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Vol 4, No I, January-March, 1995 Fragile X Mental Retardation t7

Fragile X Mental Retardation in an Indonesian Family

Sultana,l Ag. Soemantri,2 P.R.L. Lam-Po-Tang,3 Fionu Wright,a Robert Lindeman,4 Stuart purvis-Smith3

Sindronnfragile X adalah petryakit retardasi nental nenurun yang paling unun dan dikaitkan dengan adanya 'fragile site,, pada
uiung akhir lengan panjang kromoson X. Dalan penelitian penyaringan untuk nencari kelainan krotnosottr pada siswa laki-laki
retardasi mental dari sekolah luar-biasa, lcani pertatna kali nengidentifikasi keluarga dengan sindrotna
fragite X di Indonesia pada
pemeriksaanfisik, riwayat keluarga dan penteriksaan sitogenetila dan DNA nenbuktil<an dua kakak beradik laki-laki denganfenotip
abnonnal, positif fragile X nasing-nasing 3%( Dn) dan 4%(Pù. Kakak perenpuan dan ibunya nenunjukkan
fenotip dan intelektual
nonnal, tetapi secarasitogenetikpositif untukfragileX(6% padakaknkperentpuandan3% padaibunya). Analisis nolekuler nte,akinkan
bahwa kedua anak laki-lnki tersebut adalah penderita sindronafragile X, sedangkan kakak perentpuan dan ibunya adalah petnbawa

Fragile X syndrome is the nost cotnntonform offanilial nental retardation and is associated with afragile site at the end of long
arn chrotnosonre X. Whilst screening for chrontosoual abnornalities in nentally retarded nales attending special school, we have
identified thefirst fanily with afragile x syndrone, to be reported in Indonesia. ph1,sis.1 exatnination,fatnill, l1isg6r, as well as
cyto9enetic and DNA studies identified tv,o pheno4,pically abnonnal brorhers who were fragile X posirive 3% (Dn) and 4% (pr). Their
sister (Cl) and utother were pheno\,pically and intellectually nornal but were cytogenetically positive
for fragile X (6% in the sister
and 3% in lhe nother). Molecular analysis confirnted that both broîhers were afected and that their sister and tnother were carriers-

Keywords : Fragile X syndrone, ntental retardation, Indonesiry Javanese, chronosotne studies, restriction
fragtnent length
poll,norphisn, pfxa3.

INTRODUCTION to XLMR.a-6 About 4O7o of XLMR and 4% of all

mental retardation have been attributed to fragile X
Mental retardation (MR) is a common disease which l'6
syndrome. Fragile X syndrome is the most frequent
is clinically and etiologically heterogenous. MR can be known inherited cause of mental retardation, with an
divided up according to intelligence quotient (IQ) test estimated prevalence in predominantly Caucasian
scores into severe or moderate (IQ 50 or less) andmild populations ranging from 0.4/1000 to 0.8/1OOO in
(IQ 50-70),1-3 with a prevalence of about 3 per l0O0 males and from 0.2/1000 to 0.6/1000 in females.4'7
for severe mental retardation and 30 per IOOO for mild Affected individuals have minor physical abnor-
mental retardalion.2 Causes of mental retardation in- malities and may fail to thrive in infancy or childhood
clude obstetric problems, perinatal trauma, neonatal or have learning difficulties in school. These children
infection and genetic abnormalities such as Down's have a reasonably long life span, but require constant
syndrome (trisomy 21) and X-linked mental retarda- care from either the family or an institution. They
tion (XLMR). Almost all surveys of mentally retarded usually attend special schools for the mentally hand-
persons show that males tend affected more often than icapped. Fragile X syndrome is characterized by a
females. Of all mentally retarded people, 2O7o are due cytogenetically detectable fragile site which appears as

t D"port,rrnt
of Hisrology, Faculty of Medicine, Dipouegoro lJniversity/ Cytogenetics IJttit Telogorejo Hospital, Sennrang,
-a Departn of Medicine, Dip etilarartg, Itrdonesia.
'r'C)togen 4 Department of of Wales Hospital, S),dnq,, Australia 2O3I-
' Departn e of Wales Hospi 2031.
18 Sultana et al. ItreàJ Inàones

a break near the end of the long arm of the X Blood collection
chromosome. The basic defect of fragile X is expan-
sion of the number of the trinucleotide CGG repeats at Ten ml of heparinized peripheral blood were drawn
the 5' end of the FRAXA gene.8 from each individual for cytogenetic and DNA
Chromosomal disorders in mentally retarded analysis. Blood was also screened for hypothyroidism
children have not been studied in Indonesia because by blotting it on special filter paper.
of the lack of diagnostic facilities. While undertaking
a survey of chromosomal abnormalities in mentally Cytogenetic studies
retarded children in Semarang, Indonesia, we iden-
tified a family with two mentally retarded males Chromosome preparations were made by culturing 10
demonstrating the presence of fragile X chromosomes. drops of peripheral blood at 37oC for 72 hours in
This is the first reported family with fragile X in duplicate tubes each containing 5 ml Iscove's Low
Indonesia. Folate medium supplemented with 57o Fetal Bovine
Serum (FBS) and 0,025 ml Phytohaemaglutinin-P
(Gibco). To each tube, 0.1 ml thymidine (final con-
MATERIALS AND METIIODS centration 0.3 pg/ml) and 3 drops colchicine (final
concentration I pg/ml) were added respectively at 24
Patients hours and 25 minutes before harvesting. Chromosome
spreads were made according to routine procedures.
The family was identified in the course of a screening Chromosome analysis for fragile X was performed by
program of MR males who appeared to be phenotypi- scanning 50 unbanded Giemsa-stained metaphases.
cally normal. There were two brothers (Dm age 13 and When cells with chromosome breakage in group C
Pr age 8) in a special school, reported to be intellec- were found, a further 50 cells and 100 cells were
tually handicapped. They have one sister(Cl age 14) counted in males and females respectively. The coor-
who is intellectually normal and attends a normal dinates of positive cells were noted and the metaphases
school. Both parents are alive and well (see pedigree). photographed. Subsequently, the slides were destained
Findings from a physical examination including tes- in 3:1 methanol:acetic acid, and G-banded for confir-
ticular measurement (comparative palpation) using mation of the fragile X. Six further new banded cells
testicular models of known volume (orchidometer ) e were analyzed and another 20 cells were counted to
of these two brothers are shown in Table 1 . assess structural and numerical chromosome abnor-
malities (Figure 1 shows one chromosome with fragile
Table l.Clinical description of the two brothers" X in Cl),

Case I (Dm) Case 2 (Pr)

Molecular studies
Date ol birth 27l3lr98t l8i 8i 1986
Age at lime of examination l3 years
DNA was extracted from peripheral blood by using the
8 years
Head Circunrlerence 58 cm(92Væ) 53cm(>95%o)
saturated salt method.t' Eight micrograms of DNA
Weight 49kg l9kg
were digested with one unit of the restriction en-
Hcight | 58cm(95loo) ll2cnt(957æ) donuclease PstI (Pharmacia) in the buffer recom-
bng tàce Yes Ycs mended by the manufacturer together with spermidine
High forehead Ycs Yes in a total volume of 20 pl . Agarose gel electrophoresis
Prognathisnr Yes Yes was performed (0.757o agarose, xl Tris-borate EDTA
L)ng & prominent eârs Yes Yes buffer, 35 volts for 16 hours). An SPPI ladder
Macroorchidism (testicular volume ) Yes(>25m1) Yes(4nrl) (Bresatec) and normal control DNA were included in
Strabismus Yes Ycs each gel. After capillary blotting to Hybond N*1Amer-
Nystagmus Ycs No sham), prehybridisation was performed for one hour in
Cogwhecl rigidity Yr:s Ycs rapid-hyb buffer (Amersham RPN 1636) at 65oC. The
Abnormal gait & parkinsonian tremor Yes No FRAXA probe pfxa3 and an X-chromosome single
Stereotypic movenlent Ycs No locus control probe pSS (both kindly supplied by
Hypcractive No Yes
Professor G. Sutherland, Adelaide) were prepared by
Aulism Ycs No 32P-in"o.po.ation
by random hexamer pi-ing, unâ
Shyness Yes Yes
hybridisation performed in the same buffer for 16
Gaze avoidance Ycs Ycs
hours at 65"C. Washing was performed in 2x SSC and
language difficulties Ycs Yes
0.1% sodium dodecylsulphate (SDS) three times at
VoI 4, No l, January-March, 1995 Fragile X Mental Retardatiott l9

Figure I. Fragile x chrotnosone (Ieft) and nontnl X chrotnosonre (right) in

fettnle (cl)

room temperature for 20 minutes and then twice for 30 fragile X cells in 150 metaphases respectively. No
minutes ar 65oC in 0,lx SSC and 0.1% SDS. fragile sites were identified in 50 metaphases in the
Autoradiography was performed for l-3 days at _7OoC father.
using intensification screens.

Molecular Analysis
Screening for hypothyroidism
The Restriction fragment length polymorphism
Iter paper (S&S 903 (RFLP) using the pfxa3 probe detected a constant band
for hypothyroidism between 1.0 and l.l kb in normal controls. Simul_
the Oliver Latham taneous hybridisation with pSg yielded a 0.g kb band
Laboratory, Sydney. which provided an intensity control for normal X_
chromosome copy number in all individuals.
In proband son I (Dm) the normal pfxa band was
replaced by 1.60 kb and Z.6kbbands, while his brother
son II (Pr) hada2.2 kb but no normal band. Their sister
Clinical finding (Cl) was a carrier, with an abnormal 2.2 kb and a
A family of five (2 parents and 3 siblings) was normal 1.1 kb band which was also present in their
studied.Two affected brothers were examined. Both father. Their mother was also a carrier, with abnormal
1.3 kb and normal l.l kb bands (see Figure 2).
no obstetric and ms as
ata ofhypothyroidi ts and
normal on physic The Screenin g for hypothyroid ism
pedigree is shown in Figure 2 and clinical features of
the two brothers are presented in Table I and Figure 3. Screening for hypothyroidism was negative for all
five members of family.
Cytogenetic Analysis
Cytogenetic study of two affected sons identified a
break in the long arm of the X chromosome at q27.3
We report the first recorded cases of Fragile X in an
(FRAXA locus) in IOO metaphases:3To inDm and4To
Indonesian family of Javanese ethnic background. We
in Pr. The mother and daughter(Cl) had 3 % and,6To are not aware of any previous recorded cases in In_
20 Sultana et al. MedJ Indones

'yÀ: ,'J

1 I
2 3 4 5-
Figure 2. Shows five lines of a Southern blot. The band obtained by hybridising Pstl digested genonic DNA with the X chronosone
probe pS8 is used as a control (C)for densitonetry, while the nonnal band size (N) obtained using the FMXA probe is indicated.
Thefather (lane 1) has only a nornal band, while the nother (lane 2) k a cariier, since there is an additional higher band. Each
FMXA band is of reduced densiry relative to lhe control, C. The daughter (lane 3) is also a carrier, while the sons Dil (lane 4) and
Pr (Iane 5) lackthe nornral band, which has been replaced by two and one higher band respectively, Ihey are therefore affected in-
Vol 4, No l, January-March, 1995 Fragile X Mental Retardation 2l


Figure j. Two affected brothers, Dm (lefi) and Pr (right)

donesia except for a case of fragile X mental retarda- tures in these individuats.4'13 The two affected boys
tion in one Dutch-Indonesian in Hawaii, whose ethnic have different characters: one is hyperactive and the
background was not reported. I I other quiet and autistic. Both traits are seen in fragile
The affected males (Dm and Pr) showed macro- X MR syndrome
orchidism. In the case of the 8 year old the testicular The frequency of fragile X cells is 37o - 4% in
volume should be less than 4 ml. In the 13 years old the affected males (two sons) appear to be lower than
who is showing early sign of puberty such as some in other studies which reported an incidence of 2-
pubic hair the testicular size should be 4 ml but is 5O7o.r'1 Interestingly, the frequency of fragile cells was
already over 25 ml. Both have long ears and prominent higher in the carrier daughter than in her affected
jaws, shyness, no speech difficulties but both have brothers.
language deficits such as abnormally frequent use of The fragile X locus is a folate sensitive fragile
short sentences. These features were present in many site. Detection is dependent on either a culture medium
patients of fragile X syndrome.o'''tt Th.y also had deficient in folate or containing an inhibitor of
strabismus and nystagmus which are occasional fea- folate metabolism (eg.FUdR, methotrexate, excess
22 Sultanaeral. Med J Indones

thymidine) and reduced content of FBS.la'15'16 It i" head of Dharma Bhakti foundation and the teachers at
possible that although low folate medium was used the Dharma Bhakti Special School, for their willing-
there was enough folate and FBS in the medium to ness to participate in this study.
reduce the incidence of fragile X expression . Recently
two other folate sensitive fragile sites (FRAXE and
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