British Journal of Haematology, 1999, 104, 213

Review
RED BLOOD CELL MEMBRANE DISORDERS
The red blood cell membrane is a multi-component structure
that is responsible for many of the physiological functions
and mechanical properties of the cell. Defects in any of these
components can manifest as clinical disorders involving the
erythrocytes. In the past few years there have been major
advances in our understanding of the molecular basis of
these disorders, in which mutational analysis has clarified
many questions about the structurefunction relationship of
components of the red cell membrane. Some of these recent
findings will be the subject of this review. A detailed
discussion of the structure of the red cell membrane and
the pathophysiology and clinical aspects of its disorders
can be found in several recent reviews (Lux & Palek,
1995; Delaunay, 1995; Hassoun & Palek, 1996; Gallagher
et al, 1998). An updated catalogue of mutations causing
red cell membrane disorders is available at the website
http://www.kidscancer.net/membrane/.
The red cell membrane skeleton protein complex
The red cell membrane comprises a lipid bilayer, integral
membrane proteins and a membrane skeleton (Fig 1). The
red cell membrane skeleton is a multi-protein complex
formed by structural proteins including a and b spectrin,
ankyrin, protein 4.1 and actin. The membrane skeleton
proteins interact with the lipid bilayer and transmembrane
proteins to give the red cell membrane its strength and
integrity. They interact with each other to form a scaffolding
on the inner surface of the lipid bilayer. a and b spectrin
interact side-to-side to form flexible rod-like heterodimers
which self-associate head-to-head to form tetramers. The
tetramers are linked by ankyrin to the cytoplasmic domain of
the integral membrane protein band 3. Protein 4.2 binds to
band 3 at the same position and may enhance the ankyrin
band 3 interaction. Multiple spectrin tetramers interact at
their tail ends and with actin protofilaments, tropomyosin,
tropomodulin and adducin to form junctional complexes.
Protein 4.1, which also binds to the integral membrane
protein glycophorin C, interacts with b spectrin at the actinbinding domain and increases the affinity of the spectrin
actin binding.
The genes of the major red cell membrane proteins have
been cloned and their amino acid sequences deduced.
Structural and functional domains in each protein have
been characterized (Fig 2). Band 3, the erythrocyte anion
exchanger, is a 102 kD integral protein divided into
two domains. The 43 kD cytoplasmic domain binds ankyrin, proteins 4.1 and 4.2, various glycolytic enzymes and
Correspondence: Dr W. T. Tse, Division of Hematology/Oncology,
Childrens Hospital, 300 Longwood Avenue, Boston, MA 02115,
U.S.A.

haemoglobin. The 52 kD membrane domain, composed of

1214 transmembrane helices, forms the physiologically
important channel for chloridebicarbonate exchange.
Ankyrin is a 210 kD peptide divided into three domains: a
89 kD N-terminal band 3-binding domain with 24 ankyrin
repeats, a central 62 kD spectrin-binding domain and a Cterminal 55 kD regulatory domain. The b spectrin peptide
measures 246 kD and contains an N-terminal actin-binding
domain, a central rod-like structure with 17 triple helical
coiled-coil spectrin repeats, each 106 residues in length, and
a short C-terminal tail. Repeats 15 and 16 contain the
ankyrin binding site. The a spectrin peptide is 280 kD in size.
It contains 22 spectrin repeats and a C-terminal calciumbinding site with EF hand motifs. Protein 4.1 is a 80 kD
peptide that has a glycophorin C/protein p55 binding
domain and a spectrin/actin binding domain. Protein 4.2
is a 72 kD peptide that binds to both band 3 and ankyrin.
Its function is not clear.
Disruption of the interaction between components of the
red cell membrane skeleton at any contact point may cause
loss of structural and functional integrity of the membrane.
Conceptually, there are two types of interactions: vertical
interactions between the membrane skeleton and the lipid
bilayer, and horizontal interactions among components
that form the membrane skeleton meshwork (Fig 1). The
important links in the vertical interaction involve band 3,
ankyrin, spectrin and protein 4.2. The critical horizontal
interactions occur between the a and b spectrins, b spectrin
and protein 4.1, and protein 4.1 and actin (Morris & Lux,
1995). As predicted more than a decade ago (Palek & Lux,
1983; Palek, 1985), defects in horizontal interactions result
in hereditary elliptocytosis or hereditary pyropoikilocytosis, and defects in vertical interactions lead to hereditary
spherocytosis. Recent identification of the many defects that
underlie red cell membrane disorders has shown that this
model is essentially correct.
Hereditary spherocytosis is caused by defective vertical
interactions
Hereditary spherocytosis (HS) is the most common cause of
non-immune haemolytic anaemia in people of Northern
European ancestry, with a prevalence of approximately 1
in 2000. In about 75% of the cases the inheritance follows
an autosomal dominant pattern, and in about 25% the
occurrence of the disease is sporadic in nature (Agre et al,
1986; Eber et al, 1990). About half of the sporadic cases are
probably caused by a recessive form of HS and the rest by
spontaneous new mutations (Eber et al, 1990; Miraglia del
Giudice et al, 1998a, b). Clinically, HS is characterized by the
presence of spherocytes in peripheral smears with varying
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Review

Fig 1. Schematic model of the red cell membrane, with the vertical and horizontal interaction of its components indicated. Estimated frequencies
of mutations in different membrane proteins in HS and HE/HPP are as follows. Vertical interaction: hereditary spherocytosis: band 3, ,20%;
protein 4.2, ,5%; ankyrin, ,45%; b spectrin, ,30%. Horizontal interaction: hereditary elliptocytosis/hereditary pyropoikilocytosis: b spectrin,
,5%; a spectrin, ,80%; protein 4.1, ,15%. The relative position of the various proteins is correct, but the proteins and lipids are not drawn to
scale. Adapted from Lux & Palek (1995).

degrees of haemolysis and splenomegaly. There is increased

fragility of the red cell membrane, leading to vesiculation of
the membrane, loss of membrane surface area, and trapping
and destruction of the red cells in the spleen. Splenectomy
ameliorates the degree of haemolysis and is indicated in
severely affected patients, but the intrinsic abnormality of the
red cells remains after the procedure.
Early biochemical analysis of the membrane skeleton
proteins showed that spectrin is deficient in patients with HS
(Agre et al, 1985). The severity of the disease and response to
splenectomy correlates with the amount of spectrin deficiency (Agre et al, 1986; Eber et al, 1990). Cytogenetic and
genetic linkage analyses later showed that ankyrin defects
are an underlying cause of HS in some families (Lux et al,
1990; Costa et al, 1990). Subsequent work showed that most
patients with HS have combined spectrin and ankyrin
deficiency (Pekrun et al, 1993; Savvides et al, 1993). Our
current understanding is that HS is caused by defects in the
proteins involved in the vertical interactions between the
membrane skeleton and the lipid bilayer. Several surveys
using sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis have shown that 3045% of
HS patients have combined ankyrin and spectrin deficiency,
about 30% have isolated spectrin deficiency, and about 20%
have band 3 deficiency (Miraglia del Giudice et al, 1994;
Jarolim et al, 1996a; Dhermy et al, 1997; Lanciotti et al,
1997). Analysis by SDS-PAGE may, however, underestimate
the degree of ankyrin deficiency in these patients. Quantitative analyses by radioimmunoassay and ELISA show that
q 1999 Blackwell Science Ltd, British Journal of Haematology 104: 213

most HS patients have a roughly equivalent deficiency

of both spectrin and ankyrin (Pekrun et al, 1993; Savvides
et al, 1993). A high reticulocyte count may also mask the
detection of ankyrin deficiency (Miraglia del Giudice et al,
1997a). Isolated protein 4.2 deficiency is found in a small
number of American and European patients but is more
common in Japan (Inoue et al, 1994).
Ankyrin. Using a dinucleotide repeat polymorphism of the
ankyrin cDNA as a marker to distinguish between the two
ankyrin alleles, it was shown that one ankyrin allele has
reduced expression in a third of HS patients with combined
spectrin and ankyrin deficiency (Jarolim et al, 1996b). This
may be caused by reduced transcription of the gene or
decreased stability of its transcripts. By the same method, it
has been shown that de novo mutations in one of the ankyrin
alleles leading to decreased expression are frequent in HS
children with normal parents (Miraglia del Giudice et al,
1998a, c). Molecular analyses confirm that ankyrin defects
are present in roughly half of patients with HS (Eber et al,
1996).
Null mutations predominate in dominant HS. These are
nonsense or frameshift mutations that result in either
unstable ankyrin mRNA transcripts or truncated peptides.
Mutant ankyrins Bari, Bugey, Duisburg, Einbeck, Marburg,
Napoli I, Osterholz and Stuttgart are truncated in the band
3 binding domain (Eber et al, 1996; Miraglia del Giudice
et al, 1996; Morle et al, 1997; Randon et al, 1997), ankyrins Anzio, Napoli II and Porta Westfalica are affected in
the spectrin binding domain, and ankyrins Bovenden and

Review

Saint-Etienne 1 and 2 have premature termination within

the regulatory domain (Eber et al, 1996; Hayette et al, 1998).
In ankyrin Rakovnik a nonsense mutation within the
regulatory domain leads to a selective deficiency of the
major ankyrin isoform, protein 2.1 (Jarolim et al, 1995b).
A point mutation in position 204 of the promoter region in
a patient with moderate HS presumably leads to decreased
synthesis of ankyrin (Eber et al, 1996). The clinical pictures
associated with these null mutations differ markedly, ranging from mild haemolysis to severe transfusion-dependent
anaemia. This variability may be explained by different
amounts of compensation for the null mutation, either from
overproduction of the normal ankyrin allele or diminished
ankyrin degradation.
Several ankyrin defects have been found in patients with
recessive HS. Unlike the null mutations found in dominant
HS, these are usually missense or promoter mutations. A
point mutation in the ankyrin promoter 108 bases before the
translation start is particularly common. It was found in four
of seven families with recessive HS examined in one study
(Eber et al, 1996). In two of these families a second mutation
in the other ankyrin allele was identified: ankyrins Walsrode
and Bocholt. Ankyrin Walsrode was present in a patient
whose red cells were more deficient in band 3 than in
spectrin or ankyrin, which is opposite to the trend in other
ankyrin defects. It arose from a missense mutation in the
band 3 binding domain and had a decreased affinity for
band 3. Ankyrin Bocholt was found in another patient with
recessive HS and bears a missense mutation in a rare
alternative splice product that may result in aberrant
splicing of the transcript.
HS patients with ankyrin defects have prominent spherocytosis without other morphological defects. Haemolysis and
anaemia vary from mild to severe (Eber et al, 1996; Miraglia
del Giudice et al, 1996, 1998a; Morle` et al, 1997; Randon et
al, 1997; Hayette et al, 1998). In general, patients with
dominant defects are less affected than those with recessive
mutations; however, there is considerable overlap.
b spectrin. Although spectrin deficiency has long been
associated with HS, it is only recently that specific mutations
in spectrins have been identified as a primary cause of the
disorder. a spectrin is normally produced in excess in red
cells and b spectrin production is the rate-limiting step in
biosynthesis of the membrane skeleton (Hanspal & Palek,
1987). For this reason, most of the spectrin defects in HS are
in the b spectrin gene.
Frequent de novo monoallelic expression of the b spectrin
gene has been reported in children with HS and spectrin
deficiency (Miraglia del Giudice et al, 1998b), suggesting that
the defective allele is often not expressed. More than 10 b
spectrin alleles resulting in a null mutation have been
described, all associated with dominant HS. In spectrin
Promissao a mutation in the translation initiation codon
prevents translation of the peptide (Basse`res et al, 1998). In
spectrins Bergen, Houston, Ostrava and Philadelphia, frameshift mutations close to the N-terminus of b spectrin resulted
in unstable transcripts and spectrin deficiency (Hassoun et al,
1997). Nonsense mutations in spectrins Baltimore and
Tabor had the same effect. A 46 kb genomic deletion in

spectrin Durham resulted in a truncated peptide that is

inefficiently incorporated into the red cell (Hassoun et al,
1995). A splice site mutation in spectrin Winston-Salem
leads to exon skipping and an unstable truncated b spectrin
peptide, and a similar mutation in spectrin Guemene-Penfao
caused intron retention and decreased levels of b spectrin
mRNA (Hassoun et al, 1996; Garbarz et al, 1998).
Several missense mutations in b spectrin have been
described in HS. In spectrin Kissimmee a point mutation
near the N-terminus of b spectrin, close to the putative
protein 4.1 binding site (Becker et al, 1993), causes defective
binding to protein 4.1, makes the spectrin unstable and
oxidant sensitive (Becker et al, 1987), and leads to spectrin
deficiency and dominant HS. In spectrins Atlanta and
Oakland, point mutations flank the site of the Kissimmee
mutation (Hassoun et al, 1997). In one case of recessive HS
the patient has a point mutation in position 1684 of the
b spectrin chain (spectrin Birmingham), which changes an
arginine to a cystine (Hassoun et al, 1997). The presumed
mutation in the second allele has not been identified.
Clinically, HS varies from mild to severe in patients with
b-spectrin defects (Becker et al, 1993; Hassoun et al, 1995,
1996, 1997; Basse`res et al, 1998; Miraglia del Giudice et al,
1998b). Spherocytosis is prominent and is associated with a
subpopulation (815%) of dense spiculated erythrocytes or
acanthocytes that are not seen in other forms of HS.
a spectrin. a spectrin is normally synthesized three- to
four-fold in excess of that incorporated in the red cell
membrane (Hanspal & Palek, 1987). Defective a-spectrin
production in one allele is not expected to cause a disease
phenotype, and a spectrin mutations have not been
described in patients with dominant HS. Defects in both
a spectrin alleles have nonetheless been implicated in the
recessive form of HS with severe spectrin deficiency (Agre
et al, 1982; Wichterle et al, 1996; Tse et al, 1997). A patient
with a severe spherocytic haemolytic anaemia and no family
history of spherocytosis was found to have an a spectrin
allele encoding a truncated peptide, spectrin Prague, and a
low-expression a spectrin allele, spectrin LEPRA (lowexpression allele Prague) (Wichterle et al, 1996). The latter
allele contained a splicing mutation that causes premature termination of translation and a marked decrease in
a spectrin production. This low-expression allele is in linkage disequilibrium with an a spectrin polymorphism, aIIa
variant or spectrin Bughill, that is commonly found in
families with nondominant HS (Boivin et al, 1993; Tse et al,
1997). In these patients, analysis of their membrane spectrin
shows only the Bughill variant but genomic analysis reveals
the presence of both the Bughill allele and a second allele
without the aIIa polymorphism (Tse et al, 1997). These
results indicate that the allele in trans to (on the opposite
chromosome of) the Bughill allele is functionally silent in
these patients. Interaction of the a spectrin LEPRA allele
with another a spectrin allele carrying a null mutation may
be a common cause of recessive HS.
Band 3. Band 3 is the major integral protein on the red cell
membrane that interacts with the membrane skeleton.
Deficiency of band 3 is found in about 20% of American
and European patients with HS, but is more common in
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Review
Japanese (Jarolim et al, 1996a; Eber et al, 1996; Inoue et al,
1994). The disease is inherited as a dominant trait with
relatively mild anaemia and spherocytosis. Many unsplenectomized patients also have a small population (0223%)
of mushroom-shaped or pincered erythrocytes (Reinhart et
al, 1994; Jarolim et al, 1996a), which are not seen in other
forms of HS. A murine model of band 3 deficiency showed
that band 3 is essential for stability of the membrane lipid
bilayer but not for assembly of the membrane skeleton
(Peters et al, 1996).
Many changes in the band 3 gene that result in null
mutations have been identified in patients with HS. Band 3
Hodouin, Lyon, Noirterre, Osnabruck I and Trutnov have
nonsense mutations that cause mRNA instability and band 3
deficiency (Eber et al, 1996; Jarolim et al, 1996a; Jenkins
et al, 1996; Alloisio et al, 1996). Band 3 Bice`tre II, Bohain,
Bruggen, Foggia and Smichov have single nucleotide
insertions in the coding region that produce a frameshift
mutation, whereas band 3 Evry, Hobart, Napoli I, Princeton
and Worcester have single nucleotide deletions (Miraglia del
Giudice et al, 1997b; Dhermy et al, 1997). Band 3 Campinas
and Pribam result from splicing defects, and band 3 Prague
occurs because of a 10-nucleotide duplication in the gene
(Lima et al, 1997; Jarolim et al, 1994).
Missense mutations or short in-frame deletions have also
been found in HS patients. Many such mutations are located
in the transmembrane domain and probably cause poor
incorporation of band 3 into the membrane. In band 3
Bice`tre I, Dresden, Hradec Kralove, Jablonec, Prague II and
Prague III, substitution of highly conserved arginine residues
positioned at the internal boundaries of transmembrane
segments probably interferes with cotranslational insertion
of band 3 into the membranes of the endoplasmic reticulum
(Eber et al, 1996; Jarolim et al, 1995a; Dhermy et al, 1997).
In band 3 Benesov, Birmingham, Chur, Most, Napoli II,
Okinawa and Philadelphia, other highly conserved amino
acids crucial for stabilization of band 3 within the lipid layer
are substituted (Maillet et al, 1995; Kanzaki et al, 1997;
Miraglia del Giudice et al, 1997b). A 22-residue insertion in
the transmembrane domain in band 3 Milano prevents
incorporation of the peptide into the membrane (Bianchi
et al, 1997), and a single amino acid deletion in the
transmembrane domain is found in band 3 Osnabruck II
(Eber et al, 1996).
Missense mutations in the cytoplasmic domain of band 3
can interfere with its binding to other membrane skeleton
proteins, resulting in a functional defect. In band 3 Nachod,
a deletion of five amino acids from the ankyrin-binding site
disrupts this binding (Jarolim et al, 1996a). An amino acid
substitution in the cytoplasmic domain in band 3 Fukuoka
causes defective protein 4.2 binding. A compound heterozygote carrying both band 3 Fukuoka and band 3 Okinawa
has complete absence of protein 4.2 (Kanzaki et al, 1997).
Patients with band 3 Montefiore and Tuscaloosa have
spherocytic haemolytic anaemia with protein 4.2 deficiency
(Jarolim et al, 1992; Rybicki et al, 1993). Both of these alleles
have missense mutations in the cytoplasmic domain, but
symptoms are manifest in heterozygotes with band 3 Tuscaloosa and only in the homozygote with band 3 Montefiore.
q 1999 Blackwell Science Ltd, British Journal of Haematology 104: 213

The reason for this difference is unclear. Band 3 Coimbra has

a substitution in the putative second ectoplasmic loop of
band 3, causing mild HS (Alloisio et al, 1997). Several
mutant band 3 alleles modulate the severity of the disease.
Band 3 Mondega and Montefiore, which both have missense
mutations in the cytoplasmic domain, aggravate the HS
symptoms of band 3 Coimbra when either is co-inherited.
A normally silent promoter mutation in band 3 Genas
increases the clinical severity of HS in a patient with band 3
Lyon (Alloisio et al, 1996).
Since band 3 protein functions as an anion exchanger and
is also expressed in the intercalated cells in the kidney
cortical collecting ducts, patients with HS and band 3
deficiency have been tested for a defect in acid-base homeostasis. Two patients with band 3 Pribram have incomplete
distal renal tubular acidosis, but most HS patients with band
3 deficiency have no evidence of metabolic acidosis (Rysava
et al, 1997; Jarolim et al, 1998). In patients with band 3
Campinas there is increased basal urinary bicarbonate
excretion but efficient urinary acidification (Lima et al,
1997). A bovine model of band 3 deficiency exhibits only
mild acidosis (Inaba et al, 1996), and there are no obvious
metabolic disturbances in mice that have a targeted deletion
of band 3 that eliminates both the red cell and kidney
isoforms (Peters et al, 1996). Band 3 mutations have been
found in patients with dominant distal renal tubular
acidosis, but these patients have no red cell abnormality
and the mutations identified are different from those
associated with HS (Jarolim et al, 1998; Bruce et al, 1997;
Karet et al, 1998).
Protein 4.2. Partial deficiency of protein 4.2 is often seen in
patients with deficient ankyrin or band 3, secondary to the
underlying defect (Lux et al, 1990; Lanciotti et al, 1997).
Protein 4.2 is also absent in a mouse model with targeted
deletion of the band 3 gene (Peters et al, 1996). However,
some HS patients have an isolated deficiency of protein 4.2 in
their red cell membranes. The disease is recessively inherited
but clinically mild, and blood smears of patients may show
ovalocytes or stomatocytes in addition to mild spherocytosis.
In rare cases moderate haemolysis is observed (Kanzaki et al,
1995a).
Seven protein 4.2 mutations underlying the disease have
been identified. Protein 4.2 Nippon is a particularly common
mutation in Japan (Bouhassira et al, 1992). It arises from a
missense mutation that causes mild haemolytic disease in
homozygotes or in compound heterozygotes with a second
mutant allele such as protein 4.2 Fukuoka, Notame or Shiga
(Takaoka et al, 1994; Matsuda et al, 1995; Kanzaki et al,
1995b). The only two protein 4.2 mutations reported
outside the Japanese population are protein 4.2 Tozeur and
Lisboa, found in the homozygous form in Tunisian and
Portuguese patients, respectively (Hayette et al, 1995a, b).
Hereditary elliptocytosis and hereditary pyropoikilocytosis
are caused by defective horizontal interactions
Hereditary elliptocytosis and hereditary pyropoikilocytosis
(HE/HPP) are a heterogenous group of red cell membrane
disorders with a wide spectrum of clinical presentations,
ranging from asymptomatic elliptocytosis to life-threatening

Fig 3. Structure of the spectrin tetramer, showing the triple helical coiled-coil repeats of the spectrin peptides, the head-to-head spectrin self-association sites, and the nucleation sites that initiate
the side-to-side interaction between a and b spectrin chains.

Fig 2. Domain structure of band 3, ankyrin, b spectrin and a spectrin, with positions of known mutations causing HS and HE/HPP indicated beneath each protein.

6
Review

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Review
haemolytic anaemia with poikilocytosis and red cell
fragmentation. The genetic basis of HE/HPP was puzzling
in the past because patients with HPP often had family
members with HE, and the inheritance pattern was not
clear-cut. Protein analysis has shown that the basic defect
underlying most cases of HE/HPP is a failure of spectrin
heterodimers to self-associate into heterotetramers, which
are the basic building blocks of the membrane skeleton
network. In normal individuals the proportion of dimers in
the red cell membrane is < 5%. In HE/HPP patients the
percentage can be as high as 6080%, and correlates closely
with clinical severity (Lecomte et al, 1993; Silveira et al,
1997). In addition to the dimer self-association defect, HPP
patients frequently have some degree of spectrin deficiency
(Hanspal et al, 1993). It is now known that HPP patients are
often homozygotes for an HE allele, compound heterozygotes
for two HE alleles, or carriers of one HE allele and a lowexpression spectrin allele.
The mutations causing HE/HPP involve components of the
membrane skeleton responsible for interactions in the horizontal dimension: a spectrin, b spectrin and protein 4.1
(Fig 1). Mutations affecting the structural integrity of
spectrin are particularly important. The main portion of
the spectrin peptide is folded into triple helical coiled-coil
repeat units (Fig 3). The b and a spectrin chains assemble side-to-side into a heterodimer in a zipper-like fashion, beginning with a nucleation site located near the
N-terminus of b spectrin and the C-terminus of a spectrin
(Speicher et al, 1992). The b and a spectrin heterodimers
then self-associate into a tetramer by a head-to-head
interaction involving the C-terminus of b spectrin and the
N-terminus of a spectrin. The b and a spectrins contribute
two helices and one helix, respectively, to form a single triple
helical repeat unit, which constitute the spectrin dimer selfassociation site (Tse et al, 1990; Speicher et al, 1993). The
proper assembly of these repeat units is essential for the
integrity of the spectrin scaffolding, and most mutations
found in HE/HPP appear to disrupt this process.
b spectrin. More than 18 b-spectrin mutations have been
described that cause HE/HPP. Small deletions or insertions
causing frameshift mutations and premature termination of
b spectrin are found in spectrins Napoli, Nice, Tandil and
Tokyo (Wilmotte et al, 1994). Splice site mutations in
spectrins Gottingen, LePuy and Rouen and a nonsense
mutation in spectrin Nagoya result in truncated b spectrin
peptides defective in heterodimer self-association (Maillet
et al, 1996). Missense mutations in b spectrin are present in
spectrins Buffalo, Cagliari, Cosenza, Cotonou, Kayes, Linguere, Paris and Providence, in which amino acid substitutions either disrupt the triple helical coiled-coil structure that
forms the spectrin dimer self-association site or affect
residues critical to the interaction (Tse et al, 1990; Sahr
et al, 1993; Parquet et al, 1994; Gallagher et al, 1995, 1997;
Glele-Kakai et al, 1996; Qualtieri et al, 1997; Nicolas et al,
1998). Without exception, these mutations directly affect
the heterodimer self-association site of b spectrin (Fig 2).
A more proximal mutation would presumably cause mRNA
or protein instability, resulting in spectrin deficiency and a
phenotype of HS instead. This distinction is illustrated in the
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syndrome of spherocytic elliptocytosis, in which patients

have a clinical picture intermediate between HS and HE. In
spectrins Campinas and Prague, which are associated with
spherocytic elliptocytosis, splicing mutations in b spectrin
result in truncated peptides that are both unstable and
defective in dimer self-association (Jarolim et al, 1995c;
Basse`res et al, 1997). The resulting spectrin deficiency and
ineffective tetramerization may explain the combination
phenotype.
a spectrin. At least 25 mutations in a spectrin have been
found in patients with HE/HPP. Most of the mutations are
single amino acid substitutions clustered around the first few
repeat units of a spectrin, close to the site of dimer selfassociation (Fig 2). Several recently described examples are
spectrins Alexandria, Anatasia, Barcelona, Genova, Lograno, Marseilles and Ponte de Sor (Dalla Venezia et al, 1993;
Gallagher et al, 1993; Lecomte et al, 1993; Boulanger et al,
1994; Parquet et al, 1994; Perotta et al, 1994, 1995). A few
other mutations involve in-frame deletion of short segments
of the peptide. In spectrin Sfax a splice site mutation results
in a nine amino acid deletion in the fourth repeat, and in
spectrin Dayton a mobile DNA element in the a spectrin
gene causes deletion of 49 residues in the second repeat
(Baklouti et al, 1992; Hassoun et al, 1994). These mutations
presumably prevent the proper folding of the a spectrin
peptide and interfere with its capacity to interact with b
spectrin. In general, the further the mutation is from the
spectrin dimer self-association site the milder is its disruptive
effect. In spectrin Oran, for instance, a splice site mutation
causes an in-frame deletion of part of the eighth a spectrin
repeat, which is quite far from the dimer self-association
site. Heterozygote carriers of the mutation are clinically
silent and the only reported homozygous patient has mild
elliptocytosis for a homozygote (Alloisio et al, 1993).
Unlike the case of b spectrin, very few mutations that
result in truncated a spectrin peptides have been described in
patients with HE/HPP. Such a peptide would lack the spectrin
dimer nucleation site and would probably not be incorporated into the membrane. Since a spectrin is normally
produced in 34-fold excess compared to b spectrin (Hanspal & Palek, 1987), enough a spectrin subunits will be
produced by the normal a spectrin allele to bind all of the
b spectrin chains and form a complete membrane skeleton
network. This point is illustrated by spectrin St Claude, in
which a splice site mutation causes premature termination
of the a spectrin chain, abolishing the spectrin dimer
nucleation site (Fournier et al, 1997). The index patient
was homozygous for the mutation and presented with severe
haemolytic anaemia with poikilocytosis, microspherocytosis
and elliptocytosis. In contrast, his heterozygous parents and
a brother were completely asymptomatic and have normal
red cell morphology.
Low-expression allele of a spectrin. In many kindreds there
are affected individuals with severe haemolytic anaemia
consistent with a diagnosis of HPP, whereas other family
members have only mild symptoms and a clinical diagnosis
of HE. It was hypothesized that these severely affected
patients inherit a second low-expression allele, which is
silent in carriers but contributes to the severity of the disease

Review

when present in trans to a structurally mutant allele. A

common a spectrin polymorphism was found to be linked to
such an allele (Alloisio et al, 1991). In this allele, named
aLELY (low expression Lyon), a mutation in intron 45 causes
skipping of exon 46 in about 50% of the mRNAs (Wilmotte
et al, 1993). The deletion of the residues encoded by this exon
appears to abolish the nucleation site in a spectrin essential
for its side-to-side association with b spectrin and the mutant
a spectrin fails to be incorporated into the membrane
(Wilmotte et al, 1997). The aLELY allele is found in high
frequency in all ethnic groups examined. Its frequency
ranges between 16% and 31% among Caucasians, Africans,
Japanese, Chinese and Amazon Indians, suggesting that it is
of a very ancient origin (Marechal et al, 1995; Basse`res et al,
1998). Because the allele is so widespread it frequently
interacts with other spectrin mutations and may account
for much of the clinical variability seen in the HE/HPP
syndromes.
Because a spectrin peptides are normally produced in
excess and the mutation causes only partial skipping of exon
46, aLELY is completely silent by itself, even when homozygous. If the allele occurs in trans to a second allele encoding
a structurally abnormal a spectrin, however, the mutant
peptide with the structural defect will be preferentially
incorporated into the membrane, increasing the severity
of the disease dramatically (Alloisio et al, 1991). As an
example, a child with a de novo HE mutation presented with
severe congenital poikilocytosis instead of simple mild
elliptocytosis because he also inherited an aLELY allele
in trans to the HE allele (Lorenzo et al, 1993). In the index
family with spectrin Sfax the proband had prominent
elliptocytosis, haemolysis and splenomegaly even though
several other affected family members exhibited only mild
elliptocytosis (Baklouti et al, 1992). Molecular analysis
showed that the proband inherited the structurally mutant
allele from his father and an aLELY allele from his mother,
which explains his more severe clinical picture. The converse
of the above can also be seen. The aLELY allele occurring in cis
(on the same chromosome as an HE allele) ameliorates the
severity of the disease, because the peptide carrying the HE
defect is not efficiently incorporated into the membrane
(Randon et al, 1994).
Protein 4.1. 4.1() HE designates a distinct condition in
which the red cell membrane contains a reduced quantity of
protein 4.1. Heterozygotes show prominent elliptocytosis but
no haemolysis or membrane fragility. Homozygotes present
with severe haemolytic anaemia with elliptocytosis, poikilocytosis and fragmented erythrocytes. Specific molecular
defects have been identified in five different kindreds. In 4.1
Madrid and 4.1 Lille a missense mutation in the initiation
codon prevents translation of the protein (Dalla Venezia et al,
1992; Garbarz et al, 1995). A 318 bp deletion in 4.1 Algeria
eliminates the initiation codon, resulting in complete protein
4.1 deficiency in the homozygote (Conboy et al, 1993). In
protein 4.1 Annery a large 70 kb deletion of the gene occurs
(Dalla Venezia et al, 1998). Deletion of a single residue in the
spectrin-binding domain in protein 4.1 Aravis abolishes its
capacity to bind spectrin (Lorenzo et al, 1994). Unlike other
membrane skeleton proteins, missense mutations that

change codons other than the initiation codon of protein

4.1 have not been found.
Southeast-Asian ovalocytosis. Southeast-Asian ovalocytosis
(SAO) is a unique form of hereditary elliptocytosis widely
found in Malaysia, Indonesia, Papua New Guinea and the
Philippines. Microscopy of blood films shows 2050%
rounded elliptocytes or ovalocytes, some of which have
one or two transverse bars that divide the central clear space
(ODonnell et al, 1998). These distinctive red cells are not
seen in any other condition. Most individuals with SAO are
asymptomatic, but a few affected have varying degrees of
haemolysis (Reardon et al, 1993; Coetzer et al, 1996).
Homozygosity is probably lethal (Liu et al, 1994; Genton et al,
1995). In contrast to other forms of elliptocytosis, SAO is
associated with increased rigidity and decreased deformability of the red cell membrane. The molecular defect is a
deletion of nine amino acids residues at the junction of the
cytoplasmic and transmembrane domains of band 3 (Jarolim
et al, 1991). SAO band 3 has decreased anion transport
activity (Schofield et al, 1992) and an increased propensity to
form linear aggregates in the membrane (Liu et al, 1995).
The geographical distribution of SAO coincides with malaria
endemicity (Mgone et al, 1996), and there is a clear pattern
of decreasing SAO prevalence in malaria patients with
increasing disease severity (Genton et al, 1995), suggesting
that the condition conveys protection against malaria.
An unknown HE locus. One recent report describes a new
form of elliptocytosis that is different from other known
types (Jonsson et al, 1998). Four members in an English
family have a submicroscopic deletion of band q22 in the
X chromosome. All four affected individuals have Alport
syndrome, due to deletion of the COL4A5 gene located in
Xq22. The two affected male members also have prominent
elliptocytosis, even though there is no evidence of anaemia,
reticulocytosis or erythrocyte membrane fragility. These
findings indicate the existence of a previously unknown
elliptocytosis locus on the X chromosome, identification of
which will further our understanding of factors that
determine the shape of erythrocytes.
Summary
The recent discovery of the specific molecular defects in
many patients with hereditary spherocytosis and hereditary
elliptocytosis/pyropoikilocytosis partially clarifies the molecular pathology of these diseases. HE and HPP are caused by
defects in the horizontal interactions that hold the membrane skeleton together, particularly the critical spectrin selfassociation reaction. Single gene defects cause red cells to
elongate as they circulate, by a unknown mechanism, and
are clinically harmless. The combination of two defective
genes or one severe a spectrin defect and a thalassaemia-like
defect in the opposite allele (aLELY) results in fragile cells that
fragment into bizarre shapes in the circulation, with
haemolysis and sometimes life-threatening anaemia. A few
of the a spectrin defects are common, suggesting they
provide an advantage against malaria or some other threat.
HS, in contrast, is nearly always caused by family-specific
private mutations. These involve the five proteins that link
the membrane skeleton to the overlying lipid bilayer: a and
q 1999 Blackwell Science Ltd, British Journal of Haematology 104: 213

Review
b spectrin, ankyrin, band 3 and protein 4.2. Somehow,
perhaps through loss of the anchorage band 3 provides its
lipid neighbours (Peters et al, 1996), microvesiculation of the
membrane surface ensues, leading to spherocytosis, splenic
sequestration and haemolysis.
Future research will need to focus on how each type of
defect causes its associated disease, how the spleen
aggrevates membrane skeleton defects (a process termed
conditioning), how defective red cells are recognized and
removed in the spleen, and why patients with similar or
even identical defects can have different clinical severity.
Emphasis also needs to be given to improving diagnostic
tests, particularly for HS, and exploring new options for
therapy, like partial splenectomy, which can ameliorate
symptoms while better protecting patients from bacterial
sepsis and red cell parasites, and perhaps from atherosclerosis (Robinette & Fraumeni, 1977) and venous thrombosis
(Stewart et al, 1996).
ACKNOWLEDGMENTS
This work was supported in part by grants from the National
Institute of Health, U.S.A.
Division of Hematology/Oncology,
Childrens Hospital and
Dana-Farber Cancer Institute,
Harvard Medical School,
Boston, Massachusetts,
U.S.A.

W I L L I A M T. T S E
SAMUEL E. LUX

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13

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Keywords: spherocytosis, elliptocytosis, pyropoikilocytosis, spectrin,
ankyrin.