Genetic Epidemiology 20:129–144 (2001)GEPI 9945

© 2001 Wiley-Liss, Inc.

Expansion Mutation Frequency andCGG/GCC Repeat Polymorphism in FMR1and FMR2 Genes in an Indian Population

Deepti Sharma, 1 Meena Gupta, 2 and B.K. Thelma 1*

1Department of Genetics, University of Delhi South Campus, New Delhi, India2Department of Neurology, Gobind Ballabh Pant Hospital, New Delhi, India

Based on molecular screening, we estimated the frequencies of fragile X syn-drome and FRAXE syndrome in an institutionalized population (n = 130) inNew Delhi, India. Eligibility criteria for inclusion of subjects in the study weremild/moderate mental retardation, with/without family history, and the fragile Xclinical phenotype. Screening by Southern hybridization revealed an overall fre-quency of 0.077 of the syndrome in the sample population. The disorder wasobserved with a high frequency (0.1) among males as compared to females(0.027). No expansions of FMR2 allele were observed in the same study sample.CGG/ GCC allelic polymorphism of FMR1 and FMR2 were established from atotal of 392 X chromosomes, using the radioactive polymerase chain reaction–polyacrylamide gel electrophoresis method. Distinct repeat sizes, repeat ranges,and repeat modes characterised the FMR1 and FMR2 alleles. In the X chromo-somes of both MR individuals and unaffected controls, unimodal values of 29and 15 repeats in FMR1 and FMR2 genes, respectively, were observed. Allelefrequency distribution was symmetrical at the FMR1 locus whereas a significantpositive skew was observed for the FMR2 alleles. The observed heterozygosityof the FMR1 gene was 0.772 compared to 0.839 of FMR2. Correlation of CGG/GCC repeats of FMR1 and FMR2 did not show any association of repeat sizesat these two loci (correlation coefficient, ρ = 0.09). CGG/GCC repeat variationat FMR1 and FMR2 loci observed in this study sample are different from thatreported for the other Caucasian and Asian populations. Genet. Epidemiol.20:129–144, 2001. © 2001 Wiley-Liss, Inc.

Contract grant sponsor: Government of India; contract grant HG/MB/05/020/95; contract grant spon-sor: the Council of Scientific and Industrial Research, New Delhi,

*Correspondence to: Dr. B.K. Thelma, Department of Genetics, University of Delhi South Campus,Benito Juarez Road, New Delhi 110 021, India. E-mail: humgen@del3.vsnl.net.in

Received for publication June 28, 1999; revision accepted May 24, 2000

130 Sharma et al.

Key words: mental retardation; fragile X syndrome; FRAXE syndrome; New Delhi

INTRODUCTION

Inherited neurological disorders in humans caused by dynamic mutations in thegenome are persistently increasing in number. These disorders involve the amplifica-tion of various nucleotide motifs, i.e., (CTG)n/(CAG)n, (CGG)n/(CCG)n, and (GAA)n/(TTC)n, suggestive of a basic instability of such sequences in the human genome. Thelocation of the expanded unstable tract with respect to the gene transcript may, how-ever, vary among the different diseases [Richards and Sutherland, 1997].

FRAXA syndrome is the most frequent cause of familial mental retardation anddisplays an aberrant X-linked dominant inheritance. This was the first neurogeneticdisorder shown to be caused by an unstable CGG tract, lying coincident to the FRAXAfolate-sensitive fragile site at Xq27.3 [Verkerk et al., 1991]. Subsequent to cloningand characterization of the gene (FMR1), the molecular heterogeneity underlying thesyndrome has been analysed [Verkerk et al., 1991; Yu et al., 1992]. More than 95%of the FRAXA syndrome cases analysed to date are found to be due to the unstableCGG moiety which is prone to mitotic/meiotic expansion. The CGG repeat unit liesin the 5’ untranslated region (UTR) of exon 1 of the gene and occurs in three distinctranges, i.e., 6–54 (normal), 52–200 (premutation), and >200 (full mutation) [Fu etal., 1991; Oberle et al., 1991]. Phenotypic manifestation of the syndrome is observedonly in cases of full mutation and related hypermethylation of the upstream CpGisland [Verkerk et al., 1991]. With the advent of DNA diagnosis, the prevalence esti-mate of FRAXA syndrome, previously based on cytogenetic detection of a folate-sensitive fragile site in the Xq27-28 region, has decreased from 1 in 1250 males[Webb et al., 1986] to 1 in 4,000 males [Turner et al., 1996] as reported for theWestern Caucasian populations. Among the Asian populations, frequency estimatesbased on molecular diagnosis of mentally retarded (MR) individuals are availablefor Japan and China [Zhong et al., 1999].

FRAXE syndrome, is another X-linked genetic anomaly characterized by mild/mod-erate mental impairment and developmental delays [Mulley et al., 1995]. This is alsoassociated with a rare, folate-sensitive fragile site at Xq28 (FRAXE) that lies approxi-mately 600 kb distal to FRAXA [Sutherland and Baker, 1992]. Again, the mutationalmechanism involved is hyperexpansion of the GCC tract beyond 200 repeats (6–25 re-peats being the normal range) and associated hypermethylation of an upstream CpGisland of the FMR2 gene [Knight et al., 1993; Gu et al., 1996]. The prevalence of FRAXEsyndrome in the general populations is not known [Patsalis et al., 1999].

This study is a part of an ongoing genetic screening programme for the FRAXAsyndrome in the New Delhi population. The broader objectives of this project areto provide definite DNA diagnosis for FRAXA syndrome in institutionalized idio-pathic mentally retarded individuals, identify premutation carriers, and counsel fam-ily members as part of genetic service. Diagnosis was made in unrelated MR subjects(n = 130) by Southern hybridization. After this, hyperexpansion in FMR2 was evalu-ated in the same sample population. To determine repeat variability at these loci inthis North Indian population, CGG/GCC repeat polymorphism in FMR1 and FMR2genes, respectively, was analysed in all the MR cases (n = 130) and controls (n =135) using radioactive polymerase chain reaction–polyacrylamide gel electrophore-sis (PCR-PAGE).

FMR1 and FMR2 Polymorphism in Indian Samples 131

MATERIALS AND METHODSSubjects

MR individuals from five special schools for the learning disabled (with a totalnumber of 255) and one home for the mentally retarded (housing 500 inmates) atNew Delhi were clinically screened. Using the fragile X checklist [Hagerman andSilverman, 1991], institutionalized MR individuals (IQ 35–50), with/without familyhistory and scoring above 40% in the checklist (n = 130; 93 males, 37 females) butwith no definite diagnosis, were included for molecular analysis. MR cases due toapparent chromosomal anomalies, cerebral palsy, etc., were not considered. Indi-viduals without any visible physical/mental disability were considered as unaffectedcontrols (n = 135). These individuals included unrelated voluntary blood donors atthe hospitals/institutions and fathers of MR male subjects analysed in this study. Allcontrols originally were from northern India. Informed consent was obtained fromall the individuals included in the study.

Molecular Analyses

DNA isolation from blood: Heparinized venous blood (5–10 mL) was obtainedfrom each individual and DNA was extracted using the conventional phenol-chloro-form method.

Southern blot analyses: Genomic DNA (8–10 µg) was digested with the appro-priate restriction enzyme(s) (EcoRI and PstI for detection of full mutation andpremutation in FMR1, respectively; HindIII for detection of expansion in FMR2) for16–20 hours at 37°C. The digested DNA was fractionated on 1% agarose gel andtransferred to nylon membrane (Hybond N+, Amersham, Buckinghamshire, U.K.)by the conventional capillary transfer [Sambrook et al., 1989]. Each of the probes(pP2 and pX6 for FMR1 [Oostra et al., 1993], OxE20 for FMR2 [Knight et al.,1993]) was radiolabeled with 50µCi of α-32P dCTP (BRIT, Mumbai, India) by therandom priming method (NEB, Beverly, MA, U.S.A.) and column purified. Hybrid-ization was carried using 0.5mol/L phosphate buffer and 7% sodium dodecyl sulfateat 65°C.

PCR Analyses: FMR1: Radioactive PCR of the CGG repeats was carried outwith 6 pmol of each primer (c and f) as in Fu et al. [1991], using Perkin Elmer GeneAmp PCR System 2400 (Perkin Elmer, Foster City, CA, U.S.A.). The products wereresolved by electrophoresis through a 5% denaturing DNA sequencing gel. Alleleswere sized relative to the M13 sequencing ladder (NEB). Direct sequencing of theFMR1 PCR products of a few samples was carried out with primer c using the ABIPrism Model 377 Version 3.3 automated sequencer (Applied Biosystems, Foster City,CA, U.S.A.). FMR2: PCR of GCC repeats was carried out as in Knight et al. [1993].

Statistical Analyses

The weighted mean µ was calculated as Σf ixi/Σf i. Expected heterozygosity wascalculated from the overall (males and females) allele frequencies as 1– Σ(fi)

2, whereasthe observed heterozygosity was determined from the female allele frequency distri-bution only. The correlation coefficient ρ was evaluated in males as ρx,y = Cov(X,Y)/ρx.ρy using the Microsoft Excel CORREL program. Using the method described inSnedecor and Cochran [1967], g1, the measure of skewness, was evaluated to assessthe degree of asymmetry of the distribution around the mean.

132 Sharma et al.

RESULTS

Of the MR individuals analysed (n = 130; age range, 6–70 years), approximately51% were young boys and girls younger than 18 years of age. Majority (71.4%) of theselected individuals were males with characteristic dysmorphic features, and more thanhalf of the subjects presented with moderate mental retardation (IQ 35–50).

Frequency of FRAXA Syndrome

Individuals with hyperexpansion in the FMR1 gene showed bands of sizes >5.2–8.8 kb (calibrated with λ/HindIII molecular weight marker). Premutation blots gavebands in the size range of 1.1–1.6 kb. Based on these profiles, we identified nineFRAXA positive males with full mutations, from the 93 tested (frequency of 0.1)and one single female of the 37 tested (frequency of 0.027). The overall frequencywas observed to be 0.077 in this study sample. Fourteen premutation carriers wereidentified, all of whom were females and belonged to fragile X families.

Frequency and Distribution of CGG Repeats in FMR1

CGG repeat polymorphism was studied in a total of 265 individuals (MR sub-jects, n = 130; normal controls, n = 135), contributing to a total of 392 X chromo-somes. PCR amplification was successful in only 382 X chromosomes (full mutationalleles, n = 10, failed to amplify). The 14 carrier females possessed premutationalleles in the range of 64–96 CGG repeats (Fig. 1). The 368 X chromosomes in thenormal range had a total of 26 distinct alleles ranging from 19 to 50 CGG repeats(Fig. 2). The most frequent allele size in this population had 29 CGG repeats (in35.3% of the X chromosomes), with 28 repeats (27.9%) being the next frequent. Mi-

Fig. 1. Autoradiogram of FMR1 PCR products resolved on 5% denaturing polyacrylamide gel withM13 sequencing ladder; indicating allele size (bp) and the repeat number in parentheses.

FMR1 and FMR2 Polymorphism in Indian Samples 133

nor peaks were observed at 30 and 31 repeats. Our study sample did not show distinctFMR1 peaks in the 19–23 repeat range. Due to the reported technical inaccuracies inthe method of Fu et al. [1991] for FMR1 repeat sizing [Brown et al., 1993], the repeatnumbers in our study sample were confirmed by direct sequencing of a few normalFMR1 alleles on an ABI Prism Model 377 automated sequencer. The observed het-erozygosity based on allele frequency distribution in females was 0.772 versus theexpected heterozygosity 0.783 based on the overall allele distribution.

To determine discrepancies, if any, in the allele distribution patterns betweenthe MR individuals and normal controls, the allele frequencies between the two cat-egories were compared (Fig. 3). Of the 152 X chromosomes from index patients,39.4% had 29 CGG repeats, whereas 26.9% had 28 CGG repeats. Analysis of 216 Xchromosomes from controls also revealed the 29 repeat alleles to be the most fre-quent (32.4%) followed by 28 CGG repeats (27.7%). Thus, the population repeatdistributions were unimodal, with a predominant number of 29. The mean repeatsizes as well as median of the distribution were evaluated separately for the indexand the control X chromosomes (Table I).

Using the g1 statistics as a test of skewness [Snedecor and Cochran, 1967], allelefrequency distribution at the FMR1 locus was analysed for observations within the 19–42 repeat range. The distribution was observed to be symmetrical around the medianvalue of 29 (coefficient of skewness, g1 = 0.856; P = 0.05). Although the number ofobservations of 47–50 repeats was small, they contributed to a large deviation in theoverall skewness profile. Therefore, these values were not included in the test.

Frequency of FRAXE Syndrome

Southern analysis for (GCC)n hyperexpansion in FMR2 was performed on allthe MR cases testing negative for the FRAXA syndrome. However, none was foundto carry an expanded FMR2 allele.

Fig. 2. Distribution of CGG repeats (range, 19–50; filled columns) and GCC repeats (range, 3–27;shaded columns) in the X chromosomes (n = 368 for FMR1; n = 382 for FMR2).

134 Sharma et al.

Frequency and Distribution of GCC Repeats in FMR2

PCR amplification for FMR2 allele was successful in 382 X chromosomes of atotal of 392. Those samples that failed to amplify by PCR were confirmed to carry thenormal allele by the Southern hybridization method but were not included in the analy-sis. Analyses of GCC repeat polymorphism at this locus revealed a total of 20 differentalleles in the range of 3–27 GCC (Fig. 2). The modal allele in the population had 15GCC repeats (in 30.1% of the X chromosomes) with 18 repeats (18.7%) being the nextmost frequent. The observed heterozygosity of the FMR2 gene in this population wasfound to be 0.839 versus an expected value of 0.838. The mean and the median repeatsizes were also determined for this locus (Table I). Because of the apparent differencesin the modal, median, and mean values at this locus, symmetry of allele distributionwas determined by the g1 statistics [Snedecor and Cochran, 1967]. The distributionshowed a significant positive skew (g1 = 3.64) about the median value (P < 0.01).

Comparison of the FMR2 allele frequency distributions of index (MR) casesand controls revealed a unimodal repeat number of 15 in both categories (Fig. 4).Approximately 27.8% of the 157 index X chromosomes had alleles with 15 GCCrepeats compared to 31.5% in the control chromosomes. The next predominant allelehad 16 GCC repeats (17.0%) among the index chromosomes and 18 GCC repeats(21.4%) among the controls. A possible correlation between the CGG repeat size inFMR1 with the GCC repeat size in FMR2 was evaluated in males, using the MicrosoftExcel CORREL program. The correlation coefficient ρ was calculated to be 0.09,indicating lack of any significant association of repeat sizes between the two loci.

DISCUSSION

As a significant proportion of familial X-linked MR cases are a result ofhyperexpansion of the CGG moiety of FMR1, we initiated a genetic screening

Fig. 3. Distribution of CGG repeats of FMR1 in the X chromosomes from index patients (filledcolumns) and from normal controls (open columns).

FM

R1 and F

MR

2 Polym

orphism in Indian S

amples

135

TABLE I. CGG/GCC Repeats in FMR1 and FMR2 Genes in the Sample Population from New Delhi

FMR1 FMR2

Males Females Alleles AllelesCategory (n) (n) analyseda Mean Mode Median Het. analyseda Mean Mode Median Het.

MR individuals 93 37 152 29.57 29 29 0.657 157 17.13 15 16 0.851(0.394) (0.278)

Normal controls 45 90 216 28.81 29 29 0.805 225 17.24 15 17 0.830(0.324) (0.315)

Total 138 127 368 29.12 29 29 0.783 382 17.23 15 16 0.838(0.353) (0.301)

aOf 392; note this does not include full mutation and premutation alleles.Figures in parentheses indicate modal frequency.Het.; heterozygosity values based on overall (males and females) allele frequencies.

136 Sharma et al.

programme for FRAXA syndrome in the institutionalized population at New Delhi.Through selective screening of subjects on the basis of the FXS phenotype, the studyessentially aimed at identifying maximum number of FXS families. This was of rel-evance keeping in view the broader objectives of this study. Due to the greater accu-racy and reliability of DNA-based diagnosis, this method was chosen overconventional cytogenetic induction of FRAXA at Xq27.3.

Our study revealed a frequency estimate of ~8%, suggesting a high prevalenceof the fragile X syndrome among MR individuals manifesting the Martin-Bell phe-notype. As expected for this X-linked condition, the frequency of FRAXA syndromeamong selected males (0.1) was found to be higher (approximately fourfold) thanthat observed for the females (0.027), implying greater conformity of clinical mani-festation with molecular status in males. There occur a few reports on this disorderfrom the Indian population. One such study, conducted among 300 MR subjects fromWestern India and based on cytogenetic diagnosis, estimated the frequency to beapproximately 2% [Murthy et al., 1991]. Employing the DNA diagnostic methods,another report from Southern India revealed a frequency of 7%, comparable to ourresults [Baskaran et al., 1998]. However, we cannot compare our results with thosereported for most other Western institutionalized populations, which included all un-explained MR cases in their study samples [Meadows et al., 1996; Mila et al., 1997;Elbaz et al., 1998; Syrrou et al., 1998; Crawford et al., 1999; Pang et al., 1999;Patsalis et al., 1999]. Considering the stringent selection criteria employed in thisstudy, the overall frequency of FXS in India may be very similar to that reported forother Western and Asian populations [Patsalis et al., 1999].

FRAXE syndrome associated with a mild and inconsistent FRAXE phenotypethat does not necessitate institutionalization makes its detection more difficult. Ab-sence of any FRAXE-positive individuals in our sample population reiterates thatthis syndrome is very rare among institutionalized individuals with unclassified de-velopmental disabilities [Holden et al., 1996].

Fig. 4. Distribution of GCC repeats of FMR2 in the X chromosomes from index patients (filled col-umns) and from normal controls (open columns).

FMR1 and FMR2 Polymorphism in Indian Samples 137

FRAXA and FRAXE expansion mutations have been recorded in diverse popu-lations throughout the world. Besides the Tunisian Jews of Israel who are reportedlypredisposed to fragile X syndrome (due to the presence of long uninterrupted CGGstretches), no other ethnic community showing a specific predisposition to these dis-orders has been reported [Falik-Zaccai et al., 1997]. However, inter-population varia-tions are observed in the degree of allelic polymorphism and allele frequencies in theFMR1 and FMR2 genes (Tables II and III).

FMR1: The FMR1 allelic distribution profiles in our population showed an overallsimilarity to those reported for other populations, with minor differences comparedto the other Asian populations (Table II). Though the Chinese population also showsFMR1 modal peak at 29 as in our population, there seem to be some dissimilaritiesin the reported frequency distribution of other alleles (notably in the secondary modalpeak at 36 reported by Zhong et al. [1994], and the shoulder at 25–27 repeat sizes asrevealed by Chen et al. [1997]). Only 5.9% of the X chromosomes in our study had30 repeats compared to 26% in the Chinese samples [Zhong et al., 1994]. However,a direct comparison of allele frequency distribution profiles and the ethnic origin ofthe diverse populations cannot be made due to basic differences in the methodolo-gies used in repeat analysis (Table II). Our data on this North Indian region are alsodifferent from those reported for South India where the modal allele had 31 CGGrepeats [Baskaran et al., 1998]. However, the smaller sample size, inadequate de-scription of the origin of the study samples, and methodological differences in theirstudy make it difficult to compare our results with theirs.

FMR2: The overall FMR2 allele distribution is represented in Fig. 3 and is theonly report from India to date. The modal number of 15 GCC repeats observed inour study sample is similar to that in a few other Caucasian populations but differentfrom that in the Chinese (Table III).

The distinct CGG/GCC allele frequency distributions observed in our study mayrequire some explanation. It is important to consider the ethnic diversity of the present-day Indian population, which is believed to have originated from distinct geneticbackgrounds. Genetic admixture between the traditional trio of races (Caucasian,Asian, and black) has taken place over a considerable period of time in various partsof the world. India shows an exceptionally high degree of such inherited diversityand thus genetic complexity, caused essentially by primeval human migrations espe-cially into the Northern, Western, and Eastern regions of the country. MitochondrialDNA polymorphism studies confirmed the North Indian population to be essentiallyCaucasoid in origin and different from the rest of the Asian (proto-Oriental) popula-tion [Passarino et al., 1996]. The majority of the individuals included in our studyoriginally came from the northern parts of India and hence have a predominantlyCaucasoid origin. But, contrary to expectations, we do find some differences in re-peat sizes, repeat ranges, and repeat modes in the FMR1 and FMR2 genes comparedwith the other Caucasian populations (Tables II and III). However, these differencesin observations among different populations may not be totally unexpected consider-ing the highly polymorphic nature of these loci.

Statistical analyses of 382 FMR2 alleles in our sample revealed an overall meanGCC repeat number of 17.23, a median of 16, and a mode of 15. The discrete differ-ences observed between the mode and mean/median values point to the apparentskewness of the FMR2 allele frequency distribution in our population. Using the g1

TABLE II. Comparison of (CGG)n Repeat Characteristics in FMR1 Between Some Populations (1991–1999)

No. of Repeat Allelic HeterozygosityCountry (population) X chromosomes range variants Mode Meana (%) Ref. Method

United States 492 6–54 31 29 — 63 Fu et al., 1991 Fu et al., 1991(Caucasian, black,Hispanic, and Asian)

United Kingdom 309 13–49 30 29 — NR Jacobs et al., 1993 Fu et al., 1991(Caucasian)

United States 444 13–61 33 30 — NR Snow et al., 1993 Pergolizzi et al., 1992(Caucasian)

Japan 370 18–44 24 28 — NR Arinami et al., 1993 Fu et al., 1991(Asian)

United States 570 12–52 32 30 29.2 79.8 Brown et al., 1993 Brown et al., 1993(Caucasian)

Japan 125 13–39 15 28 — NR Richards et al., 1994 Kremer et al., 1991(Asian)

Central China 123 21–43 12 29 30.5 70 Zhong et al., 1994 Brown et al., 1993(Asian)

United States 997 11–56 39 30 — 76 Meadows et al., 1996 Fu et al., 1991(Caucasian)

Finland 56 20–47 14 30 30.7 88 Zhong et al., 1996 Brown et al., 1993(Caucasian)

United States 534 14–55 32 30 — 73 Meadows et al., 1996 Fu et al., 1991(Black)

Italy 141 18–47 26 30 29.8 NR Chiurazzi et al., 1996 Fu et al., 1991(Caucasian)

United States 2,500 12–59 42 30 — 79.9 Brown et al., 1996 Brown et al., 1993(Caucasian) 1,069 11–52 35 29 — NR Brown et al., 1997 Brown et al., 1993

(continued)

South China 117 17–37 16 29 28.2 NR Chen et al., 1997 Fu et al., 1991(Mongoloid)

Chile 192 19–44 19 30 28.7 72.7 Jara et al., 1998 Fu et al., 1991(Caucasian & Asian)

Southern India 161 8–44 17 31 29.6 NR Baskaran et al., 1998 Fu et al., 1991(Dravidian)

Greece, Cyprus 322 12–56 26 30 — 69 Patsalis et al., 1999 Brown et al., 1993(Caucasian)

Cyprus 600 12–59 34 30 29.9 73 Patsalis et al., 1999 Brown et al., 1999(Caucasian)

China (Hong Kong) 1,404 19–54 34 29 30.9 65.4 Pang et al., 1999 Brown et al., 1993(Asian)

Brazil 251 <10–>40 NR 21–30 — NR Haddad et al., 1999 Haddad et al., 1996(Caucasian)

Northern India 368 19–50 26 29 29.12 78.3 Sharma et al., this study Fu et al., 1991(Caucasian)

aCalculated by authors; only for those reports in which frequency distributions were available.Methods used for CGG repeat analysis: Fu et al., 1991, radioactive PCR/PAGE with primers c and f; Fu et al., 1991, radioactive PCR/PAGE with primersa and f; Pergolizzi et al., 1992, agarose gel/Southern using primers 1–21, and 203–181; Brown et al., 1993, PAGE/Southern using primers 1 and 3;Haddad et al., 1996, PAGE using primers Eag-U and Eag-L.NR, not reported.

TABLE II. (continued)

No. of Repeat Allelic HeterozygosityCountry (population) X chromosomes range variants Mode Meana (%) Ref. Method

140S

harma et al.

TABLE III. Comparison of (GCC)n Repeat Characteristics in FMR2 Between Some Populations (1991–1999)

No. of Repeat Allelic HeterozygosityCountry (population) X chromosomes range variants Mode Meana (%) Ref.

United Kingdom 86 6–25 16 15 15.8 NR Knight et al., 1994(Caucasian)

Canada 328 7–35 23 16 17.0 NR Allingham-Hawkins and Ray, 1995(Caucasian) 495 5–38 26 15 17.3 NR Holden et al., 1996

United States 416 8–39 24 16 18.2 86.8 Zhong et al., 1996(Caucasian)

Finland 92 7–25 13 16 16.8 72.5 Zhong et al., 1996(Caucasian)

China 157 9–26 17 18 17.8 87.3 Zhong et al., 1996(Asian)

United States 487 3–35 22 15 — 63 Meadows et al., 1996(Caucasian)(Black) 95 7–26 17 15 — 73 Meadows et al., 1996

African 96 NR NR NR 17.3 82.0 Ritchie et al., 1997(Black)

China 94 NR NR NR 17.6 78.4 Ritchie et al., 1997(Asian)

United Kingdom 81 NR NR NR 16.5 82.5 Ritchie et al., 1997(Caucasian)

Greece, Cyprus 159 NR NR NR 17.5 82.8 Ritchie et al., 1997(Caucasian)

India 180 NR NR NR 17.3 84.1 Ritchie et al., 1997United States 1,278 3–42 34 13 — NR Brown et al., 1997

(Caucasian)Greece, Cyprus 322 7–30 21 16 — 77 Patsalis et al., 1999

(Caucasian)India 382 3–27 20 15 17.23 83.8 Sharma et al., this study

(Caucasian)

aCalculated by authors; only for those reports in which frequency distributions were available.Method used for GCC repeat analysis: Knight et al., 1993, radioactive PCR/PAGE using primers 598 and 603.NR, not reported.

FMR1 and FMR2 Polymorphism in Indian Samples 141

test of skewness, the overall distribution was found to show a significant positiveskew (P < 0.01). Comparison of the modal, median, and mean repeat numbers in theindex chromosomes also showed a similar asymmetry of allele distribution. Theseresults are in contrast with the symmetrical allele frequency distributions reported byRitchie et al. [1997] from five different populations. In contrast to the situation atFRAXE, this study population shows a more symmetrical frequency distribution pat-tern of CGG repeats (n = 19–42; excluding gray zone alleles; n ≥ 43) at FRAXA,with comparable values for the mean (29.12) and the mode (29). Variations in themean repeat sizes at the two loci may reflect the relatively higher stability of theFMR2 locus in this population (lower mean repeat number). However, this seemsunlikely due to a higher observed heterozygosity at FRAXE (0.838) compared toFRAXA (0.783) and also the lack of interrupting “stabilizing” sequences in the GCCtract of FRAXE [Brown et al., 1997], suggestive of a greater instability (?) at thislocus. The complex relationships between mean repeat size, heterozygosity, and thelack of interspersions in the repeat tract would have to be studied to understand themechanism of dynamic mutation at this locus.

Physical/genetic proximity of the FMR1 and FMR2 genes has led to speculationsabout a possible correlation of instability between the two loci. We analyzed 117 Xchromosomes from males for a possible association of repeat lengths at the two loci.The correlation coefficient (ρ) was calculated to be 0.097, indicating no significantassociation of triplet repeats between these loci. These findings are in agreement withthose reported by Brown et al. [1997] based on 721 individuals (1,060 alleles).

To determine the effect of FMR1 expansions on repeat size at FMR2, we evalu-ated the exact FMR2 GCC repeat number for all the 10 fragile X–positive subjects.Of the 10 unrelated, full-mutation FMR1 alleles analyzed in our study sample, 50%were found to carry the 16 GCC repeat allele versus 13.2% of the normal (non-fraX)alleles. But due to our small sample size (n = 10), it would be difficult to propose apossible founder fragile X-FMR2 haplotype, which has, however, been suggestedfor the Finnish (n = 36) and Chinese (n = 13) samples [Zhong et al., 1996]. Thereported association between expanded FMR1 alleles and large FMR2 alleles andinstability at FRAXE (as evidenced by FMR2 regional deletions) [Macpherson etal., 1995; Zhong et al., 1995, 1996; Brown et al., 1997] points to the potentiallysignificant roles that flanking sequences/cis factors, or “stability” genes in the ge-nome, may play in the regional (in)stability at Xq27-28 region. We observed thehighest repeat size of 27 in FMR2 in an idiopathic mildly MR male. This individualalso had a large allele of 47 CGG repeats in FMR1. The potential inter-generational(in)stability of these alleles could not be determined due to the non-availability ofDNA samples from his family members. Extensive haplotyping of the flanking re-gions in this individual may provide some clue for the occurrence of large repeats atboth the loci. The recent discovery of another GCC locus at Xq28 (4G locus) byRitchie et al. [1997] endorses the idea that other gene(s) with a similar mutationalmechanism may lie in the distal region of the X chromosome.

ACKNOWLEDGMENTS

We thank Dr. Ben A. Oostra, Department of Clinical Genetics, Erasmus Univer-sity for providing the pP2 and pX6 probes; Dr. David L. Nelson, Department of

142 Sharma et al.

Molecular and Human Genetics, Baylor College of Medicine, for the kind gift ofprobe OxE20 and primers 598 and 603; and Dr. S.K. Brahmachari, Centre for Bio-chemical Technology, New Delhi, for allowing us access to the Automated Sequenc-ing Facility. We are also grateful to Dr. V.T. Prabhakaran, Indian Agricultural StatisticsResearch Institute, for help with the statistical analyses. Financial support from De-partment of Biotechnology, Government of India (grant HG/MB/05/020/95) to B.K.T.and M.G., and from the Council of Scientific and Industrial Research, New Delhi, toD.S., is gratefully acknowledged.

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