook
T he precise function of the NDP gene rem ains to be established. D irect inform a
tion on the role, tissue specificity and localization of the protein can com e from
studies using specific antibodies raised against the in vitro synthesized gene p ro d
u ct (M arston, 1986). T he isolation o f the m ouse gene and the construction of
transgenic m ice or o th er anim al m odels should provide a m ore detailed insight
into the function o f the N orrie disease gene.
At present, direct m utational analysis for coding changes in the second and the
th ird exons is possible, while SSCP (O rita et al., 1989) will assist in rapid screening
for additional lesions outside these regions.
T he occurrence o f small DNA deletions in 20% o f the NDP patients an d the
identification of p o in t m utations in 73% o f the cases offers the possibility o f reli
able DNA diagnosis in 93% o f all cases (B erger et al., unpublished results).
Finally, it is encouraging to note that the small size and the secretory status of
th e N orrie gene translation product raises perhaps the possibility o f therapeutic
approaches in som e instances.
ACKNOWLEDGEMENTS
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L a u d e r J. M. (1985) R oles fo r n e u ro tra n s n ritte rs in d e v e lo p m e n t: p o ssib le in te ra c tio n w ith d ru g s
d u rin g fetal a n d n e o n a ta l p e rio d s. Prog Clin Biol Res 163, 3 7 5-380.
L ib e rfa rb R. M., Eavey R. D., D e L o n g G. R., A lb e rt D. M., D ie c k e rtJ . P., H iro se T. (1985) N o rrie
disease: a study o f two fam ilies. Ophthalmology 92, 1445-1451.
L indsay S., T h is e lto n D. L., B a te m an J. B., N g o J . T., S p ark es R. S., C o le m a n M., Davies K. E. a n d
B h a tta c h a ry a SS (1992) L o calisatio n o f th e g e n e fo r N o rrie disease to b e tw ee n DXS7 a n d
D X S426 o n X p. H uman Genetics 88: 3 4 9-350.
L om ickova H ., R aska B. (1969) La m ala d ie d e N o rrie . Medecine et Hygiene 27, 1168-1169.
NORRIE DISEASE 337
W arb u rg M. (1966) N o rrie 's disease: a c o n g e n ita l progressive o c u lo -a co u stic o -ce rcb ra l d e g e n e r
a tio n . Acta Ophthalmology (C o p e n h ) 89 [S uppl] ,1-147.
W arb u rg M. (1975) N o rrie 's disease. D iffere n tia l d iag n o sis a n d tre a tm e n t. Acta Ophthalmology
(C o p e n h ) 53, 21 7 -2 3 6 .
W ie ac k e r H . F., Davies K., C o o k e H . J., P e a rso n P. L., W illiam son R., B h a tta c h a ry a S., a n d R o p e rs
H . H . (1984) T ow ard a c o m p le te lin k a g e m a p o f th e h u m a n X c h ro m o s o m e : re g io n a l assign
m e n t o f 16 c lo n e d single copy DN A se q u e n c e s e m p lo y in g a p a n e l o f som atic cell hybrids.
American journal o f H uman Genetics 36: 2 6 5 -2 7 6 .
Z h u D. P., A n to n a ra k is S. E., S c h m e c k p e p e r B. J., D ie rg a a rd e P. J., G re b A. E., M a u m e n e e I. H .
(1989) M ic ro d e le tio n in th e X -c h ro m o so m e a n d p re n a ta l d iag n o sis in a fam ily w ith N o rrie 's
disease. American Journal o f Medical Genetics 33, 4 8 5 -4 8 8 .
14. X-LINKED JUVENILE RETINOSCHISIS
INTRODUCTION
Retinoschisis is a descriptive term indicating a splitting o f the retin a into two lay
ers an d is often subdivided into congenital and acquired forms.
T he differential diagnosis o f congenital retinoschisis includes a diverse g roup of
disorders. Gorlin an d K nobloch (1972) distinguished 6 different genetic syn
drom es in which juvenile retinal d etach m en t occurs, including juvenile retin
oschisis. In m ost textbooks, the lists o f conditions to be considered in patients with
retinal d etach m en t are long. Two m ain developm ents now m ake fu rth e r subdivi
sions of the retinoschisis syndrom es easier than before. First, im proved clinical
m ethods allow distinct syndrom es to be delineated. Second, the genetic m apping
o f several congenital eye disorders provides a m eans of genetic classification.
T he subject o f this review is X-linked juvenile retinoschisis (MIM 312700, McK
usick, 1990), a disease th at belongs to the vitreoretinal dystrophies. We shall use
the designation RS ad o p ted by the H um an G ene M apping W orkshops (McAlpine
et al., 1991). A ccording to this classification RS indicates the gene an d RS the dis
ease phenotype; for simplicity this distinction is n o t always necessary. While the
first reports suggesting th at RS may reside in the distal p art of the short arm o f the
X chrom osom e appeared over 20 years ago (Eriksson et al., 1969; Race and
Sanger, 1975) its m ore precise localization has been accom plished in the last 5-10
years. This has paved the way for detailed linkage studies, carrier detection, and
pre- an d postnatal diagnostics w ithin families. Thus the concept of a disorder well
defined both by clinical and genetic criteria is em erging.
EPIDEMIOLOGY
nosed in Finland (over 300 today) the co n cep t em erged that RS is m ore preva
len t in F inland than elsewhere in the w orld (Forsius an d Eriksson, 1980; Norio,
1981).
T he idea o f an uneven geographic o r ethnic distribution now needs to be re-ex
am ined. In the past, case re p o rtin g has do m in ated the literature on RS m aking it
difficult to assess its frequency an d distribution. However, it is obvious th at RS oc
curs in m ost parts o f the world and in d ifferen t ethnic groups such as Am erican
Blacks (C onstantaras et al., 1972; Ide an d Wilson, 1973), Chinese (Zhang, 1984),
Jap anese (Kawano e ta l., 1981; M inato, 1991), Indonesians (D eutm an, 1971) and
East E uropeans (Janaky an d B enedek, 1990; Nizankowska and Wozny, 1991).
PREVALENCE
pigm entosa was 1 in 4,225 and for choroiderem ia 1 in 177,454. Even though the
authors attem pted to correct for incom plete ascertainm ent, the above figures
should be m inim um estimates.
In a sim ilar study in central Sweden, Skoog et al. (1990) en c o u n tered 9 patients
with RS am ong a total o f 487 patients referred to an opthalm ological centre for
suspected or obvious hereditary degenerative o r dystrophic eye disorders. While
no estim ates of prevalence were offered, the same series com prised 38 patients
with retinitis pigm entosa and 2 patients with choroiderem ia, respectively. These
data are com patible with the data from n o rth e rn France an d indicate that RS in
d eed accounts for a relatively large p ro p o rtio n o f these disorders. Taken together
with the Finnish prevalence figure o f 1 in 17,000 (u n corrected for incom plete as
certainm ent) we tentatively conclude that the prevalence o f RS is between 1 in
15,000 an d 1 in 30,000, o r even higher. At face value this m eans, for instance, that
there are over 30,000 RS patients in E urope an d over 300,000 in the world. T he
condition is certainly underd iag n o sed for many reasons, including the m ildness
o f symptoms in some cases, an d the difficulty ophthalm ologists have in m aking the
diagnosis. In the study by Skoog et al. (1990) for instance, only 4 o f the 9 RS pa
tients had been correctly diagnosed by the referring ophthalm ologists.
CLINICAL FEATURES
Pathogenesis
Anatomical features
In m ild cases com prising 60% of all patients changes occur in the m acular area
only. In this site a wheel-like cystic form ation about the size o f the optic disc is
seen (Figure 1). T he changes at the m acula are probably congenital but are diffi
cult to differentiate from their surroundings before the age of about 2 years.
342 ALBERT DE IA CHAPELLE, ET AL.
A radiation of superficial retinal tissue is som etim es seen outside the cystic area
in young subjects. A round the age of 40, the highly typical an d readily distinguish
able central schisis flattens out, atrophies an d becom es m ore difficult to recogn
ise. In aged patients, the pigm ent layer disappears so that the disease can no
lo n g er be differentiated from circum scribed m acular d egeneration (Figure 2). In
ad d ition to the m acular changes, m oderately severe cases show sem itranslucent
vitreous veils in the lower tem poral perip h ery (Figure 3). These veils consist of d e
tached portions o f the superficial layer o f the retina, and contain retinal blood ves
sels. O ften a dem arcation line is seen at the b o rd e r o f the schisis (Figure 4),
although this may also occur in retinal detach m en t unassociated with schisis. The
X-LINKED JUVENILE RETINOSCHISIS 343
veils can rem ain un ch an g ed for decades. Parts o f them may float freely in the
deg en erated vitreous body, bu t as a rule they ap p ear elevated from the ocular
fundus and form c o h e ren t sheets which seem to start from apparently norm al
retinal tissue close to the optic disc. In the periphery, the veil again has contact
with the fundus. In young subjects up to a few years o f age, the veil can form a
large cyst in which holes gradually appear, and the cyst collapses. In adults, the
holes often increase in size and finally only rem nants of the veil aro u n d blood
vessels may be seen. Sorsby et al. (1951) were the first to describe this p h en o m e
non. In severe cases, the veil may form a roll below the optic disc. Increasing atro
phy including degeneration o f the pigm ent layer occurs in the attached p art of
the retina. Up to m iddle age, garland-like glistening form ations nearly always
344 ALBERT DE LA CHAPELLE, ET AL.
o ccur in the perip h ery of the fundus, which otherw ise appears norm al. D rusen of
the optic disc have been described, as well as whitish streaks along the blood ves
sels and pigm ented areas; in extrem e cases, the entire retina an d choroid are dis
organised. Patients with these features can be totally blind from birth.
Some investigators have re p o rted follow-up observations of several years' d u ra
tion beginning in patients at the age o f a few m onths. Forsius et al. (1990) followed
40 patients for an average o f 22 years. An initial high bullous detachm ent is fol
lowed by hole form ation in the wall, after which the balloon-shaped detached ret
ina flattens out. Conversely, even in affected individuals as old as 50-60 years,
retinoschisis w ithout holes has been observed by some investigators.
In young subjects, visual acuity can be nearly norm al. Typically, the disease is no t
d etected until the first school year when a vision o f 0.5 to 0.7 (2 0 /4 0 -2 0 /3 0 ) is
found. However, occasionally the acuity can be very low at this stage. Am ong our
own 300 cases, two boys were nearly totally blind from birth.
X-LINKED JUVENILE RETINOSCHISIS 345
Colour vision
Com plications
B leeding in the retina an d vitreous body have been re p o rted in up to 25% of all
cases. These often constitute the cause o f the first consultation with an o p h th al
m ologist. In large series studied as p art o f family investigations, the incidence is
m uch lower, e.g. 4.7% according to K ellner et al. (1990). In our series o f 300
mostly familial cases, the figure is less than 10%. H aem orrhages occur both in the
cavity o f the schisis and in the vitreous body an d are probably caused by increased
splitting o f the ablated layer. In advanced cases, neovascularisation has been fre
quently observed in the p erip h ery bu t can also occur in the optic nerve (Pearson
a n d ja g g e r, 1989). Blood vessels can be seen in both the ablated layer and in the
p art o f the retin a th at rem ains attached to the fundus. T he bleeding is mostly
quickly resorbed b u t som etim es requires vitreous surgery (Greven et al., 1990).
T rue, full thickness retinal d etach m en t is re p o rted at highly varying frequencies
u p to 20% d ep en d in g on w hether the series em anate from hospital reports or
from studies on families. For instance, in family studies D eutm an (1971) found
retinal d etach m en t to be rare an d we have observed only 5 cases ou t o f 300. In
these cases a hole has ap peared even in the in n er retinal layer. T reatm ent is the
same as for regular retinal detachm ents a n d the results are acceptable.
T herapy
Differential diagnosis
Phenotypes o f heterozygotes
Most authors hold that even careful ophthalm oscopy o f the central and perip h
eral retin a does n o t disclose changes in RS gene carriers that can be distin
guished from those seen in norm al w om en of the same age (e.g. V ainio-M attila
et al., 1969; D eutm an, 1971). However, occasional reports o f pathological foveal
348 ALBERT DE LA CHAPELLE, ET AL.
findings such as m ottling and gliosis in the p erip h ery have appeared (Ewing and
Ives, 1969; Kaplan et al., 1991). In several carrier wom en, a consistent distur
bance o f retinal function was detected by A rden et al. (1988), who exam ined the
relationship o f cone flicker threshold to changing light intensities o f the back
g ro u n d at a single retinal site.
It may in d eed be th at some heterozygous wom en can display phenotypic effects
o f the gene m utation they carry. However, these changes are n eith er typical
en o ugh n o r do they occur in a sufficient p ro p o rtio n o f carriers so as to provide
reliable carrier detection by conventional clinical investigation.
A small nu m b er o f affected fem ales have been described (Forsius et al., 1962;
U chino and Shimizu, 1976; H am aguchi et al., 1989). These are wom en who are
hom ozygous as a result o f the m ating o f an affected m ale and a heterozygous fe
male. As expected, their phenotype resem bles th at o f affected males. T he same
was tru e o f a 45,X fem ale with RS (H om m ura et al., 1982).
MOLECULAR STUDIES
T he first attem pts to m ap the RS gene were m ade by typing RS families for the Xg
blood group. These linkage studies suggested that RS m ight be located at a m ea
surable distance from the Xg locus (Eriksson et al., 1969; Ives et al., 1970; Race
an d Sanger, 1975; Boman et al., 1976; Forsius and Eriksson, 1980). Subsequently
W ieacker et al. (1983) used a polym orphic DNA m arker (RC8) to study two RS
families which had also been typed for Xg. T h eir linkage results with RC8 (Zmax
= 1.74 at max = 0.15) gave additional support for the localisation of the RS gene
on the distal part o f Xp, although no definite location could be ascertained. O ne
o f the two RS families was fu rth e r analyzed with additional m arkers by Gal et al.
(1985). These prelim inary two-point linkage studies, re p o rted in an abstract, sug
gested that RS is also linked to DXS43, DXS16, an d DXS85.
All two-point linkage studies betw een RS and anonym ous RFLPs published to
date (with m axim um lod score values >3) are in agreem ent that RS is closely linked
to m arkers DXS16, DXS43, DXS207, DXS9, DXS274, and DXS41 on Xp22T-p22.2
(Table 1). With the exception o f one family in the initial study by W ieacker et al.,
(1983), the m arker locus DXS9 has shown no recom binations with RS in the fam
ilies studied (Alitalo et al., 1988; G ellert et al., 1988; Sieving et al., 1990). In addi
tion, no recom binations have been found between RS and DXS197 (Zmax = 4.82
at 0 max = 0.00; Alitalo et al., 1991a). T he results o f one study ap p ear divergent,
b u t all the lod scores re p o rted are sm aller than 3 (G ellert et al., 1988). W hether
the families described by W ieacker et al. (1983) and G ellert et al. (1988) rep resen t
d ifferent eye disorders, genetic heterogeneity in RS, o r indicate greater distances
betw een RS and the m arkers DXS9, DXS16, DXS43, and DXS41 than is suggested
by the o th er studies, rem ains to be resolved. Incidentally, when com paring the re
sults of various two-point linkage studies, it is good to rem em ber th at although sig
nificant lod scores can be o btained between various pairs of m arkers by two-point
X-LINKED JUVENILE RETINOSCHISIS 349
linkage analysis, the confidence intervals ten d to be wide (e.g. Alitalo et al.,
1991b). Thus, while two-point linkage analysis is a good m ethod for establishing
linkage, the genetic distances o b tained should be in terp re ted with caution. This
applies especially w hen m axim um lod score values are close to or sm aller than 3
an d the n u m b er o f inform ative m eioses is small.
7 A
Table 1 Two-point linkage data ( max at max) between RS and six markers
*1. Alitalio e ta l., 1987, 1988, 1991a; 2. Dahl e ta l., 1988,3. G ellert et al., 1988; 4. Kaplan et al., 1991; 5.
Sieving e t al., 1990
N A =not available
cM Locus
location over the second best location (Figure 6 and Table 2). It has b een estim at
ed that the genetic distance between the flanking m arkers is 7 cM and th at the RS
gene is located 2 cM proxim al to (DXS43,DXS207). T he exact o rd e r of DXS9 and
DXS197 relative to RS o r the m arkers DXS43 and DXS207 has n o t been deter
m ined due to lack of recom binations.
Diagnostic procedures
Based on all available linkage data, it is now possible to predict which m em bers of
RS families are carrier fem ales and to diagnose the syndrom e pre- an d post-
natally. Since close linkage (with Zmax > 3) betw een RS and m arkers in the
Xp22.1-p22.2 region has been observed in all families rep o rted , there is no
strong evidence for genetic heterogeneity o f RS. T he closest distal m arkers
DXS43 and DXS207 are about 2 cM from the RS gene, and the closest proxim al
X-LINKED JUVENILE RETINOSCHISIS 351
DXS207 DXS92
Locus DXS 16 DXS43 RS DXS274 DXS41
Xpter Xcen
m arker DXS274 is about 5 cM from the gene (Alitalo et al., 1991a). Because it is
known that linked m arkers flanking the disease locus provide m uch m ore diag
nostic reliability than linked b u t non-flanking m arkers, we have used these three
flanking m arkers in carrier an d prenatal diagnosis. In a few cases it has also been
necessary to screen the families with the m arkers DXS16 and DXS41, because the
diagnostic utility o f linked RFLPs is som etim es lim ited by a lack of heterozygosity
at the m arker loci in fem ale carriers. We have p erfo rm ed risk calculations using
the risk-calculadon option o f the MLINK program from the LINKAGE package of
com puter program s (L athrop et al., 1984). M icrosatellite repeats isolated from
cosm id clones correspondin g to the m arker loci DXS207 an d DXS274 are now
available, greatly im proving the inform ativeness o f these m arkers (Biancalana et
al., 1992; Rowe et al., 1992) an d thus reducing the n eed to use additional m ark
ers in diagnostics.
In diagnostic work, the population structure a n d available genealogical data
should be taken into consideration. Finland provides an exam ple. First, allele fre
quency studies (on 12 m arkers from Xp) in un related Finnish individuals have
shown that the observed heterozygosity value for DXS207 deviates significantly
from the value found in o th er populations, thus m aking DXS207 less inform ative
in Finnish families than elsewhere (Alitalo et al., 1991a). C orresponding data on
a new m icrosatellite rep eat are n o t yet available. Second, m ost o f the known RS
patients com e eith er from South West F inland an d can be traced to one super-ped
igree, o r from N orth C entral Finland a n d can be traced to a few pedigrees. H ap
lotype analysis with the m arkers DXS207, DXS43, DXS197, DXS274, and DXS41
have shown that patients from South West Finland have a haplotype association
which differs from the haplotype association found in the patients from N orth
C entral Finland. Both haplotypes differ from the m ost frequently observed haplo
type in norm al chrom osom es, which is shared by bo th groups (Alitalo et al.,
1991a). These data can som etimes be useful in genetic counselling; m oreover they
favour the hypothesis that the m utations in the two groups arose independently.
We have p erfo rm ed 17 analyses to d eterm in e carriership an d one for prenatal
diagnosis. T he results do n o t show any discrepancies with clinical an d genealogical
evidence regarding phenotype.
352 ALBERT DE LA CHAPELLE, ET AL.
With the aim o f cloning and characterizing the RS gene, ongoing an d future
studies will first attem pt to refine the genetic m ap o f the region. A nu m b er of
new m arkers have been m apped close to DXS43 an d DXS41, although these
m arkers have no t yet been used in linkage studies o f RS. Econs et al. (1990)
m ap ped GLR close to DXS43 (Zmax = 5.40 at 0max = 0.00 ), and m arkers
DXS365 an d DXS257 have been localised betw een the m arkers DXS43 and
DXS41 (Davies et al., 1991; Browne et al., 1992; Econs et al., 1992). In addition,
m arkers DXF22S4/S5 an d 1 6d/E are located close to DXS43 (Davies et al., 1991;
Rowe et al., 1992). New m arkers isolated from the Xp22.1-p22.2 region are pres
ently being m apped (B enham and Rowe, 1992; Biancalana et al., 1992).
T he next step is the physical m apping o f RS, th at is, the delineation o f a contig
uous stretch o f DNA an d the localisation o f RS within it. This will require the
building of con tigs with the aid of yeast artificial chrom osom es and cosmids. Phys
ical m apping of the RS region is in progress (Alitalo et al., 1992) with the aim of
o rd erin g the m arkers DXS9, DXS197, DXS43, DXS207 and GLR with respect to
each other, and allowing new m arkers closer to the RS gene to be isolated.
to provide leads regarding the pathogenesis of RS, and ultimately, even therapy
and prevention.
ACKNOWLEDGMENTS
O u r RS studies have been su pported by grants from the Academy of Finland, the
Sigrid Juselius F oundation, the Emil A altonen Foundation, the Jenny and Antti
W ihuri F oundation an d the Finnish C ultural F oundation. We thank the RS
patients an d families for their enthusiastic participation. Dr T iina Alitalo is pres-
endy at the D epartm ent o f Genetics, Case W estern Reserve University, Cleveland,
O hio, USA.
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X-LINKED JUVENILE RETINOSCHISIS 357
INTRODUCTION
CLINICAL CONSIDERATIONS
jThe nomenclature of Usher syndrome used is as follows: US refers to the syndrome and
the types are indicated by USla, USlb, etc, and are used to refer to the clinical syndrome;
USH refers to the corresponding locus, so that USHla is responsible for the clinical suptype
USla.
360 W. J. KIMBERLING, ET AL.
m ilder hearing loss, norm al vestibular reflexes, and retinitis pigm entosa. In fact,
type I patients are often described as d ea f and going blind while type II patients
are described as going blind with hearing im pairm ent. T he distinction between
the two is dram atic and th ere is seldom reason for confusing the two types. T he
two phenotypes seldom , if ever, occur to g eth er in the same family. This observa
tion suggested th at d ifferent loci were involved, a fact which was established once
the gene localizations were found. In fact, so far 5 different loci have been identi
fied, 3 o f which produce the type I phenotype and two which cause the type II
symptoms. T he genes and their corresponding phenotypes are listed in Table 1.
T he genes for U sher type lib and type III rem ain unlocalized. O th e r rarer, and
clinically unusual, types may exist but th eir relationship to types I and II is as yet
unclear.
hearing aids frequently benefit the U sher type II patient; som etim es the patient,
w hen fitted with bilateral hearing aids, hears an d speaks well enough th at the
hearing im p airm en t is often n o t noticeable. O ne characteristic of U sher syn
drom e has been the consistency of the audiom etric profile within subtypes. T he
extent o f the variability for both types is quite lim ited. Any deviations away from
the expected sloping audiogram , when occuring w ithout reasonable explanation,
is reason to suspect a typical type I o r II diagnosis. F urtherm ore, the hearing loss
is symmetric an d o f the same approxim ate ex ten t in both ears (M oller et al.,
1989).
Progressive hearing loss has been observed in some families with U sher syn
drom e an d may re p resen t a type III, alth o u g h its o ccu rren ce had been generally
accepted as rare. (G orlin, 1979). However, there are now several reports th at sug
gest that progressive hearin g loss may be m ore com m on than expected. For exam
ple, M erin et al. (1974), re p o rted observing a slowly progressive h earing loss in
26% of their series o f patients. Kum ar et al. (1984) observed significant differenc
es in speech discrim ination betw een type II patients with similar hearing deficits
and suggested that these differences could be due to progressive losses in som e pa
tients. Type III may be m ore freq u en t in Scandinavian countries since two o f the
rep o rts showing a high frequency are in Finnish populations (Karjalainen et al.,
1989; Nuutilla, 1970). O u r observations, however, indicate that type III U sher syn
drom e with progressive hearing loss am ong U sher type II patients is n o t com m on
in a large series o f families from the USA an d Sweden (Moller, 1989). It may be
th at the progressive loss seen by o th e r investigors is due to the norm al process of
ageing and is n o t an effect o f the gene. A nother possibility is th at the progression
o f hearing loss a p p a ren t in some type II patients is really a progressive loss in com
m unication skills due to the gradual loss in visual abilities an d which affect lip-
reading skills. N onetheless, the possibility that type III actually exists and is due to
allelic variation at o n e o f the known U sher loci m ust be considered. In fact, M erin
et al. (1974) postulated th at type III was simply a variant of type II. W hether type
III is linked to one of the known chrom osom al locations of the 4 USH loci would
answer this question b u t the possibility rem ains to be investigated.
T here has been little discussion o f the question o f progressive hearin g loss in
U sher syndrom e type I. It is generally assum ed th at a total or p ro fo u n d hearing
deficit is present at birth. T h ere have been, however, a few unpublished accounts
of residual hearing detectable in infancy by auditory evoked brain stem responses
(ABR) which disappear quickly. A lthough speech com m unication is generally not
useful for type I patients, there is variability an d one could postulate th at this is
due to the presence o f hearin g in som e patien ts. So-called co rn er audiogram s are
frequently seen in type I patients; these may be due to eith er simply the sensation
of vibration or, in some cases, to the presence o f a very m inim al residual hearing
reflex presum ably in the cochlear apex. It may be that, in type I U sher syndrom e,
the cochlea develops norm ally u ntil ab o u t the 7 -9 th m onth o f gestation and then
starts on a rapidly progressive d eterio ratio n which is usually com plete by birth.
This hypothesis is attractive because it makes cochlear dam age progressive just like
362 W. |. KIMBF.RLING, ET AL.
Hoomg WvWmJB
I s
I2S 260 SCO 1000 2CCC 0 8000
the retinitis pigm entosa. In term s of a m echanism for the neurosensory dam age,
it is m ore palatable intellectually to have both systems act in an abiotrophic m an
n e r ra th e r than having the hearing deficit ap p ear developm ental an d the re tin o p
athy being degenerative. Knowledge th at a progressive co m ponent exists, for
eith er types I o r II, will be im p o rtan t in the evaluation o f strategies to eith er re
store o r prevent the loss o f hearing in U sher syndrom e. Obviously, if the hearing
loss is progressive, then we may have an opportunity to in terfere with the deterio
ration so long as the time o f the onset o f the deterioration is known.
A uditory evoked emissions are consistent with the degree o f hearing loss in both
type I an d II patients. A uditory evoked brain stem (ABR) responses are, as expect
ed, absent in type I patients. In type II patients the results indicate that the hearing
im p airm en t is due solely to a p erip h eral lesion involving the cochlea (M oller et
al., 1989).
CLINICAL AND GENETIC HETEROGENEITY OF USHER SYNDROME 363
VESTIBULAR DEFICIT
T he second im p o rtan t distinguishing feature betw een types I and II U sher syn
drom e is the presence or absence o f vestibular responses. T he vestibular system
in type I U sher patients is virtually non-functional. Occasionally, an affected type
I individual will be seen with m inim al vestibular responses, b u t this is extrem ely
rare. This deficiency is m anifest in the adult as a non-progressive ataxia in the
form o f a swaying and swinging gait. T he ataxia is entirely o f perip h eral origin
an d there is no evidence to indicate an additional cerebellar com ponent. T he
vestibular deficit appears to be congenital, or at least occurs at a very early age,
since U sher I children are late to sit a n d walk. N one of the patients (M oller et al.,
1989) in one large well studied series walked before the age o f 18 m onths. In fact,
walking age is a good predictor o f the US subtype, since few type II children are
delayed in walking for as long.
T he postural system in hum ans relies on in p u t from three sources: vestibular, vi
sual, an d som atosensory. While the patients have com pensated well for the vestib
ular deficit, postural stability is aggravated because o f the progressive visual loss
due to RR T he result is th at U sher I patients are m ore posturally unstable than ex
pected based upon experience with o th er patients with hearing o r visual loss
alone. Type II patients, however, are essentially n orm al with regard to the vestibu
lar system. T here have b een some rep o rts of m ild vestibular abnorm alities, such as
hypo- o r hyperactivity, bu t it is unclear w hether these findings are w ithin the range
o f norm al. W hether there are vestibular deficits present in U sher II rem ains to be
definitely established.
Sophisticated vestibular studies, including rotary chair, bitherm al calorics, dy
nam ic an d posturography, are often necessary to d eterm in e if the "ataxia" of
USH1 is central or vestibular in origin and to determ ine the degree to which the
visual loss is affecting postural control. Visual im pairm ent com plicates, but does
n o t invalidate, the otoneurologic exam ination. T he RP affects the corneo-retinal
potential which is the basis o f electro-nystagm ography (ENG); the gain of the re
sponse m ust som etim es be increased an d the test p erfo rm ed in the dark since
even dim light will decrease the signals. If the ataxia is no t due to a labyrinthine
defect, then the diagnosis o f U sher syndrom e type I should be questioned. The
vestibular phenotype is highly co rrelated with the severity o f hearing loss. T he
finding o f a norm al vestibular response with p ro found deafness o r absent vestibu
lar responses with the sloping type II audiom etric profile should alert the re
searcher th at the diagnosis is n o t certain. Such patients may rep resen t new
subtypes o f U sher syndrom e or could be due to as yet unrecognized allelic varia
tion at one o r the o th er known loci.
364 W. J. KIMBERLING, ET AL.
Figure 2 B
CLINICAL AND GENETIC HETEROGENEITY OF LSHER SYNDROME 365
Figure 2 D
Visual Phenotype
Fundus changes are typical for patients with retinitis pigm entosa and currently
there is no recognized p attern o f fundus involvem ent that is correlated with any
specific subtype. In some cases the pigm entary abnorm alities are m inim al an d in
others they are extensive. Bone spicule form ation is com m on. A set o f typical fun
dus photographs are illustrated in Figure 2 for both type I an d II patients. How
ever, there are prelim inary indications o f fundoscopic: differences related to
genetic subtype, at least for type II. O ne family with U sher type II unlinked to
lq32-41 was found to have a m ilder course o f RP which had been described as RP
sine pigmento. Some bone spicule form ation was noted in the oldest affected sib
ling. O n the o th er hand, RP sine pigmento has been found in both type I a n d type
II persons and is believed to rep resen t an early stage o f the retinal deterioration
com m on to all RP (Heckenlively, 1988).
T he m ost freq u en t foveal change described by Fishm an (1979) is th at o f an atro
phic appearing lesion which was seen in 31% o f the patients. An additional 11%
showed a cystic-like foveal lesion associated with leakage from the perifoveolar ar
teries.
Several studies have shown th at electroretinography (ERG) m ight be subnorm al
as early as 2 to 3 years o f age, before abnorm alities can be seen functionally o r on
fundoscopic exam ination (M erin, 1974; De Haas, 1970; D avenport, 1978). Both
scotopic and photic responses are involved. Responses extinguish totally as the d e
g en eration progresses. T he visual evoked responses also show decreased am pli
tudes and longer latencies (Abraham , 1977). T here is no general consensus about
the severity o f the RP as a function o f subtype, bu t it has been suggested th at type
I may have a m ore severe visual involvem ent (Fishman, 1979). In type I patients,
cone an d ro d responses are usually u ndetectable by the m iddle o f the second de
cade and often in the first. Many type II patients have a recordable, b u t depressed,
cone response (Fishman, 1979). In one type II family with four affected brothers,
the gradual decline o f the ERG over two decades has been docum ented. While
electrophysiological studies provide the earliest and m ost reliable diagnosis o f re
tinitis pigm entosa, they are n o t necessary in all cases. An absent o r dim inished
ERG in at least one affected sibling is a necessary condition for the diagnosis of
U sher Syndrom e in a family.
R estricted visual fields are an integral p a rt o f the syndrom e. Im p airm en t o f p e
rip h eral vision may n o t be noticed until the teenage years b u t is probably present
early in life. Generally, visual field abnorm alities are n o t associated with scotom as
in type I patients. Several type II patients have shown a ring scotom a which may
eith er be a characteristic o f the specific type II genotype o r could simply reflect
the m ilder course of the disease for some type II patients. Searching nystagmus is
uncom m on in U sher syndrom e and the retinal d egeneration is uniform ly progres
sive from the p eripheral regions to the central.
N ightblindness is a concom itant to retinitis pigm entosa. Most patients have a
history o f nightblindness that dates back to childhood. T he nightblindness is par
CLINICAL AND GENETIC LIETEROGENEITY OF USHER SYNDROME 367
U sher types I an d II are distinct phenotypes and only rarely do the two occur in
the same family. For th at reason, it was hypothesized th at the two were genetically
distinct as well, a fact subsequently established by linkage analysis. T here is little
variability in expression within sibships for type I patients but greater intrafam il
ial variation in bo th hearin g an d visual involvem ent has been observed for type II
(Fishman, 1979; H allgren, 1959). T he origin o f the variation in hearing acuity of
type II patients has n o t been rigorously investigated. Certainly, one m ight expect
som e o f the variation to be due to the d ifferent loci known to be involved. O n the
368 W. J. K1MBERLING, ET AL,
been re p o rted to experience tem porary reactive psychosis. F u rth erm o re, the
schizophrenia-like symptoms seen in U sher syndrom e are also tem porary, a fea
ture n o t seen in true schizophrenia.
However, there is now accum ulating evidence th at there may be a cerebral le
sion associated with U sher syndrom e. Bloom e ta l. (1983) found indications o f cer
ebellar atrophy by CT scan on 6 o f 12 U sher patients. F u rth er evidence o f CNS
changes has been found by o th er investigators (Koizumi et al., 1988; Schaefer et
al., 1992). T he findings are relatively non-specific and indicate both cerebellar
an d cerebral anom alies. In one study, sim ilar anom alies were NOT seen in d eaf or
RP controls (Schaeffer et al., 1992) suggesting that the brain anom alies are no t
secondary to the sensory deficit. T he brain anom alies were seen in both type I and
II patients although they ap p eared to be m ore freq u en t in type II, which is som e
what surprising in th at type II has the m ilder sensory deficit. T he cerebellar and
cerebral anom alies have no t yet been associated with any clinical neurologic prob
lem.
P attern O f Inheritance
It has been estim ated th at U sher type I represents 90% of U sher syndrom e in the
U nited States, with type II accounting for nearly 10%, and o th er types accounting
only about 1%. However, Fishm an's g ro u p re p o rted 71 ou t o f 106 patients with
type II (Piazza, 1986). A N orw egian study o f 28 cases were distributed as: type
1 - 1 4 cases; type 1 1 -1 0 cases; an d o th er - 4 cases (G rondahl, 1986; G rondahl and
M joen, 1986)). O ur own series has alm ost 50% type II cases both from the USA
an d Sweden (unpublished d a ta ). We believe U sher type II is at least as com m on
as type I. However, type II patients are n o t usually ascertained in schools for the
d eaf because their hearing loss is n o t severe enough to place them there. Schools
and program s for the blind an d ophthalm ologic clinics have a relatively higher
p ro p o rtio n of type II patients com pared to ascertainm ent from schools for the
deaf.
T he frequency of U sher syndrom e has been estim ated at 3.0/100,000 in Scandi
navia (H allgren, 1959; N uutila, 1970) a n d at 4.4/100,000 in the U nited States
(B oughm an et al., 1983). T he prevalence o f U sher syndrom e am ong d eaf individ
uals has b een re p o rted to range from 0.6 to 28% (Vernon, 1969). Conversely, the
frequency of deafness in the retinitis pigm entosa population is estim ated to range
betw een 8.0 an d 33.3%. Overall, there are ab o u t 16,000 deaf an d blind people in
the U nited States, o f which m ore than h alf are believed to have U sher syndrom e.
Most o f these will have U sher type I. M ost U sher type II individuals, with a m ilder
h earing loss, may n o t be n o ted in surveys which have focused on the profoundly
deaf. Piazza et al. (1986) postulated that the frequency of type II is at least as great
as th at for type I.
U sher Type I
T he U sher syndrom e type I gene was first localized to chrom osom e 14q by a link
age with m arker D14S13 in fifteen families (Kaplan et al., 1992). A linkage was
suggested by the fact th at for the total sam ple the lod score m axim ized at a
recom bination fraction (9 ) o f 0.20 with lod score (Z) o f 0.83 (C hapter 1). How
ever, the test for genetic heterogeneity was significant, assum ing linkage, at the
5% level suggesting the possibility th at two or m ore d ifferent genes m ight be
involved. Ten o f the 15 families showed linkage to the 14q m arkers and 8 o f these
cam e from the same region known as Poitu-C harentes in France bu t there was no
evidence o f consanguinity. W hen these families were pooled, the two point lod
CLINICAL AND GENETIC HETEROGENEITY OF USHER SYNDROME 371
score went to 3.78 at 9 = 0 with D14S13. Based upon this location, Kaplan et al.
(1992) hypothesized th at the USH gene may be involved in an abnorm ality o f cil
iary structures of the retina, vestibule, and cochlea. T he sensory cilium o f the
rods and cones separate the o u ter segm ent of stacked discs containing the visual
pigm ent from the in n er segm ent an d it is reasonable to expect th at defective cilia
could lead to deg en eratio n of the o u te r segm ent of the retina. However, vestibu
lar and cochlear hair cells do not have tru e sensory cilium with the characteristic
9+0 m icrotubules. Vestibular hair cells have kinocilia with 9+2 m icrotubules an d
are missing the dynein arm s fo u n d in m otile cilia. D uring developm ent, the
cochlear hair cells possess a transitory kinocilium that may be responsible for
organizing the developm ent of the stereocilium characteristic o f the cochlear
hair cells. Thus, a defective kinocilium could lead to defective hair cells and
abnorm alities of hearing an d balance. Because o f the transitory n atu re o f kinoci-
lium developm ent, it w ould be expected that any hearing or vestibular abnorm al
ities would be congenital but the sensory cilia o f the eye are req u ired for norm al
retinal m aintenance th ro u g h o u t life an d a defect would be m ore likely to cause a
progressive deterio ratio n (H u n te r et al., 1986).
P h o to recep to r and sperm axonem e abnorm alities had been previously ob
served in U sher syndrom e (H u n te r e t al., 1986); nine o f the 10 U sher patients
studied had type II. In a subsequent report, B arrong et al. (1992) also observed
abnorm al connecting cilia in a type II patient. These abnorm alities are no t seen
in the general population of X-linked and autosom al retinitis pigm entosa patients
with norm al hearing. Studies o f axonem e structure o f nasal cilia showed abnor
malities associated with a group o f RP patients including U sher syndrom e bu t the
results were n o t clearly broken down into diagnostic group so it is h ard to draw
any conclusions. Sperm m otility an d structure has also been found to be abnorm al
in U sher patients (H u n te r et al., 1986; N uutila, 1970). Kaplan et al. (1992) point
out th at the gene controlling h e a rt position in the m ouse has been m apped to a
region hom eologous to hum an 14q32 an d was identified by a recessive situs inver
sus m utation, iv. T he hum an hom ologue o f the m ouse iv gene should lie exactly
between the hum an alpha-1 antitrypsin gene an d the heavy chain im m unoglobu
lin which is where the U sher gene was m apped. T hese are intriguing observations.
T here is confusion ab o u t which types o f U sher syndrom e actually have abnorm al
cilia an d m ost cases re p o rted have b een type II and n o t clinically equivalent with
the type I phenotype localized to 14q. A ciliary defect makes sense for type I U sher
syndrom e since bo th the vestibular a n d cochear functions are im paired. However,
the re p o rted ciliary abnorm alities mostly occur in type II individuals. F u rth er in
vestigation into this p h en o m en o n is w arranted and, now that the d ifferent types
can be differentiated from each o th e r at the m olecular level, the research can be
directed towards defining the differences between types. It is unreasonable, al
though no t impossible, to expect th at all types o f U sher syndrom e share a ciliary
defect. T he expectation is th at one o r m ore types would have a different biologic
basis. If this is fo u n d to be the case, th en ciliary o r axonem al structure could be
372 W. J. KIMBERLING, ET AL.
an additional m eth o d to distinguish betw een types w ithout p rio r reliance on link
age groups.
O th e r investigators have been unable to confirm the localization to 14q in fam
ilies outside o f the original French g roup (Keats et al., 1992). This analysis includ
ed a large series o f families from the USA an d N o rth e rn Europe, notably E ngland
an d Sweden. This created some initial do u b t about the linkage and raised the
question th at it may have occurred by statistical chance. However, the fact th at 8
o f the 10 linked families com e from the same region in France supports the argu
m en t for linkage an d for the existence of a locus on chrom osom e 14. Definitive
evidence will com e when those families are shown n o t to be linked to the o th er
two regions now shown to contain the U sher gene. This locus has been given the
designation o f U SH la.
HBB* -
4.0
D11S875-
2.0
D11S569.
1.0
D11S926-
3.0
D11S902-
D11S899- 3.0
5.0
USHlc D11S928*
1.0
D11S915-
1.0
D11S904*
1.0
D11S916-
12
13.1
13.2
13.3
13.4
13.5
14.1 lUSHlb
14.2 | TYRand OMP1
14.3
21
22.1
22.2
22.3
23.1
23.2
23.3
24
25
Figure 3 Map of chromosome 11 which shows the most recent evidence for localization of the
Usher lb gene to the 11 q l3-14 region.
Acadians were originally from the N orm andy area in France and there is no evi
dence th at they share ancestry with the families from Poitu-C harentes showing
linkage to 14q. It was originally th o u g h t that the use o f the Acadian isolate avoided
the com plications o f heterogeneity a n d allowed the pooling of data across sibships
w ithout m ixing types I and II. However, it was subsequently discovered that both
types I and II occur in the population. A lthough type I appears to be the m ost prev
alen t and type II may have a frequency approxim ately equal to th at in the non-Ac-
374 W. J. KIMBERLING, ET AL.
to have ways o f a priori dividing families used in fine gene m apping o f U S H la, lb,
or lc.
F u rth er refin em en t o f location will d ep en d upon the observation of critical
crossover events that will bisect an d red u ce the region o f flanking m arkers. If a
critical crossover is actually d ue to the inappropriate inclusion o f a type Ic family
in a type lb cohort, then the assessment o f location may be off by a considerable
physical distance. If the investigation is lim ited to the Acadians, th ere is little
chance o f contam ination by no n -U sh lc families. However, until we know the true
frequencies o f the no n -U sh lb families in the general population, the possibility of
e rro r will always be present fo r U S H lb. Two ways to m inim ize this effect is first to
develop clinical criteria th at allow subclassification o f the U sher type I families
w ithout resorting to m olecular data. T he second is to type all three sets of m arkers
from 11 q , l i p , an d 14q and to include only those which show definite non-linkage
to two of the three loci.
U sher Type II
U sher syndrom e type II was localized to chrom osom e lq32 (Kim berling, 1990;
Lewis, 1990). T he gene has subsequently been shown to be flanked by the m ark
ers D1S237 and D1S229. T he m ost recen t m ap o f chrom osom e lq is shown in Fig
ure 4 along with the putative location o f the U sher type II gene. T here is some
am biguity in the co rrespondence betw een the CEPH collaborative study m ap and
the second generation chrom osom e 1 m ap (W eissenbach et al., 1992), so th at the
exact position of the m arkers betw een D1S245 and D1S227 m arkers is provi
sional. D1S237 and D1S229 are definitely between D1S70 and D1S81 (u n p u b
lished data). T he best estim ate o f the physical location of the U sher II gene is in
Iq32-lq42. Localization o f the U sher II gene is narrow ed to ab o u t 4 centiM or-
gans. This region am ounts to about 4 M egabases o f DNA.
T here are few genes in th at region th at rep resen t true candidates. An autosom al
hom ologue of the X-linked choroiderem ia gene, known as hCHM L, has been lo
calized to lq. This gene shows hom ology with a bovine p rotein which regulates the
G D P/G T P exchange o f GTP binding p ro tein smg 25A. It is expressed in the chor
o id /re tin a l pigm ent epithelium an d several o th er tissues (C hapter 12). It is not
known w hether this gene is expressed in the cochlea. hCHM L has been localized
to lq 3 1 -lq te r an d hence m ust be considered a logical candidate for U sher syn
drom e.
Two o th er genes in the region are transform ing growth factor (32 (TGFB2) and
the hom eobox gene HB24 (HLX1). Both genes m ap betw een D1S81 and D1S70,
which places them in close proxim ity to the U sher II gene. Both TGFB2 an d HLX1
have been localized to lq41. TGFB2 is a m em ber o f a polypeptide family which
regulates growth an d cellular differentiation; it is expressed in a wide variety o f tis
sues. HLX1 has been fo u n d to be expressed in hem atopoietic tissues an d has been
376 W. J. KIMBERLING, ET AL.
D1S53
REN
CR1/CR2/DAF
12
11 D1S70
11 D1S245
D1S205
12 D1S217 J TGB2
D1S237 J
21 USH2a
22
D1S229
| HLX2
23 D1S227
24 PPOL
D1S8!
25
D1S48
31 D 1SI 78
32
D1S103
41
42
43
44
th at the U sher gene affects eith er DNA repair o r synthesis which provides a clue
towards the selection o f candidate genes. U nfortunately, these studies do n o t well
characterize the type o f U sher syndrom e. In fact, the case re p o rted by B lanchet et
al. (1992) h ad an atypical RP which was n o t diagnosed until the 6th decade and
an optic atrophy n o t typical o f U sher syndrom e. T here was no m ention o f vestib
ular responses. Clearly, studies of hypersensitivity to X -irradiation o r similar agents
need to be repeated with atten tio n to the clinical an d m olecular subtype. PPOL
(or ADPRT) activity is in d u ced by single strand breaks in DNA an d is req u ired for
cellular repair. It has been suggested as the gene for eith er Fanconi's anem ia or
xeroderm a pigm entosa. A gene involved in the com plem entation of the defect in
group A x ero d erm a pigm entosa cells has b een localized to lq42-lqter. T he PPOL
gene was localized lq 4 2 by in situ hybridization an d it is interesting th at a second
hybridization peak was observed on 14q24, close to the localization o f U S H la.
O ne can only speculate ab o u t the possibility of contiguous deletions involving
both the U sher II gene an d PPOL giving rise to the observation o f increase chro
m osom e breakage in some type II patients.
A potential m ouse m odel for U sher syndrom e type II has recently b een discov
ered by C hang et al. (1993). T he R BF/D nJ albino m ouse carries a recessive m uta
tion, rd-3, which causes a retinal d egeneration similar to retinitis pigm entosa.
E lectroretinogram s an d retinal appearance are both suggestive o f hum an type RP.
T here is noth in g particularly notable ab o u t the RP seen in U sher II patients which
would suggest that U sher II an d the rd-3 m ouse are the same. T he RP phenotype
is too non-specific. However, the rd-3 gene m aps to 10 centiM organs distal to the
alkaline phosphatase gene on m ouse chrom osom e 1. This region shows consider
able hom ology with hum an chrom osom e I. T he region w here the rd-3 m ouse gene
would lie is theoretically close to the position w here the m ouse hom ologue for the
U sher II gene should lie. T h ere was no re p o rt o f w hether these m ice are hearing
im paired.
O ne family with U sher type II was observed to have at least 3 double crossovers in
the lq 3 2 region betw een m arkers D1S70 an d D1S81 if th eir USH2 gene were
located in th at segm ent. A form al heterogeneity analysis using O tt's HOM O G
program was statistically significant w hen this family was included in an anlysis of
a large sam ple o f U sher type II families. T he significance disappears w hen the
family is rem oved. T he affected family m em bers had audiogram s typical o f U sher
type II along with norm al vestibular function. T he ERG was dim inished to absent.
T here was a history o f nightblindness an d visual fields were restricted. Thus, a
diagnosis o f U sher syndrom e h ad been given to the family. However, it was no ted
th at the retinitis pigm entosa was described as sine pigmento an d one ophthalm olo
gist had observed m inute white flecks in the retina. In retrospect, the retinal
findings are subtly d ifferent suggesting th at there is a good chance th at this type
o f family may be identified p rior to m olecular genetic testing.
378 W. J. KIMBERLING, ET Al
T he family did no t show linkage to the lq32 - q41 region containing the U sher
type II gene. N or does the U sher syndrom e in the family show linkage to m arkers
on chrom osom es 11 q, l i p , o r 14. This establishes the fact that a second locus for
U sher type II exists which is n o t located on lq32-41 and that the gene is no t an
allele at any of the o th er 3 U sher I loci. This new gene has been given the desig
nation USH2b, leaving USH2a to refer to the original lq locus. T he frequency of
U sher lib relative to Ila is n o t known, although it m ust be relatively less com m on
inasm uch as it has been seen only once in a series o f over 30 type II families.
CONCLUSIONS
U sher syndrom e was first described as h earin g im pairm ent with retinitis pigm en
tosa. With time, investigators realized that at least two clinical types existed and
th at they could be consistently an d reliably distinguished from each other. How
ever, linkage analysis showed that a m inim um of five d ifferent genes are involved,
th ree for type I an d two for type II. T h ere are no clear clinical differences
betw een the d ifferent subtypes within each type.
T he next stage o f U sher research is focused on identifying and cloning the re
sponsible genes. Saturation m apping o f the critical regions by chrom osom e walk
ing m ight be necessary to accom plish this task. Saturation m apping o f the
chrom osom e region will eventually pro d u ce a series o f "contigs" o f overlapping
clones th at collectively span the region o f interest, which hopefully will involve
only 1 M egabase or less. G enetic heterogeneity will rem ain a problem until the
genes are identified and sequenced. For exam ple, a family with two children af
fected with a separate type o f U sher syndrom e would show non-double crossovers
in the critical region 25% o f the tim e th at th eir m arkers are inform ative: for three
affected children, this frequency would be 6.25%. Most U sher families are small
with only 1 affected and in m ost of those cases it will be im possible to determ ine
which subtype o f U sher syndrom e is present by linkage analysis. W hen a family has
two or m ore affected m em bers, the probability that linkage can rule o u t a subtype
is related to the n u m b er o f affected children. At the p resen t time, the diagnosis of
o ne subtype o r the o th er by m olecular testing m ust rely solely on exclusion of the
alternate types. Clinical differences betw een the subtypes may exist an d the possi
bility o f being able to differentiate one type from an o th er needs to be fu rth e r in
vestigated, Resolution of the question o f clinical differences may help in the
selection o f families helpful in identifying the U sher genes.
M eanwhile, until the U sher genes are found, DNA m arkers close to the U sher
gene can be used in genetic counseling. Given tight linkage and an inform ative
family with one previously affected child, early diagnosis is possible for subsequent
children. T he m arkers showing tight linkage could also be used to d eterm in e
which of the unaffected relatives are carriers. U nfortunately, effective screening of
spouses who m arry into a family will not be possible until the ap propriate U sher
g ene is cloned.
CLINICAL AND GENETIC HETEROGENEITY OF USHER SYNDROME 379
Ultimately, the goal o f genetic research is to develop a treatm ent for U sher syn
drom e, however, the underlying defect causing it is unknow n. Most biochem ical
and physiologic studies have cen tered on the study o f retinitis pigm entosa an d the
results have been disappointingly negative in term s of elucidating the basic defect.
We would expect that the gene th at causes U sher syndrom e does so by an effect
on a substrate that is essential for som e com p o n en t of in n er ear function, balance,
and visual ability. D eterm ination o f the function o f the gene would fu rth e r o u r u n
derstanding o f the cellular an d biochem ical bases for these sensory abilities. An
u n d erstan d in g o f the underlying pathologic process occurring from gene to p h e
notype is an im p o rtan t step towards finding an effective m eth o d o f rem ediation
a n d /o r prevention. G ene cloning a n d its subsequent characterization may be the
only approach that could lead to the discovery o f the basic defect o f U sher syn
drom e.
C onsidering the fact that U sher syndrom e is the m ajor cause o f d ea fn ess/b lin d
ness, it is im p o rtan t to pursue research into the cause (s) o f U sher Syndrom e in the
hope th at som eday an effective rem edy may be possible. A bout 1 ou t o f every
20,000 individuals is affected with U sher syndrom e. T he b u rd e n caused by the loss
o f the two m ost vital hum an senses is trem endous. It isolates patients from the
m ainstream o f society, resulting in severe psychological stresses and reduces their
ability to act independently. T h e answers to many intriguing questions regarding
U sher syndrom e will no t be available until the genes are cloned. An un d erstan d
ing o f the etiologies o f U sher syndrom e will provide critical inform ation about the
underlying m echanism s o f function a n d expression o f the genes.
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16. MARFAN SYNDROME
SUMMARY
MFS is a systemic disorder o f connective tissue which prim arily involves the m us
culoskeletal, cardiovascular and ocular systems (Pyeritz and McKusick, 1979).
MFS has been re p o rte d in d ifferent races an d ethnic groups (McKusick, 1972).
T he prevalence o f MFS has b een estim ated to be approxim ately 46 p er 100,000
population in the U nited States (Pyeritz an d McKusick, 1979).
M usculoskeletal M anifestations
D olichostenom elia (long and narrow fram e) is the characteristic skeletal abnor
mality observed in MFS. Tall stature, decreased u p p er to lower segm ent ratio,
arm span in excess o f height, dolichocephaly, an d arachnodactyly o f fingers and
toes are some o f the m anifestations directly associated with dolichostenom elia
(Pyeritz an d McKusick, 1979) (Figure 1). In m ost individuals affected with MFS
the distal bones o f the extrem ities tend to be longer. Thus, a decreased ratio
(approxim ate value o f 0.85) o f u p p e r segm ent to lower segm ent is frequently
found in adults with MFS. T he face is long an d narrow, the palate is high-arched,
384 PETROS TSIPOURAS AND MICHAEL W. KILPATRICK
Figure 1 In div id u al a ffected w ith M arfan sy n d ro m e . P anel A: asym m etric p e ctu s c a rin a tu m
a t th e age o f 14 years. P an el B: sam e p a tie n t a t th e age o f 29 years.
Cardiovascular M anifestations
O cular m anifestations
Ectopia lends, alm ost always bilateral, is a clinical hallm ark of MFS (Lutm an and
Neel, 1949; M aum enee, 1981). Only 50 to 80% o f individuals with MFS have this
386 PETROS TSIPOURAS AND MICHAEL W. KILPATRICK
M iscellaneous m anifestations
A lthough infants and young children were am ong the initial patients in whom
MFS was docum ented, recognition o f the disorder in early life is n o t always an
easy m atter. O n the o th e r hand, certain infants present with a distinct set of clini
cal m anifestations which has led to the suggestion o f the existence o f a distinct
entity, term ed infantile or neonatal MFS. A characteristic aged facial appearance
due to deep-set eyes, high arched palate, arachnodactyly, pectus deform ities, flex
ion contractures, pes planus, multivalvular involvement, aortic ro o t dilatation
and ectopia lends are am ong the phenotypic m anifestations described (Morse et
a l . 1990).
INHERITANCE
T he autosom al dom inant m ode o f transm ission was clearly dem onstrated for the
first tim e by Weve in 1931. Since then, scores of pedigrees have been re p o rted
which clearly dem onstrate autosom al d o m in an t inheritance (Tsipouras et al.,
1992). A bout 80% of individuals docu m en ted to have the MFS by reasonably
strict criteria in h erited the gene from a parent; in the rem aining 20% the condi
tion arises as a result of new m utations. P aternal age effect on m utation has been
d em onstrated in the MFS . A ccording to o n e study, the average age o f fathers of
isolated cases was seven years greater than the m ean p atern al age in the popula
tion (M urdoch et al., 1972a). Most infants with the severe infantile form o f MFS
are isolated cases; for some o f these infants the possibility o f homozygosity has
been raised (Chem ke et al., 1984, Schollin et al., 1988).
MARFAN SYNDROME 387
MOLECULAR BASIS
B rief overview
Over the years several m olecules o f the extracellular m atrix o f the connective tis
sue had been considered as potential candidates in the aetiology o f MFS. Am ong
them were the d ifferent types o f fibrillar collagens, elastin, hyaluronic acid, beta-
glucuronidase, elastase an d decorin (Tsipouras, 1990). In 1986, Sakai et al., using
m onoclonal antibodies raised against microfibrils, identified a 350 kDa glycopro
tein for which they coined the term fibrillin. Indirect im m unofluorescence stud
ies on the skin and cultured derm al fibrobasts of norm al individuals and
individuals affected with MFS provided the first strong evidence o f a structural
p rotein abnorm ality in M arfan syndrom e (Godfrey et al., 1990, H ollister et al.,
1990) (Figure 2). In parallel, a consortium o f investigators attem pted to localize
the gene responsible for M arfan syndrom e by positional m apping. An exclusion
m ap was constructed that elim inated nearly 75% o f the hum an genom e as a likely
location o f the MFS gene (B lanton et al., 1990). This led to the subsequent
assignm ent o f the MFS gene to chrom osom e 15 (15ql5-q23) (K ainulainen et al.,
1990; Dietz et al., 1991a; Tsipouras et al., 1991).
T he term m icrofibril was originally used to identify m orphologically sim ilar extraj
cellular m atrix structures displaying a diam eter o f less than 20nm and lacking the
characteristic 67nm banding periodicity o f interstitial collagen "'fibres (Low,
1962). Currently, the m icrofibrils are divided into two classes according to their
average diam eter, the larger o f the two classes being the lOnm m icrofibrils also
MARFAN SYNDROME 389
FBN2 (Lee et al., 1991; Tsipouras et al., 1992). It is possible to speculate th at the
am elioration o f the clinical m anifestations o f congenital contractural arachnodac-
tyly with age may be the consequence o f a developm ental switch involving the
FBN2 an d FBN1 genes.
with MFS revealed no gross deletions o r rearrangem ents, an d to date only one ge
nom ic deletion has been identified (K anulainen et al.,1992). T he deletion results
in the loss o f 366 bases o f the fibrillin mRNA which consequently produces a tru n
cated protein. This sh o rten ed protein appears to be synthesised in norm al
am ounts, secreted efficiently from the cell, and assem bled into microfibrils. T he
m icrofibrils thus produced are structurally an d functionally abnorm al.
Seven p o in t m utations (five missense an d two nonsense) have so far b een iden
tified and characterised (Dietz et al., 1991b, 1992): four o f the missense m utations
result in cysteine substitutions an d one in the substitution o f an arginine (Dietz et
al., 1992) (Figure 4). As substitution o f a cysteine residue would be expected to
d isru p t local folding an d possibly extracellular protein-protein interactions, the
consequence o f such a m utation can be readily envisaged. Similarly the arginine
substitution, which occurs in an EGF-like repeat, m ight be expected to alter the
local secondary structure an d thus affect cysteine-m ediated bonding. O ne o f the
nonsense m utations leads to the production o f a fibrillin m olecule which lacks the
last 116 am ino acid residues and is apparently n o t secreted from the cell (Kainu-
lainen et al., 1992). Im m unofluorescence studies of fibroblasts from the individual
with this m utation reveals red u ced am ounts o f extracellular fibrillin (K ainulainen
et al., 1992). T he o th er nonsense m utation, however, results in a tru n cated m ole
cule which, although apparently synthesised in reduced am ounts, is secreted and
does participate in fibrillogenesis. (K ainulainen et al., 1992). Thus even a small
am o u n t o f structurally abnorm al fibrillin is able to elicit a clinical phenotype, thus
dem onstrating the potential for fibrillin m utations to have a d o m in an t negative ef
fect.
W ith the exception o f the originally re p o rte d missense m utation which was
fo und in two unrelated isolated cases o f MFS all the m utations re p o rted to date
have been unique (Dietz et al., 1991b). In each case, screening o f a large nu m b er
o f un related individuals affected with MFS for the presence o f the m utation has
proved fruitless. It is also o f interest to n o te th at the m utations identified to date
are distributed th ro u g h o u t the entire length o f the gene, with no evidence o f clus
tering o f m utations in particular regions o r corresponding protein dom ains.
ANIMAL MODEL
affected anim als from norm al by reduced derm al staining for elastin (Besser
et al., 1990).
A lthough the n atu re o f the genetic defect is as yet unknow n, it is possible that
this MFS-like condition will provide an excellent m odel for fu rth e r study and char
acterisation of the precise m olecular pathology o f MFS.
CLINICAL MANAGEMENT
FUTURE DIRECTIONS
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17. LOWE OCULOCEREBRORENAL SYNDROME
INTRODUCTION
CLINICAL FINDINGS
m alform ations an d b irth defects no t seen in males with OCRL m ake the diagno
sis o f OCRL uncertain. O ne fem ale p atien t was re p o rted with a deletion o f m ito
chondrial DNA, which raises the possibility th at a m itochondrial genocopy for
OCRL may exist (M oraes et al., 1991), b u t h er phenotype was that o f a progres
sive m ultisystem disease only partially overlapping with OCRL. Finally, th ere are
two fem ales re p o rte d in the literature in which clear-cut OCRL has arisen de novo
d u e to balanced X /au to so m e translocations involving the Xq25-Xq26.1 region of
the X chrom osom e (H odgson et al., 1986; M ueller et al., 1991). These patients
have an OCRL phenotype indistinguishable from that o f hemizygous affected
males because the translocations d isru p ted the OCRL locus on the translocated
X chrom osom e an d im peded random X inactivation such th at the X chrom o
som e n o t involved in the translocation is inactivated preferentially.
O cular Findings
F igu re 1 Slit lam p e x a m in a tio n o f fem ale c a rrie rs o f Lowe's Syndrom e. In p a n e l A is show n
th e typical p a tte rn o f h u n d re d s o f m ic ro p u n c ta te , grey o p acities d istrib u te d in a rad ial distri
b u tio n in th e lens. In p a n e l B is a single d e n se p o ste rio r c a ta ra c t as see n in som e O CR L h e t
erozygotes. (C ourtesy o f R. A. Lewis, M.D.)
402 ICHIRO OKABE AND ROBERT L. NUSSBAUM
though som e controversy exists in the literature concerning the specificity and
sensitivity o f the lenticular opacities as a m ethod o f carrier detection (G ardner
and Brown, 1976), reliance on strict diagnostic criteria for both appearance and
distribution of the opacities m akes carrier detection by slit lam p exam ination a re
liable an d sensitive technique (Cibis et al., 1986; Reilly et al., 1988).
Systemic Findings
Average birth weight and length o f the patients with OCRL are well within nor
mal range b u t the rate o f growth begins to decline by 3 m onths o f age. By 18
m onths, weight and height are below norm al range, an d continue to fall. T he
growth curve in the patients does n o t ap p e ar to plateau at 16 to 18 years o f age as
it does in norm al m en. Growth continues on into early adulthood b u t m ean final
weight and height are less than the third percentile o f norm al values for m en
(M cSpadden, 1991; C harnas et al., 1991).
A wide range o f intellectual ability are rep resen ted in OCRL patients (M cSpad
den, 1991; Kenworthy, Park, and C harnas, 1993); 63% have m oderate to severe
m ental retadation, 33% have a mild to bord erlin e form , with a few patients dem
o n strating intelligence within the norm al range. T hough m ental developm ent is
generally delayed, the developm ental abnorm ality is stable, in contrast to the p ro
gressive course seen in degenerative disorders.
Some behavior problem s are troublesom e, especially self-abusive behavior, non-
cooperative an d violent behavior, and eccentric, autistic-like behavior. These be
havior problem s are both m ost prevalent an d m ost severe in the 5-13 age group,
and ten d to decrease in late adolescence a n d adulth o o d (M cSpadden, 1991; Ken
worthy, Park, and C harnas, 1993).
Neuromuscular signs
H ypotonia with decreased m uscle mass and decreased d eep ten d o n reflexes are
n o ted in m ost patients. H ypotonia becom es a p p a ren t from a few m onths o f age
but is n o t progressive an d tends to im prove in association with general growth.
T he deep ten d o n reflexes are present at birth but disappear by 1 year o f age
(Abbassi, Lowe, and Calcagno, 1968). Significant nerve o r m uscle pathology is
absent and suggests th at the hypotonia a n d areflexia are o f central nervous sys
tem origin (B anerjee, Allen, an d McKee, 1982; C harnas et al.,1988).
Seizures occur in 50% o f OCRL patients (C harnas, 1989). All three m ajor types
o f seizure have been reported: febrile, p etit mal, and grand mal. T he average age
o f first seizure for these three types of seizure is 2 years, 5 years, 9 years o f age re
spectively. T he grand mal type is the m ost com m on seizure (M cSpadden, 1991).
As in the general population, severe, early onset seizures are a p o o r prognostic
sign for intellectual developm ent and seizure control.
LOWE OCULOCEREBRORENAL SYNDROME 403
Renal dysfunction
Skeletal signs
M usculoskeletal com plications in OCRL can result eith er as a prim ary feature of
the disease or as secondary consequences o f renal and neurom uscular abnorm al
ities o f the illness, i.e., Fanconi Syndrom e a n d hypotonia. Tenosynovitis and
arthritis or arthropathy are freq u en t an d appear to be prim ary com plications of
OCRL. Jo in t involvem ent can p resen t as n o n te n d e r jo in t swelling involving both
small and large joints, focal nodules on the finger, or bilateral p lan tar masses. On
occasion these masses becom e painful an d require resection (C harnas and Gahl,
1991). It seems likely th at these changes are the m anifestation of abnorm al,
excessive growth o f fibroblasts from periarticular tissue or tendons. Secondary
404 ICHIRO OKABE AND ROBERT L. NUSSBAUM
Other features
Prognosis
Pathological Findings
Ophthalmic pathology
Lenses are small, flattened, an d discoid. T h ere is a diffuse cataract with no differ
entiation o f cortex and nucleus, and p ro n o u n ced degeneration in the posterior
p o lar region (C urtin, Joyce, and Ballin, 1967; Tripathi, Cibis, an d Tripathi, 1980).
T he an terio r subcapsular epithelial hyperplasia an d nodular excrescences of the
lens capsule are present; however, these are no t necessarily specific for OCRL
(Z im m erm an and Font, 1966; C urtin, Joyce, and Ballin, 1967; Tripathi, Cibis, and
Tripathi, 1980; Tripathi, Cibis, an d T ripathi, 1986). An anom alous a n terio r cham
b er angle with an terio r displacem ent o f rudim entary ciliary processes, which
could contribute to glaucom a, m ight be secondary to a m icrophakia in which
zonular fibres drag the ciliary body anteriorly (Zim m erm an and Font, 1966; Cur
tin, Joyce, and Ballin, 1967; Tripathi, Cibis, and T ripathi, 1980; Cibis, Tripathi,
LOWE OCULOCEREBRORENAL SYNDROME 405
and T ripathi, 1990). T he small islands o r folds o f retinal tissue, which m ight also
be dragged anteriorly by the zonules,are p resen t in pars ciliaris retinae (Zim m er
m an an d Font, 1966; C urtin, Joyce, an d Ballin, 1967; Garzuly et al., 1973). O th er
wise, the retina including optic nerve, sclera, and choroid appears unrem arkable
in the light m icroscopic exam ination (C urtin, Joyce, and Ballin, 1967; Garzuly et
al., 1973).
Renal Pathology
BIOCHEMICAL FINDINGS
MOLECULAR STUDIES
Gene M apping
Linkage studies o f OCRL were first re p o rted in 1982 (H ittner, C arroll, and
Prchal, 1982) which ru led ou t close linkage of OCRL to eith er the Xg blood
g roup (Xp22.3-pter) or to glucose-6-phosphate dehydrogenase (Xq28). Using
restriction fragm ent length polym orphism s (RFLPs) on eight X chrom osom e
loci, Silver et al. (1987) analyzed fo u r large families with OCRL and identified
two loci, DXS10 at Xq26 an d DXS42 at Xq24-26, which were tightly linked to
OCRL and supported a m ap assignm ent to Xq24-q26. Evidence for placing
OCRL at Xq24-q26 also cam e from two fem ale patients with an X;3 translocation
b reakpoint at Xq25 (H odgson et al., 1986) and an X;20 translocation breakpoint
at Xq26.1 (M ueller et al., 1991). DXS42 and DXS10 have been shown to flank the
Xq25 breakpoint by analysis o f som atic cell hybrids g enerated from this t(X;3)
p atient (Reilly, Lewis, an d Nussbaum , 1990). No recom bination was found
between OCRL and the proxim al m arker DXS42 in two studies with a com bined
lod score o f 11.8 (Reilly, Lewis, a n d Nussbaum , 1990; Wadelius, Fagerholm ,
Pettersson, an d A nneren, 1989).
Positional Cloning
Nelson et al. (1991) isolated yeast artificial chrom osom es whose hum an inserts
spanned the breakpoint o f a fem ale patient with an X;3 translocation. Using
these yeast artificial chrom osom es as probes, a candidate cDNA, OCR1.-1, was iso
lated which is in te rru p te d by both the t(X;3) an d t(X;20) breakpoints (O kabe et
al., 1992; A ttree et al., 1992). G enetic evidence th at OCRl^-1 is the gene for OCRL
relies on the discovery of m utations in the gene in patients with the disease. T he
transcript o f this gene is absent in both fem ale OCRL patients with X;autosom e
translocations an d is absent or ab e rra n t in size in some u n related m ale patients
with no detectable re arra n g em en t (Attree et al., 1992). D irect sequencing of
reverse transcribed mRNA from m ale patients with detectable message has
revealed p oint m utations leading to prem ature term ination or missense m uta
tions as well as an exon skipping m utation an d a three base deletion of a single
am ino acid (Leahey, C harnas, an d Nussbaum , 1993).
T he function of OCFLL-1 is unknow n. O ne clue, however, is th at the protein p re
dicted to be encoded by OCR!. I has 71% similarity to hum an platelet inositol poly
408 ICHIRO OKABE AND ROBERT L. NUSSBAUM
THERAPY
C u rre n t therapies for OCRL are mainly sym ptom atic treatm ent, such as extrac
tion o f cataracts, m anagem ent of glaucom a, bicarbonate replacem ent for renal
tubular acidosis, anticonvulsant drugs, o rth o p ed ic care for bone fracture, physi
cal rehabilitation, a n d special education. P hosphate a n d vitamin D supplem enta
tion may also be n eed ed to control rickets. Identification o f the gene for OCRL
facilitates the u n d erstanding of m olecular pathogenesis, and will contribute to
the developm ent of new strategies for treatm en t as well as accurate carrier detec
tion an d prenatal diagnosis.
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18. CRYSTALLIN GENES AND CATARACT
1Department of Genetics, Research Institute, The Hospital for Sick Children, 555 University
Avenue, Toronto, Ontario M5G 1X8, Canada
2Department of Molecular and Medical Genetics, University of Toronto, Toronto,
Ontario M5S 1A8, Canada
3Department of Biologica l Sciences, Washington Singer Laboratories, University of Exeter,
Exeter EX4 4QG, United Kingdom,
4Department of Molecular Biology, University of Nijmegen, Toernooiveld,
6525 ED Nijmegen, Netherlands
ABSTRACT
Crystallins constitute over 90% o f the water-soluble proteins in the ocular lens.
They are subdivided into d ifferent classes according to their m olecular proper
ties. Early biophysical studies indicated that crystallins are im p o rtan t for confer
ring, as well as m aintaining the transparency o f the lens. M ore recent inves
tigations have focused on the isolation and characterization of the genes encod
ing these proteins; these studies have resulted in the identification of m utations
associated with a n u m b er o f cataracts in m ammals, thus strengthening the
involvem ent o f the crystallins in lens transparency. Moreover, the phenotype o f at
least one o f these m utants suggests th at crystallins are also im p o rtan t for p ro p er
lens developm ent.
INTRODUCTION
Crystallins are the m ajor soluble proteins in the eye lenses o f vertebrate animals.
Most of the crystallins are contained in the fibre cells which occupy the bulk of
this m ulticellular organ (] gure 1; for a review o f em bryology and m orphology of
the lens, see Zigman, 1985). T he u n iq u e spatial arran g em en t and short range
ord er of these m olecules are tho u g h t to be im p o rtan t for the m aintenance of the
rem arkable transparency an d refractive properties o f the lens (Delaye and Tar-
dieu, 1983).
T he functional im portance of crystallins is also reflected by their evolutionary
conservation and their role in disease conditions. Loss o f lenticular transparency,
com m only known as cataract, can be due to a variety o f reasons, such as aging,
traum a, o r m etabolic im balance, but at least two congenital form s have already
been shown to be due to m utations in crystallin genes (see below). Since cataracts
414 MIREILLE CARTIER ET AL.
zone of
primary fibre cell
anterior lens fibres differentiation
lens vesicle epithelium
secondary
lens fibres
range from slight, dust-like opacities which do no t disturb the vision to com plete
opacification o f the lens, som etim es accom panied by loss o f the norm al anatom y
o f the organ, a careful analysis o f these in h erited cataracts should help in u n d er
standing the various roles o f crystallins in cataractogenesis.
D ifferent classes of crystallins have been described for a n u m b er o f vertebrates
and, m ore recently, for some invertebrate species. Most of the initial knowledge
ab o u t crystallins was derived from the calf, simply because of their accessibility.
T h re e m ajor classes of crystallins, a , p an d y, were originally described. They
are distinguished on the basis of their chrom atographic properties, with the high
m olecular weight fraction being the a-crystallins, the interm ediate m olecular
w eight fraction, the f$-crystallins and the low m olecular weight fraction, the y-crys-
tallins. A lthough the proteins contained in each of the th ree fractions are ra th e r
h eterogeneous, they are antigenically closely related. As we now know, the com
plexity of the crystallin proteins is not only due to the variety of genes that encode
these polypeptides, bu t is also caused by post-translational m odifications o f the
p roteins themselves (for a review of the crystallins, see H arding and Dilley, 1976;
B loem endal, 1981, 1985; Piatigorsky, 1984; Lindley et al., 1985; Wistow and
Piatigorsky, 1988; B loem endal and de Jong, 1991).
While a-, P- and y-crystallins can be found in alm ost all vertebrate lenses,
u nique "taxon-specific" crystallins have been identified for many species (for re
view, see de Jo n g et al., 1989; Piatigorsky an d Wistow, 1989, 1991). For exam ple,
lenses from birds and some reptiles contain a d ifferent class o f proteins called the
CRYSTALLIN GENES AND CATARACT 415
5-crystallins. In hum ans, there is no evidence for crystallins o th er than the a-, (3-
and "/-cr ystallius.
T he various crystallin species are n o t uniform ly distributed within the lens; a
specific concentration grad ien t for each protein is established through tem poral
and spatial regulation o f gene expression as well as post-translational modifica
tions. T here is, however, a general increase o f p rotein concentration towards the
cen ter (nucleus) o f the lens, prim arily due to the increasing concentrations o f |3-
an d y-crystallins. As a consequence, the refractive index increases towards the core
o f the lens, com pensating for the changing curvature of the lens (Fernald and
W right, 1983). In addition, since the term inally differentiated fibre cells are never
lost from the lens, but ju st m igrate towards the lens center while gradually losing
their nucleus an d subcellular organelles, older proteins are located at the nucleus
o f the lens and newly synthesized ones are located in the o uter cortex region. T he
o lder crystallins in the lens nucleus cannot, therefore, be replaced a n d m ust last
the lifetim e of the organism . H ence, crystallins are very stable proteins, able to re
sist d en atu ratio n by oxidation, UV radiation (sunlight) as well as racem ization, all
processes th at occur with age.
In the following sections, we provide a discussion on each of the three m ajor
classes of crystallins found in the hum an lenses and o u r c u rre n t knowledge on the
involvem ent of these proteins in cataractogenesis.
a-Crystallins
(Tardieu et al., 1986), while others propose that the protein has a m icellar struc
ture (Walsh, Sen and C hakrabarti, 1991; Rad lick and Koretz, 1992).
T h e a-crystallins may also have o th er functions than that o f being an a b u n d a n t
structural p rotein o f the lens. They show sequence hom ology to the small heat
shock proteins (Ingolia and Craig, 1982). In fact, aB-crystallin has been found in
o th er tissues than the lens an d its synthesis can be induced by stress (such as heat
shock and incubation in Cd+2) in cultured fibroblasts (Bhat and N agineni, 1989;
Dubin, Wawrousek and Piatigorsky, 1989; Klemenz et al.. 1991). This protein also
accum ulates in the brains o f hum ans suff ering from A lexander's disease (a degen
erative neurological disorder characterized by the presence of R osenthal fibres or
g ran u lar inclusions within astrocytes) an d in the brains o f scrapie-infected ham
sters (Duguid, Rohwer and Seed, 1988; Iwaki et al., 1989). M ore recently, a-crys
tallins have been found to have chaperone activity (Horwitz, 1992), raising the
possibility that they may stabilize o th er crystallins in the lens.
Genomic organization
As best dem onstrated in the ham ster, the two a-crystallin genes have the same
genom ic structure; both have three exons an d the introns are located at equiva
len t positions in the coding sequence, suggesting a close evolutionary relation
ship between these two genes (Quax-Jeuken et al., 1985b; van d en Heuvel et al.,
1985). In hum ans, a partial sequence has been obtained for the aA gene (McDe-
vitt et al., 1986) and a com plete sequence for aB (D ubin et al., 1990). T heir
stru ctu re is similar to th at o f the ham ster genes. In addition, the hum an A-crystal-
lin gene is localized to chrom osom e 21 region q22.3 (Table 1 and Figure 3; Quax-
Jeu k e n et al., 1985c; Hawkins et al., 1987) and aB-crystallin to chrom osom e 11,
betw een bands q22.3 and q23.3 (Table 1 and Figure 3; B rakenhoff et al., 1990b).
Expression pattern
D uring developm ent of the lens, at least in rodents, a-crystallins are the first crys
tallins to appear. They are p resen t in the lens vesicle before form ation o f the pri
m ary lens fibre cells (McAvoy, 1978; van Leen e ta l., 1987a, b). T he synthesis o f a-
crystallins com m ences mainly in the epithelial cell layer and early stages o f fibre
cell elongation. In the rat, the levels o f both aA- and aB-crystallin mRNAs are rel
atively constant at all ages o f growth and developm ent (Aarts, Lubsen and
Schoenm akers, 1989; V oorter et al., 1990), suggesting an even distribution o f a-
crystallins in all cells. Relatively little is known about the developm ental xegula-
tion o f a-crystallin gene expression in hum ans, except that aA-crystallin mRNA
can be detected in the fetal lens (B rakenhoff et al., 1990a).
CRYSTALLIN GENES AND CATARACT 417
T ab le 1 L o ca tio n o f th e h u m a n crystallin g en es
a The symbols for the p-crystallins may be changed in the near future to better reflect the name of the
protein: PA1/A3 would be CRYBA3/A1, PB2 would be CRYBB2 (and the pB2 pseudogenc: CRYBB2P1)
and pB3 would be CRYBB3.
[3-Crystallins
Protein structure
T h e stru ctu re o f the calf pB2 dim er has been solved by X-ray crystallography (Bax
et al., 1990). T he core of each m olecule contains four antiparallel P-sheeted (P~
b arrel) structures known as "Greek key motifs, organized into two similar
dom ains o f two motifs each (Figure 2). T he dom ains are flanked by a N- and a C-
term inal arm . T he two dom ains are separated by a connecting peptide in an
ex tended conform ation, such that the N -term inal dom ain o f one pB2 chain inter
acts with the C-term inal dom ain of the second PB2 chain and vice versa. All
known p-crystallin sequences can be m odeled in the same tertiary conform ation
and it is generally assum ed that all p-crystallins fold into the same four m otif/tw o
dom ain structure. The m ost distinct feature for individual P-crystallin m olecules
is located at their N- an d C-term inal arms. In addition to sequence variations, the
N -term inal arm s differ significantly in length (from about 55 am ino acid residues
in pBl to 15 am ino acid residues in PA1). T he C-term inal arms, varying between
13 an d 18 am ino acid residues in length, are only found in the basic P-crystallins
(for review, see Lubsen, Aarts an d Schoenm akers, 1988).
Genomic organization
T h e com plete set o f calf P-crystallin sequences has been derived from cDNA
clones (G orin and Horwitz, 1984; Q uax-Jeuken et al., 1984; Hogg et al., 1987; van
Rens et al., 1991a). PA1 an d pA3 are encoded by the same mRNA, using the same
reading fram e, bu t with d ifferent initiation codons, resulting in a pAl polypep
tide sh o rter than pA3 by 17 am ino acid residues (Quax-Jeuken et al., 1984). This
double initiation o f the p A l/A 3 mRNA has been preserved in evolution and is
fo u n d no t only in calf, but also in chicken and in m an (Hogg et al., 1986; Peter
son an d Piatigorsky, 1986). Further, on the basis of cDNA hybridization patterns,
CRYSTALLIN GENES AND CATARACT 419
orthologous m em bers o f the calf p-crystallin gene family probably exist in hum an
(van Rens e ta l., 1991b).
T he structure o f the p-crystallin genes has been derived from representative
m em bers in hum an, rat, a n d m ouse (Inana et al., 1983; den D u nnen et al., 1985b;
Hogg et al., 1986). A lthough the paralogous P-crystallin genes no longer share suf
ficient sequence identity to recognize each o th er in hybridization experim ents,
the genes are clearly evolutionarily related. A typical P-crystallin gene contains six
exons. T he first exon o f the pR l gene is non-coding, whereas the first exon o f the
PA3/A1 gene contains the start codon for the PA3 protein. T he rem ainder o f the
gene structure is rem arkably sim ilar am ong all P-crystallin genes, with the second
exon coding for the N -term inal arm an d each o f the 4 following exons (exons 3 to
6) encoding a G reek key motif. In addition, for the basic chains, exon 6 also en
codes the extension o f the C -term inal arm .
Because o f the similarity in gene structure, it is generally believed th at all P-crys-
tallin genes were derived from a single ancestor sequence. D uplication of this an
cestral gene an d subsequent divergence would have led to the proto-acidic and
proto-basic P-crystallin genes which would then have been duplicated fu rth e r to
form the 6-m em ber gene family seen today. In m an, all P-crystallin genes except
the pA 3/A l and possibly the PA2 are located on chrom osom e 22 (Table 1 an d Fig
ure 3; Law et al., 1986; Sparkes e t al., 1986; H ogg et al., 1987; H ulsebos et al.,
1991). T hree o f the genes, pB2-l, pB2-2 and pB3, have been m apped within a dis
tance o f 120 kb (Bijlsma et al., 1991), but the pB2-2 sequence probably co rre
sponds to a pseudogene (B rakenhoff et al., 1992). It is o f interest to note that only
a single copy o f the PB2 gene can be fo u n d in the rat, although th at gene is also
closely linked to pB3 (Aarts et al., 1987). Moreover, chrom osom e duplication has
420 MIREILLE CARTIER ET AL.
Figure 3 Ideogram o f the hum an chrom osom es showing the Giemsa banding patterns. T he
locations of the m ajor genes expressed in the lens and loci responsible for dom inantly inher
ited congenital cataracts are indicated. S tandard gene nom enclature according to H um an
G ene M apping 11 (McAlpine et al., 1991) is used. T he gene symbols used are as listed in Ta
bles 1 and 2, except for CRYB2 which, here, represents the whole cluster of (i-crystallin genes
m apped to chrom osom e 22: (3A2, [3B2, pB3 an d (3B4 an d MIP, which is the symbol for the ma
jo r intrinsic protein o f the lens fibre m em brane.
CRYSTALLIN GENES AND CATARACT 421
b een proposed as a m echanism for P-crystallin gene duplication; the close prox
imity o f the PA1/A3 gene to the von R ecklinghausen neurofibrom atosis locus
(NF1) on chrom osom e 17 (Fain et al., 1989) an d the co-existence o f the pR2 and
PB3 genes and neurofibrom atosis 2 (NF2) on chrom osom e 22 (H ulsebos et al.,
1991) are tho u g h t to be m ore than coincidental.
T he developm ental regulation o f the P-crystallin genes has been well docu
m ented for the chicken (Ostrer, Beebe an d Piatigorsky, 1981; H ejtm ancik et al.,
1985) and rat (Aarts, Lubsen an d Schoenm akers, 1989). T he latter study shows
th at the pBl and pB3 genes are both active d u rin g early lens form ation an d that
their respective transcripts reach th eir m axim um levels aro u n d birth. In contrast,
the PB2 mRNA only accum ulates after birth b u t rem ains p ro m in en t at least up to
one year o f age. T he PA3/A1 gene shows an interm ediate p attern o f expression,
as its transcript is m ost ab u n d a n t betw een one an d three m onths after birth. Rel
atively little is known ab o u t the developm ental profile for the hum an p-crystallin
genes. T he p attern may be sim ilar to th at o f the rat, however, because hum an PB3
is also expressed early and PB2 is p resen t in later stages (B rakenhoff et al., 1992).
Based on the mRNA profile, it may be assum ed that there is a differential gradi
en t of p-crystallin species across the lens. For exam ple, the nucleus is expected to
contain relatively high levels of PBl and PB3 whereas the cortex would include
prim arily PB2. C onsistent with this assum ption and the fact that the form ation of
PH requires the presence o f pB l, th ere is a clear shift of pH in the rat lens nucleus
to p in the cortex (Siezen, Anello an d Thom son, 1986).
y-Crystallins
T he y-crystallins are found in the low m olecular weight fraction o f the soluble
proteins from the lens. T he 6-7 m onom eric proteins o f about 20 kDa included in
this family can be divided into two branches. T he older branch com prises a single
protein called ys, which is p resen t in all vertebrate species exam ined thus far.
This protein was once tho u g h t to be a m onom eric P-crystallin (thus previously
nam ed Ps), mainly because its N -term inal residue is acetylated, as is the case for
the P-crystallins, but n o t for the o th er y-crystallins. C loning and sequencing stud
ies, however, revealed th at the coding sequence and e x o n /in tro n organization of
the ys gene are m ore related to those o f the y genes than those o f the P genes
(Quax-Jeuken et al., 1985a; van Rens et al., 1989). T he o th er branch o f the y-crys-
tallin gene family contains u p to six closely related proteins (whose genes are
clustered to g e th e r), which are known as yA to yF. In contrast to the a - , P- an d y s-
crystallins, these yA to yF-crystallins are n o t present in all vertebrate species;
while these low m olecular weight proteins can be found at varying levels in fish,
frogs and m am m als, they are absent in birds an d som e reptiles.
422 MIREILLE CARTIER ET AL.
Protein structure
Genomic organization
All y-crystallins are specific to the lens; they are located prim arily in the nucleus
region, presum ably to provide the dehydrated environm ent required. In rodents,
the precise spatial an d tem poral regulation o f individual y-crystallin genes has
been well d o cum ented and, as expected, m ost o f the genes are expressed at early
stages o f developm ent (M urer-O rlando et al., 1987; van Leen et al., 1987ab;
Goring, B reitm an and Tsui, 1992). T ranscription of the yA to yF genes is
restricted to the term inally differentiated fibre cells and can be readily detected
in day 12 em bryonic lenses w hen the prim ary fibre cells begin to form . T he
steady state level o f mRNA for m ost y-crystallin mRNAs increases gradually and
reaches a peak at aro u n d day 10 after birth. In addition, the yD, yE and yF are
relatively m ore ab u n d a n t than the o th er species, whereas the yB gene rem ains
active long after the o th ers have been shut down. A sim ilar expression p attern is
fo und in the calf, w here yB is the p ro m in en t cortical y-crystallin (Slingsby and
Croft, 1973; Slingsby an d Miller, 1983).
T h ere are th ree to four times less y-crystallins in the hum an lens than in the ro
d e n t lens (Siezen et al., 1988; T hom son an d Augusteyn, 1985). In addition, al
though the hum an yA, yB, yC and y D genes are transcriptionally active, only the
latter two yield ab u ndant mRNA and p rotein (Russell et al., 1987; Siezen et al.,
1987; B rakenhoff et al., 1990a). T he yC an d yD genes are relatively active in the
fetal lenses bu t are differentially shut-off after birth. At 22 m onths, only the yD
mRNA can be detected an d it rem ains present till at least 10 years o f age (B raken
h o ff et al., 1990a). T he yE gene is transcribed at a low level; nevertheless, a small
am ount o f its predicted (truncated) protein product has been detected in the h u
m an lens (N.H.L., u n p u b l.). T he yF gene lacks a TATA box and is probably silent
(M eakin, B reitm an an d Tsui, 1985).
T he exceptional position o f the ys gene in the y-crystallin gene family is also re
flected by its expression pattern: in the ra t lens, the ys gene is active only after
b irth (Aarts, Lubsen and Schoenm akers, 1989). However, although the develop
m ental expression o f ys has not been followed in m an, it is known that the tran
script o f this gene is p resen t in a 9 week old hum an fetal lens (N.H.L., unpubl.).
CATARACTS
Since lens opacity can occur for many reasons, it is impossible to define a com
m on pathway o f lens disorganization leading to the various form s o f cataracts.
Nevertheless, num erous studies have described the m orphological differences
betw een the norm al an d cataractous lens (see review by B erm an, 1991). T he gen
eral features of a cataractous lens, aside from the opacity, may include the pres
ence o f vesicles, lens fibre swelling, gaps betw een the fibre cells which may be
filled with liquid o r debris, and loss of m em brane integrity.
CRYSTALLIN GENES AND CATARACT 425
At the biochem ical level, cataractous lenses generally display high m olecular
weight proteinaceous aggregates as well as some proteolysis products, unbalanced
concentrations o f ions such as calcium , sodium and potassium and loss o f glu
tathione an d soluble crystallins. T he high m olecular weight protein aggregates are
th ought to be caused by the unfolding o f the various crystallins, leading to the ex
position of hydrophobic groups which may then interact together.
Some o f the high m olecular weight protein aggregates p resen t in the hum an
cataractous lens have been isolated an d fractionated, to reveal peptides of m olec
ular weights aro u n d 43 kDa, 20 kDa an d 10 kDa (Spector et al., 1979; Garner,
G arner and Spector, 1979). While the 43 kDa p rotein seems to be an extrinsic
m em brane protein, the 20 kDa is th o u g h t to be a crystallin and the heterogeneous
10 kDa fraction is com posed o f y-crystallin (as d eterm in ed by im m unological re
action) (Takem oto, Straatsm a and Horwitz, 1989). It has been postulated that
such 10 kDa fragm ents may occur from cleavage at the connecting peptide be
tween the two dom ains o f the p rotein. Indeed, this 10 kDa fragm ent, which also
accum ulates with age, has been identified as the C-term inal dom ain o f yD (Srivas-
tava, Srivastava an d Silney, 1992). F urtherm ore, in a study o f 13 cataractous lenses,
10 were fo u n d to contain y-crystallin proteins in close association with the m em
b rane (all ten cataracts presenting nuclear sclerosis), while 4 displayed (3-c,rystal-
lins associated with the m em brane (two o f which had no nuclear involvem ent) and
n o n e showed a-crystallin association with the m em brane (Kodam a and Takem oto,
1988). From this study, it may be concluded that y-crystallins can play a role in the
cataractogenic process, especially in cases of nuclear cataracts. Such a role may be
attrib u ted to th eir high concentration of thiol groups on the surface of the m ole
cule, which may be oxidized, leading to a destabilization o f the protein with con
com itant unfolding, a n d /o r participate in interm olecular disulfide bonds with the
m em brane.
All o f these changes, which have b een studied mainly in senile cataracts, point
to sites for potential m utations in in h erited cataracts, such as the crystallin genes
and the m em brane proteins including the pum ps and channels responsible for
the osm otic balance. F urtherm ore, o th e r genes known to play a role in lens devel
op m en t may be im plicated in hered itary congenital cataracts.
n a n t transm ission is the m ost com m on m ode o f inheritance, but autosom al reces
sive an d X-linked form s are also known to occur.
Morphologies
Genetic factors
Lens opacification may result directly from a b e rra n t functions expressed in the
lens itself, such as alteration o f structural proteins (e.g. crystallins) o r proteins
th at serve to pro tect the lens from dam age and to preserve clarity o f the lens
m atrix (e.g. glutathione S-transferase). Alternatively the defect may lie in a m eta
bolic pathway resulting in accum ulation an d deposition o f insoluble m aterial in
the lens (e.g. galactokinase activity).
In general, it is anticipated th at a gene defect causing a disruption o f the struc
tural proteins would have a d om inant phenotype w hereas an enzymatic defect
m ight be recessive. T herefore cataracts caused by defects in the crystallins an d ma
j o r intrinsic proteins of the lens fibre cells may be predicted to exhibit dom inant
p atterns o f inheritance, since the presence o f a m utant p ro d u c t is likely to result
in loss o f lens clarity. O n the o th er hand, proteins involved in protecting the lens
from dam age are likely to rem ain effective even at reduced levels o f activity, at least
CRYSTALLIN GENES AND CATARACT 427
Linkage studies
T he genetic linkage approach may be used to identify the basic genetic defect
underlying the various in h erited lens opacifications in hum ans. So far, all o f the
gene m apping studies on in h erited congenital cataract loci have considered only
those exhibiting a d o m in an t m ode o f inheritance. T he genetic loci known to
cause dom inantly in h erited congenital cataracts are listed in Table 2 and local
ized on Figure 3. T he possible genetic heterogeneity an d the small family size
re n d e r the m apping approach m uch less attractive in the case of the recessive
form s o f cataracts.
T h ere are at least two disease syndrom es w here cataracts appear frequently,
potentially as a result o f abnorm al crystallin gene expression. First, about 60% of
patients with Down's syndrom e, which is due to trisomy 21, suffer from cataracts.
It has b een postulated that lens opacity in Down's syndrom e may be due to the
over-expression of the aA-crystallin gene located on th at chrom osom e. Second,
neurofibrom atosis type II (NF 2) has been associated with posterior capsular cat
aracts (K aiser-K upfer et al., 1989). Since the NF2 locus is close to the cluster of
CRYSTALLIN GENES AND CATARACT 429
While positive linkage data have b een d o cum ented in som e family studies, nega
tive findings an d inconclusive results have b een obtained in m any o th er studies.
H am m erstein and Scholz (1974) fo u n d no evidence for linkage between a he
reditary central cataract and 11 m arkers, including FY in a six generation family.
No linkage was detected betw een the locus responsible for an autosom al dom i
n a n t nuclear cataract an d 21 inform ative m arker loci in a large family studied by
H untzinger, W eitkamp an d Roca (1977).
In a study on a single large Italian family with dom inantly in h erited pulverulent
cataracts, there was no evidence for linkage of the locus responsible for the cata
racts to FY (Stabile et al., 1983).
B atem an et al. (1986) investigated a large family with dom inantly in h erited con
genital cataracts exhibiting considerable variation between affected m em bers of
the family, but with opacification o f the em bryonal nucleus as a com m on trait to
all the cataracts available for exam ination. Linkage was excluded conclusively with
11 genetic m arkers including FY an d HP. A fu rth e r 15 polym orphic loci yielded no
evidence for linkage.
B eaum ont et al. (1989) and Birdwood et al. (1992) investigated a large English
family with autosom al d o m in an t lam ellar cataracts for linkage with chrom osom e
Iq21-q23 m arkers and with the y-crystallin gene cluster. T here was no evidence for
linkage to either o f these regions.
These studies em phasize the high degree o f genetic heterogeneity underlying
dom inantly inherited cataracts. F uture studies on the m apping o f loci responsible
for in h erited cataracts should investigate the segregation o f alleles of the genes
identified as potential candidates for defects in lens clarity, the crystallins and m a
jo r intrinsic proteins o f the lens.
ANIMAL MODELS
T he Philly Mouse
T h e Philly m ouse suffers from a dom inantly inherited, progressive cataract which
appears approxim ately 15 to 30 days after birth, dep en d in g on w hether the ani
m al is homozygous or heterozygous for the m utation (Kador et al., 1980; C arper
et al., 1982). T he cataract first appears as a faint an terio r subcapsular opacity
aro u n d the lens sutures which, within 1 m onth, develops into a dense an terio r
subcapsular and nuclear cataract. In the m utant lens, the first cytologically
detectable aberration is the appearance of dense bodies in the an terio r portio n
o f the newly elongating fibre cells at one week after birth (Uga, Kador and Kuwa-
bara, 1980). This change is followed by the failure of the fibre cells to elongate as
well as a general swelling o f the lens cells. This swelling initially led to the conclu
sion that the Philly cataract was due to a defect in osm otic regulation.
A m arked decrease in the levels o f soluble (3- and y-crystallin proteins was n oted
in the m utant lens whereas the levels o f a-crystallins were apparently norm al
(Piatigorsky, K ador and Kinoshita, 1980). In addition to this general decrease of
soluble crystallin levels, often observed in cataractous lenses, a specific p-crystal
lin related antigen ap p eared to be com pletely lost (Zigler, C arper an d Kinoshita,
1981). S ubsequent in vitro RNA translation studies showed that a 27 kDa P-crystal-
lin p rotein, now known as PB2, was no t p roduced from hom ozygous Philly lens
RNA (C arper et al., 1982). Sequence com parison o f the pB2 cDNA isolated from
b o th norm al and m utant lenses revealed th at the cDNA found in the Philly lens
carried a 12 nucleotide deletion at the 3' e n d o f its coding region (C ham bers and
Russell, 1991). This deletion would result in a m utant pB2 protein lacking 4
am ino acids at the end o f the fourth G reek key motif. C onsistent with the DNA
finding, a p rotein slightly sm aller than pB2 was in fact detected in the Philly lens
with a polyclonal antibody directed against PB2, b u t no t with a m onoclonal anti
body raised against the C-term inal p o rtio n of the norm al protein (N akam ura et
al., 1988). This result suggested that the m u tan t Philly phenotype may be caused
by the presence o f an altered PB2 polypeptide.
CRYSTALLIN GENES AND CATARACT 431
In a norm al m ouse, the am ount o f [3B2 polypeptide is alm ost undetectable at 1-5
days after birth bu t increases m arkedly thereafter (C arper et al., 1982). This pat
tern of expression is consistent with the im m unocytochem ical localization o f the
pB2-crystallin to the newly elongating fibre cells in the neonates (Carper, Smith-
Gill and Kinoshita, 1986). Biochemically, the m utant pB2-crystallin differs from
its wild-type co u n terp art in two ways. First, whereas the wild type PB2 protein is
very heat-stable, the m u tan t protein is heat-labile (N akam ura et al.. 1988). Sec
ond, the m u tan t p rotein is found as high m olecular weight aggregates but the
wild-type protein is found as an octam er, in the pH fraction (Russell an d C ham
bers, 1990). Since failure o f the fibre cells in the cortical layers to elongate
appears to be an early sign o f the defect in the Philly lens, pB2-crystallin may be
im p o rtan t for norm al secondary fibre cell elongation.
T he Philly lens also differs from a n orm al lens in its phase-separation tem pera
ture (Tc), o r the tem p eratu re at which the lens cytoplasm moves from a transpar
ent state (single phase) to a condition of opacity (dual phases). For every lens, this
Tc varies with age, since the com position o f the lenticular cytoplasm is continually
changing. T he Tc o f the hom ozygous Philly lens is approxim ately 17C h ig h er than
that o f the wild type lens at day 1 after birth (Clark and Carper, 1987). Thereafter,
the Tc of the Philly lens varies in a fashion sim ilar to that o f the wild-type lens until
aro u n d day 27, when the Tc of the Philly m ouse suddenly soars, resulting in the
appearance o f a cataract. Meanwhile, the Tc o f the wild-type lens continues to de
cline. It seems probable th at the increase in Tc results from the lack o f heat stabil
ity an d the abnorm al aggregation p attern o f the m utant PB2 p rotein, bu t how this
alteration interferes with fibre cell elongation rem ains to be elucidated.
T he Elo Mouse
W hen eye rudim ents were taken from the Elo m ouse an d cultured in vitro, the
lens fibre cells failed to elongate properly, suggesting that the defect was not
caused by an extraocular com p o n en t (W atanabe et al., 1980). In addition, chi
m eric anim als derived from m ixing BALB/c and C 3H -Elo/+ em bryos were fo u n d
to have lenses with a spectrum o f phenotypes, ranging betw een wild type, n orm al
sized b u t opaque an d Elo-like. T he severity o f the lens defect was pro p o rtio n al to
the relative contribution o f lens fibre cells from the m u tan t strain (Yoshiki et al.,
1991). In fact, im m unohistological analysis showed that the necrotic fibre cells
originated from the C3H strain. These studies thus argued strongly that the Elo
phenotype was expressed in a cell autonom ous fashion.
T he Elo m utation is located on chrom osom e 1, close to the -crystallin gene clus
ter (yA to yF) (Masaki and W atanabe, 1989; Q uinlan et al., 1987). T he specific in
volvem ent o f the y-crystallin locus is fu rth e r suggested by the m arked reduction of
the general y-crystallin mRNA an d p rotein levels when com pared to those o f p-
an d y-crystallins (Masaki and W atanabe, 1988; Q uinlan et al., 1987). M ore recen t
ly, a single base pair deletion has been fo u n d in the yE-crystallin gene of the Elo
m ouse (Cartier, B reitm an and Tsui, 1992). T he deletion, which is located in the
m iddle of the last exon o f the gene, would result in a truncated polypeptide miss
ing the last G reek key m otif o f the norm al protein (replaced by 11 am ino acids
specified by a different reading fram e). T h e causal relationship between this dele
tion an d the Elo phenotype is fu rth e r su p p o rted by the genetic linkage data show
ing no recom bination between the two in 274 meioses. Since mRNA from the
m u tan t allele is readily detectable in the Elo lens, it seems probable th at the failure
o f the lens fibre cells to elongate is caused by the presence o f a putative tru n cated
polypeptide. T he cu rren t hypothesis, therefore, about the Elo phenotype is that
its small lens would be due to the presence o f a tru n cated yE polypeptide. Al
though the m olecular m echanism by which this m u tan t y-crystallin polypeptide
causes a small lens is unknow n, this result suggests a role for y-crystallins in fibre
cell differentiation.
13 /N Guinea Pig
liver and kidney. In the hom ozygous m u tan t guinea pig the protein is absent
from all three organs, while it is p resen t at h alf the norm al levels in the liver and
kidney o f the heterozygous anim al (H uang et al., 1990; Zigler an d H uang, 1988).
Thus, an association betw een the in h erited cataract condition and the absence of
^-crystallin has been postulated.
M olecular analysis shows that the ^-crystallin gene in the cataractous guinea pig
harbors a deletion at the splice-acceptor site o f exon 7 (Borras et al., 1990;
R odriguez et al., 1991). As a result, the m ajor mRNA transcript from this gene
lacks the entire exon 7 sequence and, since this exon contains 102 base pairs
(bp), an internally deleted polypeptide is predicted. In addition, however, the
m utation has apparently activated a cryptic splice-acceptor site, giving rise to a
m inor mRNA species with a fram eshift caused by the insertion of a 20 bp intron
sequence in place o f the missing exon. T he presence of these putative m u tan t
polypeptides may explain the fully p e n e tra n t an d d om inant phenotype o f the cat
aract m utation. N either o f the m u tan t form s are predicted to form the tetram eric
structure postulated for the norm al p ro tein (Rao an d Zigler, 1992).
Studies on the genetic causes o f cataract have also been perfo rm ed with m ouse
m utants obtained after parental m utagenic treatm en t (Kratochvilova, 1981; Graw
434 MIREILLE CARTIER ET AL.
et al., 1986; Kratochvilova and Favor, 1988). Fifteen of the m utants o btained have
been tested for allelism an d found to segregate into seven d ifferent groups
(Kratochvilova and Favor, 1992). T he largest group com prises five alleles and,
based on m apping results from two o f the strains, the com m on im itation locus
appears to be located on chrom osom e 1, close to the y-crystallin cluster (J. Favor,
M.F. Lyon an d J. Loster, personal com m unication). These d o m in an t cataracts
may, therefore, be caused by m utations in the y-crystallin genes.
CONCLUSION
Crystallins are the m ain soluble proteins o f the eye lens. As such, they play a
m ajor role in the two specific properties o f this organ: accom odation an d trans
parency. It is, therefore, th o u g h t that th eir recru itm en t by the lens was due to
th eir appropriate biophysical characteristics. This view is fu rth e r stren g th en ed by
the fact th at different anim al species, with varying lenticular requirem ents, have
recru ited num erous proteins as crystallins to fit their particular needs. For exam
ple, rodents, which are n octurnal anim als, have evolved a hard, dehydrated and
spherical lens with high levels of y-crystallins. Meanwhile, birds, requiring high
accom m odative power, n eed a m ore flexible and hydrated lens, obtained by
replacing the y-crystallins with the 8-crystallins, which allow m ore hydration.
T he study of inherited cataracts has fu rth e r dem onstrated the im portance of
the crystallins for lenticular function, since fo u r m utations in crystallin genes have
been associated with lens disorders so far: the hum an y-crystallin cluster associat
ed with the Coppock-like cataract, the m urine pB2 and yE im plicated in the m u
tan t phenotypes of the Philly an d Elo m ice and the ^-crystallin m utated in the
cataractous N /1 3 strain o f guinea pigs. F urtherm ore, two o f these crystallin m uta
tions, the ones present in the m urine (JB2 an d yE genes, affect the fibre cell elon
gation process and may, therefore, im plicate these crystallin proteins in the
developm ent o f the lens. Such results are p art o f the growing data im parting a
function o th er than structural to the d ifferent crystallins. For exam ple, m ost tax-
on-specific crystallins were derived from enzymes and still possess an enzymatic ac
tivity which may be necessary for p ro p e r lens function. Also, as discussed above,
the a-crystallins have recently been fo u n d to possess some ch ap ero n e activity.
T h erefore, although the three-dim ensional structure and biophysical properties
of the crystallins are probably the main reasons for the recru itm en t o f these p ro
teins by the lens, their enzymatic activity may have also played an im p o rtan t role
in this process and may still be req u ired for the p ro p e r function o f the organ.
A nother p oint which should also be stressed is the advantage o f the genetic ap
proach to investigating the role of crystallins in cataractogenesis. In d eed , al
th ough biochem ical studies had im plicated these m olecules in the actual
lenticular opacity, it was never possible to d em onstrate w hether the crystallin in
volvem ent was a prim ary or secondary event. In the case o f in h erited cataracts,
however, if the m utation responsible for the disease lies in a crystallin gene, it is
CRYSTALLIN GENES AND CATARACT 435
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19. ANIRIDIA
INTRODUCTION
Phenotype
A niridia is a congenital in h erited disorder o f the eye which derives its nam e from
the noticeable iris hypoplasia seen in m ost cases. T he nam e is som ething o f a mis
n o m er since there is always som e iris tissue present although this may be so m ini
mal as to be invisible to the naked eye. T he nam e also draws attention to the m ost
dram atic feature o f the phenotype from the observer's point o f view, when in
reality the effects on the rest o f the eye are far m ore visually disabling for the
patient. T he associated eye an d visual defects include some or all o f the follow
ing: corneal opacification, cataract, lens dislocation, ciliary body hypoplasia,
foveal dysplasia, optic nerve hypoplasia, strabism us and nystagmus (H ittner,
1989). To this long list o f what ap p ear to be mainly prim ary disorders resulting
from m aldevelopm ent o f the eye an d from altered control o f eye m ovem ent by
the central nervous system, we m ust add the com plication o f glaucom a. Very
rarely this is actually p resen t at birth, indicating th at it may be caused by a pri
m ary dysgenesis o f the aqueous outflow system but m ore com m only it has its
onset in pre-adolescence o r later, w hen it appears to be due to obstruction o f the
drainage angle by adhesions between the lens and the trabecular m eshwork
(Margo, 1983). T he wide range o f tissues affected has justifiably led to aniridia
being described as a panocular disorder (Nelson et al., 1984).
O ne striking feature o f aniridia is the variable expressivity. T he phenotype can
vary dram atically betw een individuals even within the same family. For exam ple,
the iris hypoplasia ranges from a readily visible alm ost com plete absence o f the
iris, th ro u g h enlargem ent an d irregularity of the pupil m im icking a colobom a, to
small slit-like defects in the an terio r layer seen only on transillum ination with a slit-
lam p (Figure 1; H ittn er etal., 1980). T he m ildest phenotype is detectable as subtle
abnorm alities o f the iris an d m acula which can be seen only by fluorescein
angiography (H ittn er et al., 1992). T he effect o f aniridia on visual function is also
very variable. Some families have been re p o rted in which m ost affected m em bers
only had iris hypoplasia an d alm ost n orm al visual acuity whereas in o th er cases the
presence of various com binations of defects elsewhere in the eye has caused a se
vere reduction in acuity (Grove, Shaw and B ourque, 1961; Elsas et al., 1977; H itt
n e r et al., 1980). In addition, all aniridia patients are at risk o f developing
glaucom a. T he incidence o f this com plication has been variously re p o rted as be
tween 6% and 75% which may reflect differences in the selection o f patients stud-
446 ISABEL HANSON ET AL.
ied an d th eir ages at the tim e o f assessm ent (Nelson et al., 1984). T he glaucom a
o f aniridia is notoriously difficult to m anage and if n o t treated successfully will d e
stroy any residual vision an d can cause intractable ocular pain necessitating rem ov
al of the eye.
Genetics
T he iris hypoplasia and associated ocular anom alies seen in aniridia provide no
simple im m ediate biological inform ation ab o u t the underlying functional defect.
However, the existence of a m utation can be used to identify the gene responsible
for these abnorm alities. D eterm ination o f the genom ic position o f the m utant
gene can lead us by positional clo n in g (Collins, 1992) to the aniridia gene.
Genom ic position can be ded u ced e ith e r from linkage studies in familial cases or
from the analysis o f cytogenetic (karyotypic) abnorm alities associated with the
448 ISABEL HANSON ET AL.
Cytogenetics
A key initial observation in m apping the aniridia gene cam e from cytogenetic
analysis o f patients with the WAGR syndrom e. T he visible constitutional intersti
tial deletions of the short arm o f chrom osom e 11 in these patients h ad a com m on
region o f overlap in band p l3 which suggested th at 11 p 13 contained a gene or,
m ore likely, a n u m b er of genes which, w hen hemizygous, caused the WAGR p h e
notype (Riccardi et al., 1978; Francke e ta l., 1979).
T he 11 p i 3 assignm ent was strongly su p p o rted by evidence from three pedigrees
in which aniridia segregated with a chrom osom al translocation. Simola et al.
(1983) described a family in which three affected m em bers from three genera
tions carried a balanced translocation t (4; 11) (q22;pl3). M oore et al. (1986) re
p o rted a family in which four affected m em bers spanning three generations
carried t( l 1;22) (p 13;q 12.2). Pettenati, Weaver and B urton (1989) described an af
fected fath er an d d au g h ter with t(5 ;l 1) (q l3 .1 ;p l3 ). Wilms' tum our was absent
from all three pedigrees, suggesting that Wilms' tum our and aniridia are caused
by separate loci within 11 p 13. T he chrom osom e 11 aniridia locus was designated
AN 2.
Linkage
m ining at the m olecular level the proxim al (centrom eric) an d distal (telom eric)
limits of the deletions in m any patients, it was possible to define the smallest
region of overlap (SRO) o f the deletions in which the critical genes m ust lie (Fig
ure 2).
CC cc
C9 O .
< < < CD
5 5 5 5
x< cc
<
Q
15
HVBS1
FSHB
14
D11S16
495, AN
13 p5
WT
12
CAT
11
D11S9
T he genes for catalase ( CAT) and the p-subunit o f follicle stim ulating h o rm o n e
(.FSHB), both o f which m ap to 11 p 13, were found to be deleted in many bu t not
all WAGR patients, placing them close to, b u t no t within, the SRO (Figure 2; Gla
ser et al., 1986; P orteous et al., 1987; Seawright et al., 1988). Cytogenetic com par
ison o f the extent o f deletions in patients lacking o n e m arker bu t n o t the o th er
revealed th at FSHB was distal to G1Y with the SRO betw een the two. T he CAT-FSHB
interval was subdivided into regions which m ust contain the Wilms' tu m o u r and
aniridia genes by d eterm in in g the positions of the breakpoints in key patients with
incom plete WAGR phenotypes. Since aniridia shows com plete penetrance, the
characterization o f the deletion in a p atien t with reduced catalase activity, Wilms'
tu m o u r and genitourinary abnorm alities, bu t not aniridia, placed the Wilms' tu
m our gene proxim al to the aniridia gene (Patient DAR, Figure 2; Davis et al.,
1988). T he precise position o f these breakpoints was defined using random DNA
m arkers. This approach pro d u ced a detailed consensus breakpoint m ap (Junien
and McBride, 1989).
O nce a high density of m arkers becam e available, the construction o f a long
range restriction m ap by pulsed-field gel electrophoresis (PFGE) was possible. T he
high resolution physical m ap o f the 11 p 13 region pro d u ced in this way showed for
the first tim e the distances, in kilobase pairs, between m arkers, and the positions
o f the key disease-associated chrom osom al rearrangem ents (Figure 3; C om pton et
al., 1988; Gessler an d Bruns, 1989; Rose et al., 1990). T he sites for rare cutting re
striction enzymes used for PFGE m apping also m ark the position o f regions which
are relatively rich in unm ethylated CpG dinucleotides. Such 'CpG island' sites are
often associated with the 5' ends o f genes, an d are in fact found upstream o f both
the Wilms' tu m o u r gene (Call et al., 1990; Gessler et al., 1990) an d the aniridia
gene (Ton e ta l., 1991).
WT1 PAX6
t# <#
p5 495 D11S16 FSHB
I \ I I
N N N N N
SIMO (A) ............. . . .. ;,|------ - ' .................. ..................... > -...t :---------------:
d g -85 (A) i z^ a M c a a i
Figure 3 Physical m ap o f the m inim al WAGR region in band 11p i 3 constructed by pulsed
field gel electrophoresis (C om pton et al., 1988; Gessler and Bruns, 1989; Rose et al., 1990).
Sites for the infrequently cutting restriction enzyme Notl are shown (N ). Positions of some of
the probes used to construct the m ap are rep resen ted by vertical arrows. T h e breakpoints in
key patients which helped subdivide the m ap into regions containing the genes for Wilms'
tum our and aniridia are shown below the map. T he phenotypes of these patients are desig
nated as for Figure 2. DG-85 is described In Davis et al. (1988), C om pton et al. (1988), and
Rose et al. (1990); DAR in Call et al. (1990), and van Heyningen et al. (1991); SATO in Ton
et al. (1991); and SIMO in Gessler, Simola and Bruns (1989), Davis et al. (1989), an d Ton et
al. (1991). T he Wilms' tum our (WT1) an d aniridia (PAX6) genes are rep resen ted by horizon
tal arrows (Call et al., 1990; Gessler et al., 1990; Ton et al., 1991).
breakpoint, they cloned the ju n c tio n o f the two AV//I fragm ents and since this po
tentially m arked a CpG island, it was decided to search for coding sequences in
this region. O ne genom ic fragm ent, conserved across species, was successfully
used to isolate cDNA clones from an 8.5d em bryonic m ouse library and a hum an
adult retinal library. T he m ouse cDNA was used in tu rn to isolate a cDNA from a
h u m an foetal eye library (Ton et al., 1991).
Sequencing of the cDNA clones revealed th at the candidate gene was a m em ber
of the Pax gene family. Pax genes are characterised by the presence of a m otif
known as a p aired box which encodes a 120-amino acid DNA-binding dom ain. T he
p aired box was first described in the Drosophila segm entation gene paired (Frigerio
et al., 1986; Bopp et al., 1986) and has since b een found in o th er developm entally
im p o rtan t Drosophila genes (B aum gartner et al., 1987; Bopp et al., 1989). Subse
quently, a family of paired-box containing genes have been described in m ouse,
m an, zebrafish and chicken (D eutsch an d Gruss, 1991). T he expression patterns
o f the Pax genes, as identified by RNA in situ hybridisation, an d the involvem ent
o f these genes in developm ental m utations in Drosophila, m ouse and m an has ar
gued for a key role for them in p attern form ation (Deutsch an d Gruss, 1991; Gruss
and Walther, 1992). T he vertebrate Pax gene family has been best characterised in
m ouse and so far contains eight m em bers (W alther et al., 1991; Gruss an d Walther,
1992). O n the basis o f predicted am ino acid sequence, the candidate aniridia gene
is the hum an hom ologue of th e m ouse Pax-6 gene; in d eed the predicted protein
products of the m ouse ( Pax-6) and hum an (PAX6) genes differ by only one am ino
acid. As well as the paired motif, the PAX6 gene also encodes a paired-type ho-
m eobox, an o th e r phylogenetically conserved m otif found in developm entally im
p o rta n t genes an d also known to encode a DNA binding activity (W alther and
Gruss, 1991; D eutsch an d Gruss, 1991).
T he candidacy of PAX6 as the aniridia gene was strengthened by RNA in situ hy
bridisation studies designed to d eterm in e the pattern of expression o f the PAX6
gene. This approach revealed that PAX6 was strongly expressed in the developing
eye, at a stage preceeding iris form ation, in both hum an an d m ouse em bryos (Ton
et al., 1991; W alther and Gruss, 1991). Interestingly, the dom ain o f expression in
th e eye was no t confined to the rim o f the optic cup which would ultim ately give
rise to the n eu ro ecto d erm al com ponent o f the iris, but also encom passed the n eu
ral retina, the lens and the overlying surface ectoderm which contributes towards
the cornea. All these structures can be affected in aniridia, and the in situ results
were therefore consistent with PAX6 being the aniridia gene. However, to show
th at a candidate gene isolated by positional cloning is really responsible for the
disease it is necessary to dem onstrate intragenic m utations in affected individuals.
T he first genetic evidence th at PAX6 was involved in eye developm ent cam e from
analysis of a m ouse m utation called Small eye.
Small eye is a sem i-dom inant m utation affecting eye developm ent in the m ouse.
T h ree alleles have been described, two spontaneously occuring (Sey an d Sey^)ey)
ANIRIDIA 453
Mouse m utations
Because the Small eye m ouse was such a plausible m odel for aniridia an d biologi
cal m aterial is m ore readily available from laboratory anim als than patients,
m utation analysis o f the Pax-6 gene was first carried o u t in the mouse.
454 ISABEL HANSON ET AL.
Dosage analysis and interspecific backcrosses dem onstrated that Pax-6 was delet
ed in the Sey m ouse (Hill et al., 1991). A m ore im p o rtan t goal however was to
dem onstrate intragenic m utations o f Pax-6 in Small eye alleles which were predict
ed to be p oint m utations on the basis o f phenotype. At this stage little was known
about the genom ic organisation o f Pax-6 so the m ost feasible approach to m uta
tion analysis was th ro u g h mRNA. From the expression p attern it was clear th at Pax-
6 was m ost highly expressed in neural tissue in em bryos betw een 8 and 18 days ges
tation. T herefore RNA was extracted from the heads o f 15d em bryos, reverse tran
scribed, PCR-amplified and analysed for the presence o f p oint m utations by
chem ical cleavage o f m ism atch (Hill et al., 1991). This RNA-PCR approach re
vealed a single base change in the Sey Pax-6 gene which created a term ination
codon in the open reading fram e. T he predicted effect of this would be to tru n
cate the p rotein p ro d u ct before the hom eodom ain and thus to im pair the func
tion o f the Pax-6 protein (Figure 5b).
A dditional evidence th at Pax-6 is m u tated in Small eye came from analysis o f the
EN U -induced S e ^ m m utation (Hill et al., 1991). A lthough allelism of this m uta
tion with the Small eye locus had n o t b een form ally dem onstrated, genetic m apping
suggested that it m apped to the ap p ro p riate region o f chrom osom e 2, an d the
phenotype was virtually indistinguishable from that of Sey. RNA-PCR analysis of
Pax-6 in Sey revealed an unusually large Pax-6 transcript which was shown upon
sequencing to include an intron. Sequencing o f the genom ic DNA of SeyNeu
showed that the invariant G residue o f the splice d o n o r site was m utated, thus p re
venting intron excision from the mRNA. T he predicted effect o f this m utation
would be to term inate translation at a stop codon within the intron and thus to
truncate the protein before the serine-threonine rich C-term inal dom ain (Figure
5 b ). A lthough the function o f this dom ain is no t known, it is sim ilar in am ino acid
com position to the trans-activation dom ain o f the transcriptional regulators Oct-
1 and Oct-2 (Tanaka an d H err, 1990). Analysis o f Small rye alleles therefore provid
ed strong evidence for the involvem ent of Pax-6 in eye developm ent.
H um an m utations
Sey (Mouse)
S e y '* 1(Mouse)
HZAMT (Human)
RUBAI (Human)
that m ism atched heteroduplexes are form ed when the region containing the
m utation is am plified. This approach was used successfully to d etect h etero d u
plexes of anom alous mobility in the PCR p ro d u c t derived from exon H o f one
aniridia patient. N ucleotide sequencing o f the PCR pro d u ct revealed a two nucle
otide insertion which changed the reading fram e and would be predicted to
cause chain term ination ju s t distal to the hom eodom ain (Patient HZAMT, Figure
5b;Jo rd a n e ta l., 1992).
A second strategy which has b een used to find PAX6 m utations is th at of nested
RNA-PCR, which is carried out essentially as described for the Small eye analysis but
with extra rounds o f PCR am plification to com pensate for the very low break
th ro u g h levels o f PAXdRNA prod u ctio n in the lym phoblastoid cell lines which
are the only available m aterial from the patients. Reverse transcription is followed
by two rounds o f PCR am plification using two ,PAX6-specific p rim er pairs, o n e nest
ed inside the o th er (Roberts et al., 1991). W hen this m ethod was applied to the
hom eobox region o f the gene in a panel of patients, an RNA-PCR product o f re
duced size was detected in addition to the norm al p ro d u ct in one patient. Se
quencing revealed that the short transcript did no t contain exon G, b u t no
deletion was detectable at the genom ic level. T herefore the m utation in this pa
tien t is m ost likely a point m utation o r sub-visible deletion at the DNA level, prob
ably at or n ea r to a splice site, which causes exon G to be spliced ou t o f the m ature
PAX6 mRNA product. T he predicted protein p ro d u ct o f this m u tan t allele would
term inate after the first third o f the hom eodom ain (Patient RUBAI, Figure 5b;
Jo rd a n et al., 1992). These analyses strongly suggest that PAX6 m utations cause
aniridia in at least som e patients.
CLINICAL SIGNIFICANCE
These early results o f PAX6 m utation studies open up the possibility o f genetic
counselling an d prenatal diagnosis, bo th o f which are often requested by aniridia
patients. T he frequency and spectrum of PAX6 m utations can now be assessed in
a large cohort o f patients. T he existence of w ell-docum ented deletion aniridia
patients whose phenotypes are clinically indistinguishable from familial cases,
who would be predicted to have p o in t m utations, suggests th at aniridia results
from .PAX6 haplo-insufficiency i.e. loss o f one active copy o f the PA X 6gene. Since
many different m utations could lead to loss of gene function, we expect to see a
wide spectrum of m utations both within the coding region an d in control regions
outside the transcribed sequence. This implies that each sporadic p atien t o r fam
ily will have to be assessed de novo before genetic counselling can be given. T he
way is already open for m utation analysis at the RNA level which will reveal point
m utations, small insertions o r deletions, or ab e rra n t splicing, all of which can
458 ISABEL HANSON ET AL.
lead to deleterious changes in am ino acid sequence (Figure 5b; Hill et al., 1991;
Jo rd a n et al., 1992).
In patients w here RNA-PCR analysis does n o t reveal a m utation, it is possible
th at one copy o f the PAX6 gene is n o t being expressed. This could result from a
deletion o f all o r p art o f the PAX6 gene or a m utation in a control region. N othing
is known at present about the elem ents which control PAX6 expression bu t these
will have to be defined, m ost probably in the m ouse, before m utation analysis is
possible. Loss o f expression o f one copy of PAX6 could be verified by searching in
the genom ic DNA of patients for heterozygosity at a transcribed site an d then
showing at the RNA level that only one allele is expressed. A fu rth e r possibility in
patients w here no PAX6 m utation is detectable is that they have m utations in a sec
o n d aniridia gene. T he existence o f a second gene is suggested by the t(4; 11)
breakpoint, which is associated with aniridia bu t which does n o t physically disrupt
PAX6 (Gessler, Simola and Bruns, 1989; Ton etal., 1991;Jordan etal., 1992). How
ever the aniridia in this family could ultim ately be explained by a position effect
w here the translocated portio n o f chrom osom e 4 silences PAX6 at a distance, p er
haps by altering chrom atin structure. Again, expressed heterozygosity would be
valuable in d eterm in in g w hether the copy o f PAX6 on the translocation chrom o
some is transcribed.
A nother clinically significant aspect of this work is the assessm ent o f the risk of
W ilm s tu m o u r in new borns with sporadic aniridia. In contrast to aniridia, both
Wilms' tu m o u r an d the associated g en itourinary abnorm alities show red u ced p en
etrance so that even sporadic cases of isolated aniridia may carry a deletion, pu t
ting them at risk o f developing nephroblastom a. D uring the m apping o f the
WAGR region, cosm id clones were isolated for WT1, PAX6 an d two intervening
m arkers (Fantes et al., 1992). These can be used in fluorescent in situ hybridisation
(FISH) analysis o f m itotic chrom osom es to d eterm in e w hether o r n o t a deletion
is present. If a deletion is indicated, assessm ent o f incipient Wilms' tu m o u r by re
nal u ltrasound should be carried ou t at regular intervals. We have validated this
technique retrospectively in several subm icroscopic WAGR deletions. In one u n
expected an d presum ably very rare case a m o th er with sporadic isolated aniridia
gave birth to a son with aniridia who later died as a result o f Wilms' tu m o u r which
arose in a horseshoe kidney. FISH analysis showed that both were deleted for WT1
and PAX6 (Fantes et al., 1992).
A great deal of phenotypic variability has been observed am ong aniridia patients,
even within families who are presum ably segregating for the same m utation (H itt
n er et al., 1980). This variability is particularly striking in the Small eye m ouse
where there are m arked differences in eye phenotype even within the same
in b red strain (Roberts, 1967). T he phenotypic heterogeneity is o f clinical im por
tance in assessing familial patterns o f inheritance an d the associated genetic risks
because some individuals are so mildly affected th at they may be m isdiagnosed
(H ittn er et al., 1980; Lyons et al., 1992). In view o f the heterogeneity am ongst
ANIRIDIA 459
aniridia patients it is possible that PAX6 m utations are also responsible for o th er
disorders which are usually considered to be clinically distinct but may be part of
a wide phenotypic spectrum that encom passes aniridia. These include atypical
colobom a (Figure 1), early onset cataracts an d a range of an terio r segm ent
defects such as Peters' anomaly, all o f which have been observed in families segre
gating for aniridia an d which are consistent with the pattern o f PAX6 expression
in the eye (H ittn er et al., 1980; B eaucham p, 1978). It will be interesting to see
w hether PAX6 is m utated in these disorders and, if so, how the spectrum of m uta
tions com pares to that defined for classical aniridia.
In the majority o f aniridia patients n o neurological or brain abnorm alities have
been noted. It was therefore som ew hat surprising to find th at PAX6 is expressed
n o t only in the developing eye b u t also in the brain and neural tube (Ton et al.,
1991; W alther an d Gruss, 1991). However in cases of the rare Gillespie syndrom e,
aniridia is associated with cerebellar ataxia. M ight this phenotype also be account
ed for by m utation in PAX6? It may be significant in this respect th at in addition
to the eye the cerebellum is the m ajor expressing tissue in adult mice (Ton, Miwa
and Saunders, 1992).
D ourain, 1988; Servetnick and Grainger, 1991). T he optic sulcus is the first m or
phological indication of the em ergence o f the prim ordial eye from the optic pla
code on the surface of the developing neural plate. It extends by invagination to
form the optic stalk, the en d o f which th en swells to develop the optic vesicle and
finally invaginates to produce the optic cup. T he posterior layer of the iris, the
epithelial layers o f the ciliary body and the whole o f the n eu ro retin a all develop
from the optic cup. T he surface ectoderm proceeds through acquisition o f a lens
form ing bias to specification of the lens placode. O n contact with the optic vesi
cle this placode invaginates to form a lens vesicle which eventually separates from
the surface ectoderm and moves into the optic cup which is form ing sim ulta
neously. T he corneal epithelium will form from the rem ains of the lens placode
(O'Rahilly, 1966; Kaufm an, 1979). It is therefore quite conceivable th at the pri
m ary defect in aniridia lies entirely within ectoderm and this idea is su p p o rted by
the discovery th at PAX6 is expressed in the rim of the optic cup, the developing
n euroretina, the lens and the corneal epithelium . It rem ains to be d eterm in ed
ju st what PAX6 is doing in this respect.
It is tem pting to speculate that PAX6 is som ehow involved in the induction o f the
lens. T he absence o f any functional Pax-6 transcripts i.e. in Sey/Sey m ice, results
in the absence o f lens form ation in the presence o f a correctly positioned optic
vesicle which fails to form an optic cup an d undergoes involution (H ogan et al.,
1986). Existing data show that lens vesicles can form in the absence o f the optic
vesicle an d that the optic vesicle is no t capable of producing lenses from n o n
specified surface ectoderm (M uthukkaruppan, 1965; Grainger, H enry and H en d
erson, 1988). These findings contradict the results o f earlier induction experi
m ents. T he m ost recent experim ents in this field point to the existence o f a
crucial interaction between the neural plate and the adjacent surface ectoderm
in o rd e r to achieve lens placode specification (H enry and Grainger, 1990). It is
interesting to note that m any of the so-called free lenses seen in earlier tissue
m anipulation experim ents i.e. lenses occurring in the absence o f an optic vesicle,
were found in association with the developing nose, since we now know that both
the olfactory bulb and the nasal placode express Pax-6 during developm ent
(Saha, Span and Grainger, 1989; Ton et al., 1991; W alther and Gruss, 1991).
These findings are evidence o f a critical role for Pax-6 in the form ation o f the
lens, perhaps by the early specification o f the lens placode. To u n derstand this
and o th er roles o f Pax-6 will require additional experim ents using both tradi
tional tissue m anipulation m ethods and m o d ern m olecular techniques. By assem
bling conjugates o f tissues from different stages of developm ent an d with
d ifferent degrees o f Pax-6 expression, it may be possible to define developm ental
windows an d tissue interactions. Somatic cell chim eras in which m ixed popula
tions o f Pax-6 expressing an d non-expressing cells are ju x tap o sed in the develop
ing em bryo will tell us w hether or no t the gene acts cell autonom ously o r if its
ANIRIDIA 461
function can be rescued by the presence of adjacent norm al cells (H atta et al.,
1991). T he free lens evidence argues for the latter. It is to be h o p ed th at such
experim ents will also provide valuable insights into the m olecular events which
lie beh in d the norm al developm ent o f the eye.
genom ic DNA fragm ents an d PCR-amplifying the b o und fragm ents to create a li
brary o f Pax-6 recognition sequences (Kinzler and Vogelstein, 1989).
A lternative splice form s o f transcriptional regulators may be im p o rtan t in the
control o f target genes. For exam ple, alternative splice form s affecting the struc
ture o f the zinc finger DNA binding region o f WT1, the Wilms' tum our predispo
sition gene, can alter its binding specificity, thus raising the possibility of
in teraction with m ore than one physiological dow nstream target (Bickm ore et al.,
1992). A lternative splicing in the paired box o f PAX6, which results in the inser
tion o f an additional fourteen am ino acids into this DNA binding dom ain, has
been dem onstrated in m ouse, zebrafish an d m an, b u t its functional significance
rem ains to be dem onstrated (W alther an d Gruss, 1991;Jo rd a n etal., 1992;Puschel
et al., 1992). RNA in situ hybridisation using probes specific for the alternate splice
form s may reveal spatial or tem poral heterogeneity in their expression.
CONCLUSION
NOTE:
T he com plete genom ic organisation o f the hum an PAX6 gene has now been
d ed u ced (Glaser, W alton an d Maas, 1992). Intragenic PAX6 m utations have been
characterised in eight additional aniridia patients (Glaser, W alton and Maas,
1992; H anson e ta l., 1993)
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Wilms' tu m o u r an d aniridia: clinical a n d cytogenetic features. Archives of Disease in Childhood
5 7 , 685-690.
Shaw, M. W., Falls, H. F. and N e e l,J. V. (1960). C ongenital aniridia. American Journal o f Human
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Sim eone, A., G ulisano, M., A cam pora, D., Stornaiuolo, A., R am baldi, M. an d B oncinelli, E.
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467
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20. MITOCHONDRIAL DNA MUTATIONS
AND THE EYE
O phthalm ic dysfunction is one o f the most comm on clinical observations associated with mitochondri
al DNA (mtDNA)-based hum an disease. Ocular pathology in these diseases includes optic atrophy, pig
mentary retinopathy, retrochiasmal visual loss, and myopathy involving the extraocular muscles and
the eyelids. The wide spectrum of symptoms involving the eye demonstrates the dependence of the
ophthalmic system on mitochondrial energy production.
^ C o rre s p o n d in g a u th o r
470 MICHAEL D. BROWN ET AL.
D-Loop Region
12s
rRNA
0/16*#
r 16s
rRNA
Complex I genes
(NADH dehydrogenase)
Complex IV genes
(cytochrome c oxidase)
Complex V genes
(ATP Synthase)
KCOIIIStgS
W M r
K ATPase8
and AGG codons are term ination codons (instead o f arginine codons), a n d the
AUA codon is used for m ethionine (instead of isoleucine).
Five features o f the hum an mtDNA distinguish mtDNA genetics from nuclear
genetics (Wallace 1992a,b). First, m itochondria are m aternally in h erited (Giles
et al. 1980; Case and Wallace 1981). At fertilization, the egg contains greater than
105 m itochondria while the sperm contribution is less than 0.1% o f zygote
mtDNA (Gyllensten et al., 1991). Thus, mtDNAs are in h erited m other-to-daugh
ter, m eaning that pedigrees o f mtDNA diseases with m ultiple affected family
m em bers will show exclusive m aternal transm ission o f the phenotype. Second,
mtDNAs random ly segregate to d au g h ter cells u p o n mitosis o r meiosis (replica
tive segregation) (Wallace, 1986). T he high copy nu m b er o f mtDNA genom es
allows for the presence of both m u tan t an d wild-type mtDNAs (heteroplasm y)
within an organelle or cell. At cell division, the p ro p o rtio n o f m u tan t an d wild-
type mtDNAs stochastically segregate to d au g h ter cells and, over time, the p o p u
lation o f mtDNAs within a cell can drift towards eith er p u re (hom oplasm y)
m u tan t or wild-type genom es. Since the m agnitude o f an OXPHOS deficiency is
frequently co rrelated with the fraction o f m u tan t mtDNAs within a tissue, h etero
plasmy due to replicative segregation is an im portant feature o f mtDNA-based
disease. T hird, phenotypic expression o f an mtDNA defect is d e p e n d e n t on a tis
sue-specific OXPHOS threshold. Each tissue in the body depends on OXPHOS
for energy production to a d ifferent extent. For exam ple, central nervous system
tissue and skeletal muscle are am ong the m ost highly oxidative tissues in the body
and therefore are highly reliant on OXPHOS. Disease results when the ATP-gen-
erating capacity o f an organ drops below a m inim um OXPHOS threshold neces
sary for tissue m aintenance. Thus, clinical m anifestation o f an OXPHO S defect is
a p ro d u ct o f the n ature o f the mtDNA m utation, the fraction o f m u tan t mtDNA
genom es within a tissue, and the O XPHO S requirem ents of the affected tissue.
F ourth, ATP production within a tissue naturally declines with age (T rounce et
al., 1989; Yen et al., 1989). This process parallels an increase in som atic mtDNA
dam age in post-m itotic tissue with age (L innane et al., 1989; Cortopassi an d Arn-
heim , 1992; Corral-Debrinski et al., 1992). Oxygen radicals are produced at rela
tively high concentrations at the m itochondrial in n e r m em brane as a result of
respiratory chain redox activity, causing the mtDNA to accum ulate oxidative
dam age at a rate 16 times higher than nuclear DNA (R ichter et al., 1988). Thus,
the age-related decrease o f OXPHOS capacity may be co rrelated with the age-
related increase in mtDNA dam age. This p h en o m en o n would help explain why
some mtDNA diseases are expressed in mid-to-late life an d progress with age.
Fifth, new mtDNA m utations are fixed at a rate 10-20 times hig h er than are
m utations occurring in nuclear-encoded OXPHO S genes (N eckelm ann et al.,
1987; Wallace et al., 1987). Because mtDNA OXPHOS genes are essential for aer
obic m etabolism and subject to equivalent selective pressure as nuclear-encoded
OXPHOS genes, deleterious mtDNA m utations are expected to arise with signifi
cant frequency.
M ITOCHONDRIAL DNA MUTATIONS AND THE EYE 473
Because the retina, optic nerve and extraocular m uscles controlling eye move
m en t are am ong the m ost A T P-dependent tissues in the body, it is n o t surprising
that those mtDNA p o in t m utations an d deletio n s/d u p licatio n s which im pair
OXPHOS capacity often result in o cular dysfunction. T he m ost com m on o p h
thalm ic m anifestations o f mtDNA diseases include optic neuropathy, pigm entary
retinopathy, an d ophthalm oplegia with ptosis (Table 1).
MtDNA
Mutation Phenotype Ocular involvement
I. Point Mutations
A. Missense Mutations
1. Electron transport chain LHON Optic neuropathy
(Complexes I-IV)
2. ATP synthase N ARP/Leighs Pigmentary retinopathy
(Complex V) Disease
B. tRNA Mutations
1. Lysine MERRF Occasional optic atrophy, pigmentary
retinopathy
2. Leucine MELAS Occasional optic atrophy, pigmentary
retinopathy
3. Leucine MMC None
4. Leucine MM None
5. Isoleucine FICM None
II. Rearrangements
A. Deletions
1. Spontaneous CPEO O phthalm oplegia/Ptosis, occasional
pigmentary retinopathy
KSS Ophthalm oplegia/Ptosis, pigmtary
retinopathy
Pearsons Can progress to KSS
2. Heritable KSSa ophthalm oplegia/ptosis, bilateral
cataract formation
Diabetes plusb None
B. Duplications Variable Ophthalm oplegia/Ptosis, pigmentary
retinopathy
Pathogenic mtDNA p o in t m utations have been found in both tRNA genes and
protein-coding genes (Wallace, 1992a,b). T ransfer RNA m utations are associated
with severe m itochondrial m yopathies such as Myoclonic Epilepsy an d Ragged-
Red Fiber disease (MERRF1, an A to G transition at n p 8344 an d a T to C transition
at n p 8356 in tRNALys) (Shoffner et al., 1990; Zeviani et al., 1992); M itochondrial
Encephalom yopathy, Lactic Acidosis an d Stroke-like symptoms, (MELAS, an A to
G transition at nucleotide pair 3243 in tRNALeu) (Goto et al., 1990a); M itochon
drial Myopathy and Cardiom yopathy (MMC, an A to G transition at nucleotide
p air 3260 in tRNALeu) (Zeviani et al., 1991); an d Fatal Infantile Cardiom yopathy
(FICM, an A to G transition at nucleotide pair 4317 in tRNAIie) (Tanaka et al.,
1990). T ransfer RNA m utations usually alter nucleotides conserved betw een vari
ous species an d result in a protein synthesis defect. Despite the heterogeneity of
neuro-m uscular symptoms associated with these diseases, ophthalm ic involve
m en t is relatively uncom m on an d generally lim ited to occasional optic atrophy or
pigm entary retinopathy (MERRF an d MELAS) or retrochiasm al visual loss
(MELAS).
In contrast, diseases attrib u ted to mtDNA missense m utations feature o phthalm
ic involvem ent (Table 1). Both Leber's H ereditary O ptic N europathy (LHON)
an d N eurogenic Muscle Weakness, Ataxia a n d Retinitis Pigm entosa (NARP), are
caused by mtDNA p o in t m utations in OXPHO S polypeptides.
<TAT AG E 24
III
AGE 15
IV
AG E 17
male. Thus, although mtDNA m utations have a prim ary etiological role in the dis
ease, o th er factors m ust influence LHON expression. Such factors likely include
additional genetic determ inants, sex-limiting physiological determ inants, and u n
specified environm ental determ inants.
T he genetics o f LH O N have proven to be com plex and heterogeneous as twelve
mtDNA p o in t m utations have now b een associated with LHON (Brown et al.,
1992a; Johns, N eufeld and Park, 1992) (Table 2 ). Eleven o f these are missense m u
tations in genes contributing to respiratory Com plexes I and III. T he twelfth m u
tation alters the translational term ination codon o f the gene encoding
cytochrom e c oxidase subunit I (C om plex IV). These m utations appear to im pair
respiratory chain function to d ifferent degrees, a variation reflected in the risk
each m utation represents in causing LHON. Risk can be estim ated on the basis of
four criteria: (1) n ature o f the am ino acid change, (2) w hether o r n o t a m utation
can cause blindness by itself, (3) the expressivity o f the disease in LHON pedi
grees, an d (4) the pen etran ce o f the m utation in the general population. Amino
acid substitutions are eith er conservative o r radical replacem ents and, if function
ally significant, usually alter evolutionarily conserved am ino acids. Expressivity is
d eterm in ed by the p ro p o rtio n o f b lind individuals in a hom oplasm ic pedigree and
by the pro p o rtio n o f affected males within a pedigree. Since males are m ore com
m only affected than females, m ore severe LH O N m utations m ight be expected to
affect an increased p ro p o rtio n of females. T he p enetrance of a m utation in the
population involves three param eters, heteroplasm y (indicative o f a new m uta
tion), the occurrence o f a m utation on a single versus m ultiple mtDNA haplo-
types, and the frequency with which a m utation is found in unaffected controls.
Based on these criteria, m utations associated with LHON can be classified as high,
m oderate, o r low-risk LH O N m utations.
476 MICHAEL D. BROWN ET AL.
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M ITOCHONDRIAL DNA MUTATIONS AND THE EYE 477
Individuals harboring mtDNA m utations at nps 3460, 4160 an d 11778 are at the
greatest risk for expressing LHON. All of these m utations cause non-conservative
am ino acid substitutions a n d alter am ino acids which are conserved am ong many
species. These m utations do no t occur together in LHON pedigrees and have not
been d etected in unaffected controls, indicating that they are sufficiently delete
rious by themselves to cause blindness. Further, pedigrees harb o rin g these m uta
tions have a relatively high p ro p o rtio n o f affected individuals.
T he 4160 (ND1 gene, Com plex I) m utation appears to be the m ost severe LHON
m utation as roughly 80% o f the m em bers o f the single 4160-positive LHON p edi
gree are blind and nearly one-half o f the affected individuals are female (Howell
et al., 1991a). Atypical o f m ost LH O N pedigrees, additional neurological symp
toms such as dysarthria, ataxia, an d severe infantile encephalopathy are prom i
n e n t in this family. Further, the analysis o f OXPHO S enzyme activity from platelet
m itochondria revealed a m arked reduction in Com plex I specific activity in all
family m em bers assayed (Parker, Oley an d Parks, 1989).
The 3460 (ND1 gene, C om plex I) a n d the 11778 (ND4 gene, C om plex I) m uta
tions appear to be the n ex t m ost severe LHON nucleotide substitutions (Wallace
et al. 1988; H ouponen et al. 1991; Howell et al. 1991b). C om plete mtDNA se
quence analysis o f probands carrying eith er the 3460 m utation o r the 11778 m u
tation has revealed no o th e r mtDNA m utations which ap p ear to play a p rom inent
pathogenetic role in disease expression (Wallace et al., 1988; Brown et al., 1992a).
Also, OXPHO S biochem ical deficiencies have been detected in individuals har
boring these m utations. M ajander an d colleagues (1991) re p o rted an 80% reduc
tion in the rotenone-sensitive electron transfer activity o f Com plex I in 3460-
positive patients (M ajander et al., 1991). A com parable reduction in Com plex I
enzyme activity has n o t been observed in 11778-positive patients, however reduced
oxidation o f Com plex I-linked substrates has been detected with polarography
(Larsson et al., 1991; M ajander et al., 1991). Finally, the 11778 m utation is found
on strikingly d ifferent mtDNA genotypes, having been detected in African, Asian
an d Caucasian LHON pedigrees. Thus, this m utation has occu rred m ultiple dif
feren t times an d each occu rrence associated with LHON, providing convincing ev
idence th at the 11778 m utation is a prim ary causal LHON m utation (Singh et al.,
1989).
LHON m utations at nps 3394, 7444, an d 15257 appear to im part an interm ediate
risk for the expression of LHON. All th ree m utations have some characteristics of
the high-risk LHON m utations such as the radical substitution of highly con
served am ino acids and a relatively high fraction o f pedigree m em bers who
develop optic nerve atrophy. However, each m utation has been detected at a low
frequency in the unaffected control population and these LHON m utations are
478 MICHAEL D. BROWN ET AL.
of a high-to-m oderate risk LHON m utation, yet, in some cases, this m utation alone
may be insufficient to cause LHON.
T he n p 15257 m utation is alm ost invariably found with a specific subset o f lower-
risk mtDNA LHON m utations. MtDNA sequence an d restriction endonuclease di
gestion analysis has indicated that patients harboring the np 15257 m utation have
very sim ilar mtDNA haplotypes which include the frequently-observed low-risk
LHON m utation at np 13708 (ND5, gene, Com plex I), and the lower risk LHON
m utations at nps 15812 (cytochrom e b gene, C om plex III) and 5244 (ND2 gene,
C om plex I) (Brown et al., 1992b). Such sim ilar mtDNA genotypes cluster upon
phylogenetic analysis, dem onstrating a LHON mtDNA lineage which is defined by
the linked n p 13708 + 15257 m utations. T he np 15812 m utation is fo u n d in rough
ly one-half o f the individuals with the 13708 + 15257 genotype an d the 5244 m uta
tion has been fo u n d in a single individual with the 13708 + 15257 + 15812
genotype. As this lineage sequentially accum ulated these four m utations, the nu m
ber o f unaffected individuals decreased and the pro p o rtio n of blind individuals in
creased. Blindness occu rred in the lineage only after the occurrence o f the 15257
m utation. Thus, the n p 15257 m utation is the m ost pathogenic mtDNA m utation
in this lineage, bu t in some cases the risk for developing LHON is apparently in
creased with the presence of additional mtDNA m utations. T he additional m uta
tions in teract synergistically with the 15257 m utation to decrease OXPHOS
efficacy below an energetic threshold necessary for optic nerve function (Johns
and B erm an, 1991; Brown et al., 1992a,c).
Six mtDNA m utations appear to im part a low-risk for LHON expression: nps
4216 (N D l), 4917 (ND2), 5244 (ND2), 13708 (ND5) and 14484 (ND6) in Com
plex I genes and np 15812 in cytochrom e b o f Com plex III (Johns and B erm an,
1991; Brown et al., 1992a,b; Johns, N eufeld and Park, 1992). These m utations can
alter poorly o r highly conserved am ino acids and can be found in statistically sig
nificant frequencies in LH O N families. However, they can also be found in rela
tively high frequencies in unaffected controls, the exceptions being the
heteroplasm ic 5244 m utation which has several high-risk LHON m utation char
acteristics (Table 2) b u t has only been found in one 15257-positive LHON patient
an d no controls, an d the 14484 m utation which also has n o t been found in con
trols. Both of these m utations may therefore rep resen t a m ore significant risk for
developing LHON, bu t m ore data is necessary to draw this conclusion. In gen
eral, however, the low-risk LHON m utations probably cannot cause LHON as sol
itary etiological agents, bu t instead are either non-pathogenic mtDNA
polym orphism s linked to a LHON-causing mtDNA genotype o r contribute to dis
ease expression as a m utation of low pathogenicity which interacts synergistically
with m ore severe mtDNA m utations. Some of these m utations can be found asso
ciated with various o th er m ore severe LHON m utations and som e are specifically
associated with a second LHON m utation, such as the np 15812 m utation which
has only been fo u n d linked to the m ore severe np 15257 m utation in LHON
patients.
480 MICHAEL D. BROWN ET AL.
>6 26
n *
% Mutant mtDNA 0 0 86 >95 >95 >95 >95
Age (years) 2 14 11 1
RP + nd + + +
Leigh's - + - - +
Optic atrophy - - - - +
Migraine + - - - -
MR - + + + +
Ataxia - nd + + nd
ADD . nd + + nd
10.4 kb deletion
ch ildhood disease. Those children who survive the early stages o f Pearson Mar
ro w /P ancreas syndrom e often progress to KSS.
MtDNA Deletions
inheritance. Spontaneous deletions are com paratively m uch m ore com m on and,
as a group, share certain general genetic characteristics.
Spontaneously-occurring deletions account for the majority o f CPEO, KSS and
Pearson M arrow /Pancreas Syndrom e cases seen clinically. Patients harboring
spontaneous deletions are observed sporadically as such deletions are somatic m u
tational events and are therefore no t transm itted to successive generations.
Over 120 d ifferent spontaneous, pathogenic mtDNA deletions have been
m apped and some com m on genetic features are evident (Wallace et al., 1991).
First, although such deletions vary widely in position and size, ranging from
roughly 2 kb to 9 kb, the two origins o f DNA replication are usually m aintained,
thus confining spontaneous deletions to the two m ajor arcs o f the mtDNA defined
by the position o f the two origins (M oreas et al., 1989; Goto et al., 1990b; Wallace
et al., 1991) (Figure 4). Second, b o th tRNA an d protein-coding genes are com
m only deleted (Wallace et al., 1991). T hird, sequence analysis o f the breakpoints
of over 50 spontaneous deletions revealed th at these deletions m ost often occur
within directly re p eated mtDNA sequences (Shoffner et al., 1989; Schon et al.,
1989; Mita et al., 1990; Wallace et al., 1991). D irect repeats are com m on in mtD
NA, in part because o f the disproportionate n u m b er o f H -strand g uanine residues.
Some direct repeats, which range from th ree to 13 base pairs in length, are
hotspots for deletion form ation (Wallace et al., 1991). T he best exam ple of such
a hotspot is a 13 base pair perfect re p eat ( 5 - ACCTCCCTCACCA 3'), found in the
ATPase 8 gene (from n p 8470 to np 8482) an d the ND5 gene (from np 13447
to np 13459) (Wallace et al, 1991; Wallace, 1992a). This re p eat is found at the
b reakpoint o f the m ost com m only-occurring pathogenic deletion of 4977 base
pairs, which has o ccu rred independently over 100 times (Wallace et al., 1991)
(Figure 4).
T he association of spontaneous deletions with direct repeats suggests a com m on
m utagenic m echanism . H om ologous recom bination has no t been proven to exist
in m itochondria and therefore probably does not account for somatic deletion
events. However, a "slipped replication" m odel as proposed by Shoffner et al.
(1989) does n o t rely on strand cross-over, yet is d e p e n d e n t on direct repeats for
deletion form ation. U n d e r the "slipped replication" m odel, the upstream , dis
placed, H -strand direct rep eat base-pairs with the com plem entary dow nstream L-
strand re p eat exposed by the DNA replication fork. A strand breakage event ju st
dow nstream from this pairing generates a 3'-OH for subsequent H -strand synthesis
an d results in the degradation o f the single-stranded H -strand dow nstream o f the
breakage event back to double-strand DNA. Thus, slipped replication resolves one
wild-type an d one deleted mtDNA m olecule. Since a m inority o f deletion events
occur in the absence of directly re p eated sequences, slipped replication cannot ac
count for all large spontaneous deletions. O th er m echanism s, such as illegitim ate
recom bination and topoisom erase II-m ediated deletion form ation, have been
proposed to account for those deletions n o t associated with direct repeats (Mita
e ta l., 1990).
484 MICHAEL D. BROWN ET AL.
protein (mRNA coding region spans the deletion breakpoint) can be detected
u pon SDS-PAGE o f S-Met labeled m itochondrial proteins. However, in cybrids
containing greater than 60% deleted mtDNA, overall m itochondrial translation
decreased dramatically, as did synthesis o f the fusion protein. Thus, a generalized
translation defect due to tRNA deficit is likely in those cells harboring m itochon
dria with high prop o rtio n s o f deleted mtDNA. Such a pathogenic m echanism
would explain why ocular m yopathy is a com m on phenotypic result am ong pa
tients harboring m arkedly d ifferent deletions.
Pathogenic mtDNA deletions can also be inherited. These deletions are less
com m on than spontaneous deletions an d can be fu rth e r subdivided on the basis
o f m aternal versus M endelian inheritance. O ne well-characterized m aternally in
herited deletion o f 10.4 kb was d etected in a three generation pedigree exhibiting
adult-onset diabetes m ellitus and hearin g loss (Ballinger et al., 1992). All mater-
nally-related family m em bers studied h arb o red the heteroplasm ic deletion in m ul
tiple tissues. Unlike pathology associated with spontaneously-occurring deletions,
m em bers of this pedigree did n o t p resen t with m ild or severe form s o f ocular my
opathy. Also, unlike spontaneously-occurring deletions, this large deletion elim i
nates the light-strand origin of DNA replication (Figure 4). It is possible th at the
absence of the light-strand origin decreases DNA replication efficacy an d thereby
does no t allow the sm aller deleted m olecule a replicative advantage. Thus, p ro
gressive en rich m en t may n o t occur o r be greatly reduced in p atien t tissues, ac
counting for the unique pathology an d inheritance associated with this deletion.
Several pedigrees exhibiting the autosom al dom inantly transm itted propensity
for harboring mtDNA deletions have b een identified (Zeviani et al., 1989; Zeviani
et al., 1990; C orm ier et al., 1991). KSS an d bilateral cataract form ation are com
m on clinical m anifestations in many o f these pedigrees. In contrast to both spon
taneous and m aternally-inherited deletions, affected individuals h arb o r m ultiple
mtDNA deletions. D eletion breakpoints can vary am ong family m em bers, strongly
suggesting th at a predisposition to de novo deletion form ation is inherited, n o t the
deletions themselves. These deletions always spared both origins o f DNA replica
tion and occurred within d irect repeats. Zeviani et al. (1989, 1990) have identified
a m utational hotspot for deletion form ation near the tRNAPr/D -loop ju n ctio n
(nps 16070-16080). T he non-coding D-loop region contains im p o rtan t sequence
elem ents (including PL, PH, an d O h ,) that interact in trans with nuclear gene
products to allow mtDNA replication. Thus, it is quite possible th at a m utation in
a nuclear-encoded, trans-acting factor involved in mtDNA replication prom otes
deletion form ation, perhaps by facilitating slipped replication or intram olecular
recom bination. Such deletions have been found in both leukocyte and skeletal
m uscle mtDNA and the n u m b er o f deletions can increase with tim e in an individ
ual (C orm ier et al., 1991; Servidei et al., 1991). This would explain the progressive
clinical course seen in affected family m em bers an d is consistent with the consti
tutive presence o f a nuclear-encoded m u tan t gene product.
486 MICHAEL D. BROWN ET AL.
MtDNA Duplications
MtDNA duplications have also been associated with progressive ocular m yopathy
an d Pearson M arrow /Pancreas Syndrom e (P oulton et al., 1989a,b; Rotig et al.,
1990). All patients h arb o r a single, partially-duplicated mtDNA m olecule present
in heteroplasm ic ratios in m ultiple tissues. Rotig et al. (1991) have described the
m aternal transm ission o f a 26 kb mtDNA m olecule from a m other to two daugh
ters. In this pedigree, the m o th er had ocular m yopathy an d extrem ely low levels
o f rearra n g ed mtDNA while the daughters had m itochondrial myopathy, diabetes
m ellitus, proxim al tubulopathy and ataxia an d h arb o red higher am ounts o f the
partial duplication. As is the case with mtDNA deletions, disease courses are p ro
gressive in these patients, possibly due to two sets o f DNA origins found on these
rearra n g ed m olecules. Both the genetic m echanism producing the partial dupli
cations and the pathogenic m echanism p roducing the phenotype are unknow n.
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GLOSSARY
Aacceptor splice site: T he ju n c tio n betw een the 3' e n d o f an in tro n an d the 5'
e n d o f an adjacent exon.
*Alu PCR: T he use o f sequences from Alu repeats (see below) as prim ers for PCR
(see polym erase chain reaction). Generally the sequences are selected from Alu
segm ents th at have evolved after speciation so that hum an sequences can be
detected in d ependently of DNA from o th er organism s. Because there are so
m any Alu repeats within the genom e, short DNA sequences betw een adjacent Alu
repeats can be am plified from many regions o f the genom e. T he technique has
b een used to identify an d characterize radiation hybrids, somatic cell hybrids and
YACs containing hum an genom ic DNA.
AAlu repeats: T here are an estim ated 3-5 x 10 repetitive elem ents in m am m alian
DNA th at are p a rt of a family re ferred to as Alu repeats. These repetitive ele
m ents are 300 bp in length and were first recognized when genom ic DNA was cut
with the restriction endonuclease, Alul. They are scattered th ro u g h o u t the
genom e an d because subfamilies o f the Alu repeats have arisen since m am m alian
speciation, specific Alu prim ers can be used to selectively amplify hum an geno
mic DNA from a m ixture o f yeast or ro d e n t DNA (see Alu PCR).
Aavidin: A tetram eric protein derived from egg whites that tightly binds biotin
(see strep tav id in /b io tin ).
Abase pair: A pair o f hydrogen-bonded nucleotide bases (i.e a purine and pyrim
idine each attached to a ribose an d phosphate groups). T he sizes o f double
stranded nucleotide chains are often indicated by the # o f base pairs that are
shared. A 500 base pair (bp) DNA fragm ent consists o f two strands o f DNA, each
containing 500 nucleotides an d a total m olecular weight o f approxim ately 330
kilodaltons.
*bubble PCR: A m plification of specific fragm ents of DNA w hen only one prim er
o f known sequence can be constructed. T he genom ic DNA is cleaved into pieces
(either specifically with restriction endonucleases or nonspecifically) and then
special DNA adaptors are ligated on to the exposed ends. T he special adaptors,
known as bubble adaptors, have m ism atching internal sequences but m atching
ends th at allow for stable ligation. T he bubble prim er is m ade to be identical (not
com plem entary) to one of the strands o f DNA within the bubble. W hen PCR is
perform ed, the sequence-specific PCR prim er allows the Taq polymerase to syn
thesize a com plem entary strand o f DNA. T he polym erase synthesizes the com ple
m en t to one o f the strands within the bubble. In the next ro u n d of prim er
annealing an d am plification, the bubble p rim er is able to anneal to this newly
synthesized DNA and allow a new com plem entary strand to be m ade. W ithout
the first synthesis step directed by the sequence-specific prim er, the bubble
p rim er is unable to anneal and prim e DNA synthesis. Using this technique, n o n
specific am plification o f DNA fragm ents th at contain identical ligated adaptor
m olecules is avoided.
494 GLOSSARY
*cDNA synthesis and cloning: cDNA synthesis begins with eith er total or
polyA+RNA and an oligonucleotide p rim er that will anneal to the 3 polyA tract
of the RNA (oligodT) o r a set of random prim ers that will an n eal/hybridise
along the length of the RNA. Reverse transcriptase is used to synthesize a com ple
m entary DNA (see above). By a variety of m ethods, the RNA of the RNA/DNA
hybrid is replaced by DNA so that a double-stranded DNA contains the original
genetic inform ation that was encoded in the RNA. T here are num erous varia
tions in the m ethod, including the use o f prim ers th at are already attached to a
vector, using PCR to amplify the double-stranded DNA, different techniques to
prim e the second strand synthesis, and different strategies for inserting this syn
thesized DNA into a vector. O nce the process o f building an d incorporating the
cDNA into the vector is com pleted, then the recom binant m aterial is in troduced
into E. coli eith er by transfection (for bacteriophage vectors), transform ation
(introducing un packaged DNA into cells) or electroporation (creating transient
holes in the cellular m em brane with an electric shock so that the DNA can en ter
the cells). Cells are spread an d grown so that individual colonies can be estab
lished. A ntibiotic selection is used to ensure that only cells that have taken up the
vector will grow. T he individual colonies can then be screened by a variety of
m ethods to identify those that contain a specific cDNA of interest.
AcentiM organ: A unit of m easure of recom bination frequency betw een two m ark
ers such that one centim organ equals a 1% chance th at a recom bination event
would occur in a single generation. While th ere is a rough correlation o f physical
distances (Mb) and genetic distances (cM), with 1 Mb equivalent to about lcM
in m an, there are regions o f red u ced o r increased recom bination that lim it direct
com parisons.
GLOSSARY 495
Acentiray: A unit of m easure sim ilar to cendm organ bu t the centiray is based on
the probability o r frequency of DNA breaks induced by radiation. This m easure
o f genetic distance between m arkers is used in radiation hybrid m apping and
m ore closely correlates with physical distance than recom bination frequencies.
In general, one centiray equals approxim ately 50 kb o f DNA.
Achem ilum inescence: T he pro d u ctio n of light by a chem ical reaction. An enzyme
(e.g. horseradish peroxidase) th at can catalyze a chem ilum inescent reaction is
covalently linked to a m olecule (avidin o r an antibody) that also binds a DNA
"probe" (such as a specific DNA labelled with biotin or digoxygenin). Specific
d etection o f DNA fragm ents com plem entary to the probe can be achieved after
hybridisation by the addition of ap p ro p riate substrate m olecules an d the detec
tion o f light on photographic film. A lthough it is a m ore com plicated procedure
than autoradiography, the m ethod has the advantage o f creating stable reagents,
high sensitivity, rapid exposure times, an d avoidance of radioactivity.
Achrom osom e: A self-replicating genetic elem ent within the nucleus o f a eukary
otic cell that is com prised o f a single double-stranded m olecule o f DNA and a
large n u m b er o f proteins. Each chrom osom e contains a centrom ere w here spin
dle fibers attach during cell division an d telom eres at the ends o f each chrom o
some th at are req u ired for replication. All of the genetic inform ation in the
nucleus o f hum an cells is contained within a set of 23 pairs o f chrom osom es
which range in size from 50 to 250 Mb (average 150 M b), com prising a total of
three billion base pairs of haploid DNA. T he size o f the hum an m itochondrial
chrom osom e is 16,569 base pairs.
^chrom osom e banding: W hen cells in m etaphase are lysed an d spread on glass
slides, the chrom osom es are easily visible by light microscopy. Because the struc
ture an d com position o f the chrom osom es are n o t hom ogeneous, d ifferent dyes
(such as Giemsa and quinacrine) can be used that will create a standardised
b anding pattern along the length of each chrom osom e. These patterns can be
used to identify specific chrom osom es, establish a physical ru le r along the chro
m osom e by which genes can be m apped, and aid in the detection o f chrom o
somal rearrangem ents such as deletions and translocations.
chrom osom e painting: Alu-PCR (see Alu-PCR) can be used to amplify hum an-
specific genom ic DNA from flow-sorted chrom osom es or som atic cell hybrids (see
flow cytom etry and som atic cell hybrids), so that a hybridisation reagent can be
produced that will specifically label all or part o f specific chrom osom es. FISH
analysis can be carried o u t (jw fluorescence in situ hybridisation) by using fluo
rescence detection of the labeled PCR m aterial that will p ain t all or p a rt o f a
chrom osom e. These reagents are now com m ercially available an d are widely used
to d etect subtle chrom osom al rearrangem ents that were previously undetectable
by standard cytogenetic m ethods including chrom osom e banding.
496 GLOSSARY
Acoding region: T he coding region is that po rtio n of the mRNA transcript (the
processed RNA synthesized from the gene) that contains the genetic inform ation
for the am ino acid sequence o f the p rotein product, see exon, intron, splicing,
m essenger RNA, open reading fram e.
Acolony: A population o f cells that have arisen from a single p ro g en ito r cell. In
plasm id, cosm id o r yeast libraries, individual colonies can be identified by spread
ing the library on agar plates. Libraries gen erated in bacteriophage vectors can
also be spread on plates to identify hom ogeneous, clonal populations o f viruses
which cause discrete spots o f bacterial lysis, known as plaques.
Acosmids: A type of cloning vector that is derived from plasm ids bu t contains
the COS site of lam bda bacteriophage so that the recom binant vector can be
packaged an d transfected into E. coli. Cosmids are generally used for cloning
genom ic DNA in the size range of 25-40 kb because o f their greater efficiency at
en terin g bacteria by transfection com pared to the transform ation o f bacteria with
plasm ids containing large inserts.
AC ot-l DNA: M uch of the DNA within the m am m alian genom e is repetitive (see
repetitive DNA) and this DNA can be pari tally purified by d en atu rin g total
genom ic DNA and allowing it to reanneal to itself. Because repetitive sequences
are present in m any copies, the majority o f the DNA that initially reanneals will
contain these repetitive elem ents. DNA th at has rean n ealed u n d e r certain
GLOSSARY 497
defined conditions is called Cot-1 DNA and contains the m ost highly repetitive
DNA within the genom e. This m aterial can be used in hybridisations to com pete
o u t repetitive DNAs on target m olecules, allowing unique sequences that are re p
resented in the hybridisation probes to be detected.
#cR: w c e n tiR a y
Acrossing over: Crossing over occurs w hen two paired chrom osom es exchange
m atching regions o f their DNA. see recom bination
90 etc*
AdATP, y- P o r a- P labelled: D eoxyadenosine triphosphate with the y- posi-
09
don phosphate containing P is used for 5 end-labeling o f DNA or oligonucle-
otides using T4 polynucleotide kinase, a - P-dATP is used for labelling DNA
m olecules by incorporation o f the nucleotide into the polynucleotide chain using
a DNA polymerase.
Adigoxygenin: A steroid derived from the digitalis p lan t that can be covalently
linked to DNA by incorporation o f derivatized nucleotides. T he labelled DNA
can then be detected by a variety o f m ethods (chem ilum inescence, enzymatic
498 GLOSSARY
Adonor splice site: T he ju n ctio n between the 3' end o f an exon an d the 5' en d of
an adjacent intron.
*electroporation: A m ethod for perm eabilizing cell m em branes to allow for the
passage o f large m olecules such as DNA into the cell. T he cells are suspended in
a special cuvette and exposed to a b rief electrical pulse that causes transient holes
in the m em brane. T he electrical conditions vary considerably am ong different
cell types. T he technique can be used for eukaryotic cells or bacteria.
Aexpressed sequence tag: Equivalent to the sequence tagged site (STS) except
th at the un iq u e sequence that is used for construction o f the prim ers is within an
exon o f a gene.
Aexpression vector: A special vector (see Vector) that allows for the expression of
a foreign gene in an appropriate host. Expression vectors are used when a gene
can only be recognized by detection of its protein p roduct (im m unologically or
functionally), o r as a m eans o f m anufacturing the protein for biochem ical and
cell biology studies.
*flow cytom etry: Cells or subcellular fractions such as chrom osom es can be anal
ysed an d sorted by dispersing individual elem ents into m inute, electrostatically-
charged w ater droplets, d eterm in in g the optical or fluorescent pro p erties o f the
elem ent w ithin the droplet, an d then sorting the d roplet using a controlled mag
netic field. By this approach, individual chrom osom es can be isolated to 80-90%
purity a n d then used for the construction o f chrom osom e-specific genom ic
libraries.
# G 1: T he growth phase after mitosis during the cell cycle (see cell cycle).
*gel electrophoresis: see agarose gel electrophoresis, polyacrylam ide gel electro
phoresis
500 GLOSSARY
*gel re tard atio n assays: A m eth o d to identify proteins that bind to specific nucle
otide sequences within the pro m o ter regions of genes. D etection of specific bind
ing is achieved by dem onstrating th at the mobility o f a labeled DNA fragm ent in
a gel is specifically decreased by the binding o f one o r m ore proteins to an inter
nal sequence. U nlabelled oligonucleotides that contain the target sequ-ence can
be used to com petitively block this binding and allow the labeled DNA to m igrate
as if the binding protein was n o t present.
Agenetic m ap: T he linear ord erin g and spacing o f m arkers and genes along the
chrom osom es based upon the frequency o f recom bination betw een the linked
elem ents.
Agenom e: T he en tire set o f genetic inform ation contained within a single (hap-
loid) set o f chrom osom es.
Ah eterogeneous nuclear RNA: RNA that is found within the nucleus an d consists
o f unprocessed and partially processed transcripts, as well as small nuclear RNAs.
H eterogeneous nuclear RNA is rapidly processed by polyadenylation an d splicing
o u t o r removal o f the introns so th at the final transcript, the m essenger RNA,
only contains the exon regions o f the gene a n d can be exported to the cytoplasm.
Ah orseradish peroxidase: An enzyme that, w hen com bined with the appropriate
substrate, creates a chem ilum inescent or insoluble product th at can be visually
detected. By coupling the enzyme with an ap p ro p riate m olecule, e.g. an antibody
or avidin, it can be used for the detection o f specific DNA fragm ents o r proteins
(see chem ilum inescence, in situ hybridisation).
*in situ hybridisation: In situ hybridisation (ISH) is a m ethod for directly visual
izing the cellular o r subcellular location o f o ne or m ore DNA or RNA sequences
using hybridisation with a labeled probe a n d a suitable detection m ethod. In situ
hybridisation is used for the localisation o f one or m ore m arkers to specific chro
m osom es arrayed on a glass slide (see Fluorescence in situ hybridisation). ISH is
GLOSSARY 501
also used to identify the location o f cellular or viral transcripts o r viral DNA on
histologic preparations o f cells and tissue and to evaluate the developm ental
a n d /o r tissue-specific expression o f specific genes.
jum ping libraries: A genom ic library that has been constructed by cutting
genom ic DNA with a rare-cutting restriction endonuclease. T he large fragm ents
(100-200 kb) are allowed to circularize an d ligated. T he ligated circles are then
cut with a second enzyme an d the fragm ents are cloned. Clones are selected for
the presence o f the first, rare-cutting restriction endonuclease site. These clones
contain small fragm ents th at were originally separated by the length of the origi
nal fragm ent. They provide the m eans for ju m p in g across a distance th at is
g reater than can be norm ally contained within a single cosm id o r phage clone.
Akaryotype: T he display o f a com plete set of chrom osom es from a given individ
ual or cell (see B anding).
Amessenger RNA: T he RNA in the cell that encodes the processed transcript
from a gene and is the tem plate for p ro tein synthesis. Most, but no t all, of the
m essenger RNA in eukaryotic cells is polyadenylated (see polyA+RNA).
GLOSSARY 503
N orthern blot: A m ethod for the analysis of RNA. An RNA m ixture is fraction
ated by size using d en atu rin g agarose gel electrophoresis. D enaturing the RNA is
necessary to elim inate secondary stru ctu re and to allow the RNA m olecules to
m igrate in accordance with their size. These gels generally em ploy form aldehyde
or glyoxal, an d rarely, methyl m ercuric hydroxide, as the denaturants. T he RNA
is then transferred to a filter or support so as to preserve the size fractionation
achieved by gel electrophoresis. Specific RNA species can then be detected and
assessed for size and am ou nt using a hybridisation probe.
Aopen reading fram e: A region of genom ic DNA that potentially encodes for an
am ino acid sequence o f a protein . T he region is characterized by a stretch of
nucleotide sequence th at begins with an initiation codon, followed by a series of
triplets th at w ould encode for different am ino acids, an d ends with a term ination
codon that would en d peptide synthesis.
Aplasm ids: An extrachrom osom al genetic elem ent found in bacteria th at is capa
ble of self-replication. N aturally occurring plasm ids were first identified by their
ability to confer antibiotic resistance to bacteria. Plasmids are double-stranded
circular DNA's th at range in size from 1 to 200 kb. Most plasmids that are used as
cloning vectors range in size from 2.5 to 7 kb an d contain a nu m b er of unique
restriction sites for insertion of foreign DNA, drug-selection gene(s), and o th er
DNA sequences that allow for replication in the bacterial host, gene expression
or in vitro transcription.
ApolyA+ RNA: RNA that contains a 3 tail of adenine nucleotides (polyA). The
enzymatic addition of ad enine nucleotides occurs shortly after transcripation as
part o f the processing o f the prim ary RNA transcript in the cell nucleus. This
RNA represents the majority o f the m essenger RNA within a cell. Poly A+ RNA is
purified from total cellular RNA by selection with oligo dT (an oligonucleotide
consisting of 15-30 thym idine residues) th at can be b o u n d to cellulose, m em
branes, o r beads.
*polym erase chain reaction: A m eth o d (p aten ted by Perkin Elm er Cetus C orpo
ration) for amplifying a defined segm ent of double-stranded DNA by using two
DNA prim ers, one m atching a unique sequence at the 5' en d o f the sense strand
an d the o th er corresponding to the sequence o f the 5' en d o f the antisense
strand (Figure 1). W hen the DNA tem plate is d en a tu red by heat and allowed to
cool, the prim ers can specifically anneal to the com plem entary DNA strands and
serve as starting points for DNA synthesis by a therm ostabile DNA polym erase
(e.g. Taq polym erase). As a result, two new strands o f DNA are synthesised that
are com plem entary to the original DNA tem plate and two double-stranded m ole
cules are g enerated where previously there was only one. T he process of d en a tu r
ation, prim er annealing and DNA synthesis is then repeated a n d both DNAs can
serve as tem plates. W ith each successive ro u n d o r cycle, there is a doubling o f the
synthesised m aterial. W ithin a small n u m b er o f cycles (30-35) the initial tem plate
can be am plified 10-106 fold. As the process continues, a nearly hom ogeneous
506 GLOSSARY
po pulation o f DNA fragm ents o f a defined length are synthesised (Figure 1).
T hese fragm ents can then be cloned o r directly analysed for m utations or by
nucleotide sequencing. For Alu-PCR, the prim ers are derived from hum an-spe-
cific regions o f the highly repetitive Alu sequences. For reverse transcription-
PCR o r R T/PC R , the initial step consists o f synthesising a com plem entary DNA
(cDNA) to an RNA tem plate using reverse transcriptase and then using the cDNA
as the tem plate for a standard polym erase chain am plification.
Ap rom oter: T h at portion o f the gene that is involved in the control o f gene
expression, including tissue an d developm ental specificity an d regulation. T he
p ro m o ter includes the site at which the RNA polym erase binds a n d initiates the
transcription o f RNA from the gene.
nique is used for physical m apping o f genom ic DNA using long-range restriction
endonucleases and for the analysis o f YACs and yeast chrom osom es. T he cells to
be analysed are em b ed d ed in agarose plugs, lysed and de-proteinised with pro
teolytic enzymes because very large DNA m olecules are highly vulnerable to
shearing in aqueous solutions. T he extracted DNA m olecules are then separated
in an agarose gel using altern atin g directions o f electrical cu rren t that are at vary
ing angles to the final direction o f DNA m igration. This alternating application
of electric fields causes the DNA m olecules to shift their orientation and confor
m ation so th at they can gradually pass th ro u g h the gel m atrix and be separated
on the basis of size a n d shape (see FIG E).
*random primer labeling: T here are several m ethods of generating labeled DNA
that can be used as a probe for hybridisation. R andom p rim er labeling involves
the use o f small oligonucleotides (e.g. hexam ers) th at can anneal to the tem plate
DNA and serve as the starting points for DNA synthesis using a DNA polym erase
and nucleotides th at are radioactively labelled (35S, 32P,33P ). T he probe can then
be detected using autoradiography. If the nucleotides are m odified to contain
biotin, then a detection m ethod based upon the binding o f biotin an d an avidin-
conjugate can be used (horseradish peroxidase stain or chem ilum inescence)
O th e r DNA labeling m ethods include nick translation, incorporation o f ;~P on to
the 5' en d o f the probe or p rim er using T4 polynucleotide kinase, or the addition
of radioactive or derivatized nucleotides on to the 3' e n d o f a DNA probe using
term inal deoxynucleotide transferase.
elem ents (long interspersed nuclear elem ents, also known as LI repeats) that
range in size from 1 to 6 kb. T here are also h u n d red s o f thousands o f short rep et
itive elem ents known as m icrosatellite repeats (see m icrosatellite repeats, Cot-1
DNA).
AMPLIFICATION
[primer annealing (55C)
and extension (72C)]
M B
' [---------------------------------T -
#S phase: T he DNA synthesis phase o f the cell cycle (see cell cycle).
Asense strand: Also known as the coding strand, the sense strand is the strand of
duplex DNA whose sequence corresponds to the nucleotide sequence o f the
mRNA transcribed from that gene. T he com plem entary strand to the sense
strand is often referred to as the antisense strand o f DNA. T he antisense strand of
genom ic DNA is the tem plate for RNA transcription.
510 GLOSSARY
Asequence tagged site: A specific location in the genom e that is defined and
identified by m eans of flanking PCR p rim er sequences th at specifically amplify
that locus. T h e site can therefore be identified using PCR within genom ic clones,
som atic o r radiation cell hybrids. T he PCR products re p resen t the sequence
tagged sites, STSs, and are used for the construction o f genetic and physical
maps. T he presence of a given STS in two o r m ore genom ic clones is an indica
tion that the clones overlap and form part o f a contig (see PCR, physical maps,
contig m apping).
Ashuttle vectors: G enetic elem ents that allow for the propagation o f foreign
DNA in m ore than one cell type. For exam ple, a plasm id that allows for propaga
tion o f a foreign gene in bacteria and can also be stably transferred to a yeast host
o r to a eukaryotic cell for gene expression (see v ecto r).
^single strand conform ation polym orphism : Also called single strand conform a
tion analysis, this m ethod detects single base differences betw een two segm ents of
DNA (usually u n d e r 400 bp in length) by observing the subtle differences in
mobility o f the DNA when the single-stranded m olecules are allowed to form
intra-strand base pairing as they m igrate through a non-denaturing polyacryla
m ide gel. T he differences in mobility are due to the slightly d ifferent folding pat
terns shown by partially base-paired single-stranded DNA m olecules as a result of
nucleotide differences.
*som atic cell hybrid m apping: Somatic cell hybrids are cell lines th at contain the
m ajority o f their DNA from one species an d a sm aller am ount of DNA (a few
chrom osom es o r chrom osom al fragm ents) from an o th er species. Panels o f these
hybrid cells, containing a broad distribution of hum an chrom osom es or chrom o
somal fragm ents, can be readily used to localise new m arkers o r genes to specific
hum an chrom osom es o r chrom osom al regions.
Asplicing, in tro n /ex o n : T he process by which the initial transcript o f the gene
(called h eterogeneous nuclear RNA o r hnRNA) is edited to remove the intron
regions and appropriately connect the exons that make up the final mRNA tran
script (see intron, exon).
GLOSSARY 511
ATATA Box: A nucleotide sequence, e.g. TATAAAA, within the pro m o ter region,
usually located 19-27 bp from the transcription start site of eukaryotic genes.
Avariable number tandem repeats: DNA segm ents that contain variable num bers
o f short tandem ly repeated segm ents with a particular sequence. G enom ic DNA
is cut with a restriction enzyme outside th e rep eated sequence, the specific frag
m en t is size fractionated using gel electrophoresis and then d etected by hybridis
ation. Because o f the variable nu m b er of repeats, the fragm ents d em onstrate dif
feren t lengths (polym orphism s) in d ifferent individuals which can be used as
alleles for that locus.
Avector: A genetic elem ent th at is used to carry a foreign piece o f genetic infor
m ation into a cell and will support the propagation of that foreign DNA within
the cell. Vectors can refer to plasmids, cosm ids an d phage which su p p o rt foreign
DNA in bacteria, as well as YACs in yeast a n d viral elem ents that in troduce DNA
into m am m alian cells.
Ayeast artificial chrom osom e: An artificial chrom osom e that contains large frag
m ents o f foreign DNA (0.1-1.5 Mb) within a linear vector m olecule that can stably
propagate itself and segregate within the yeast Saccharomyces cerevisiae. A YAC con
tig (see contig) can define a large region of the genom e with a lim ited nu m b er of
clones. T he use o f these vectors is h am p ered by occasional instability o f the
inserted DNA, cloning artifacts such as rearrangem ents an d jo in in g o f unrelated
sm aller pieces o f DNA (chim aeric clones), an d difficulties in obtaining large
am ounts o f the purified YAC free of background yeast chrom osom es. YACs pro
vide a source of DNA for the identification of genes, Alu-PCR, fluorescence in situ
hybridisation and chrom osom e painting, lim ited genom ic libraries, an d contig
m apping o f large genom es (see Alu-PCR, chrom osom e painting, cloning, contig
m apping, fluorescence in situ hybridisation, and library).
zinc linger proteins: Proteins th at contain one o r m ore segm ents that are capa
ble o f binding Zn to two closely-linked cysteine residues followed by two histidine
residues. T he binding of Zn creates loops or fingers th at are capable of binding
DNA and potentially regulating transcription.
INDEX