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European Journal of Medical Genetics 52 (2009) 170–173

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European Journal of Medical Genetics

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Original article

Fragile X syndrome screening of families with consanguineous andnon-consanguineous parents in the Iranian population

Ali Reza Pouya a,1, Seyedeh Sedigheh Abedini a,1, Neda Mansoorian b, Farkhondeh Behjati a,Nooshin Nikzat a, Marzieh Mohseni a, Sahar Esmaeeli Nieh c, Lia Abbasi Moheb c, Hossein Darvish a,Gholamreza Bahrami Monajemi a, Susan Banihashemi a, Kimia Kahrizi a, Hans Hilger Ropers c,Hossein Najmabadi a,b,*

a Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iranb Kariminejad–Najmabadi Pathology and Genetics Center, Tehran, Iranc Max Planck Institute for Molecular Genetics, Berlin, Germany

a r t i c l e i n f o

Article history:Received 18 January 2008Accepted 27 March 2009Available online 8 April 2009

Keywords:Fragile X syndromeMental retardationConsanguineous marriage

* Correspondence to: Genetics Research Center, UnRehabilitation Sciences, Koodakyar St, Daneshjoo BlvIran. Tel./fax: þ98 21 22180138.

E-mail address: hnajm12@yahoo.com (H. Najmaba1 These authors contributed equally.

1769-7212/$ – see front matter � 2009 Elsevier Massdoi:10.1016/j.ejmg.2009.03.014

a b s t r a c t

Fragile X syndrome is the most common form of inherited mental retardation (MR). It is caused by theexpansion of CGG triplet repeats in the fragile X mental retardation 1 (FMR1) gene. In mentally retardedmales, the frequency of fragile X syndrome is approximately 2–3 percent, but little is known about itsproportion in mentally retarded patients from countries where parental consanguinity is common.The objective of this study was to estimate the frequency of fragile X syndrome (FXS) in mentallyretarded patients from Iran. We examined a total of 508 families with MR that had been referred to theGenetics Research Center (GRC) in Tehran of which 467 families had at least two mentally retardedchildren. In 384 families, the parents were related and in 124 they were not related of which most ofthem had putative or established X-linked inheritance pattern. Full FMR1 mutations were found in 32 ofthe 508 families studied (6.3%), in 19 out of 124 families with apparently unrelated parents (15.3%), andin 13 of the 384 consanguineous families (3.4%). Thus, in Iran, the relative frequency of FXS seems to behigh, and in patients with unrelated parents is much higher. We also show that even in families withconsanguineous parents, FXS has to be ruled out before assuming that familial MR is due to autosomalrecessive gene defects. Molecular studies are in progress to explain the high proportion of FMR1mutations in mentally retarded offspring of unrelated Iranian parents.

� 2009 Elsevier Masson SAS. All rights reserved.

1. Introduction The number of CGG repeat in the general population is highly

The prevalence of mental retardation (MR) in developed coun-tries is thought to be in order of 2–3% [2,23], although estimatesvary widely, particularly for mild mental retardation [20]. Accord-ing to recent estimates [29], roughly 10 percent of males withsevere MR have X-linked genetic defects. Fragile X syndrome (FXS)is the most common form of X-linked MR (XLMR) with a prevalenceof one in 4000–6000 males and 1 in 7000–10,000 females [22], andit may account for 10–12% of all families with XLMR [17]. In mostcases, FXS is caused by the expansion of an unstable trinucleotideCGG repeat, located in the 50 UTR (untranslated region) of the FMR1(fragile X mental retardation 1) gene [16,36].

iversity of Social Welfare andd, Evin 1985713834, Tehran,

di).

on SAS. All rights reserved.

variable; the majority of FMR1 alleles have 29–30 repeats [18].When the expansion reaches the threshold of 200 repeats (fullmutation), the whole promoter region becomes hypermethylatedand the FMR1 gene is silenced, leading to deficiency of the FMR1protein (FMRP) and fragile X syndrome [6,14]. In contrast, carriersof pre-mutations (55–200 CGG repeats) typically have normalintelligence; however some of the individuals with a pre-mutationhave lower FMRP levels than normal [10,11]. As a result, they mayhave mild mental retardation or learning disabilities, social phobiasor anxiety disorders and mild versions of the physical features seenin fragile X syndrome [10,11,15]. Individuals with a pre-mutationwere shown to be associated with premature ovarian insufficiency(POI) in women and late onset tremor/ataxia syndrome in men[10,13,15,31]. In rare cases, FXS is caused by point mutations [21] orby a deletion of all or part of FMR1 gene, which may vary in sizefrom 650 bp to 9 Mb [25,26].

The clinical phenotype of the FXS is variable, but often includestall stature, macrocephaly, a long and narrow face with prominent

Table 1Sex distribution among the total of mentally retarded families.

Sporadic cases Familial cases Total cases

Male # Female # Male # Female # Male # Female #

Consanguineous marriage 15 8 853 584 868 592Non-consanguineous

marriage16 3 294 167 310 170

Established XLMR – – 124 40Putative XLMR – – 170 127

Total families 31 11 1147 751 1178 762

A.R. Pouya et al. / European Journal of Medical Genetics 52 (2009) 170–173 171

forehead and mandible, large ears, and macroorchidism (25–100 ml) [11]. The phenotype is more subtle in children. Mentalretardation is mild to severe, and behavioral abnormalities mayinclude autistic features or hyperactivity [7,19,22].

Several studies have assessed the prevalence of FXS, e.g., in theNetherlands, Australia [7,8,35], China [37], India [9], Taiwan [12]and other recent studies have revealed that FXS accounts for 2–3percent of the mentally retarded males [2,23]. In contrast, no suchdata are available for countries where parental consanguinity iscommon. High consanguinity rates are often associated with anelevated prevalence of birth defects and MR [3,4], and a plausibleexplanation for this association is a higher risk for recessivedisorders.

In Iran, about 40 percent of all children have consanguineousparents [30], and in some parts of the country, the consanguinityrate is even higher. Here, we have employed PCR and Southern blotanalysis to determine the frequency of FXS in an Iranian cohortcomprising 1460 mentally retarded patients (384 families) withconsanguineous parents, as well as 480 patients (124 families)whose parents were not related.

2. Materials and methods

2.1. Patients

Our study included a total of 508 families comprising at leastone mentally retarded patient. All had been referred to GeneticResearch Center (GRC), Tehran, Iran as part of a large collaborativestudy into the causes of familial MR. These families belonged tovarious ethnicities from different provinces of Iran.

Physical, cognitive and behavioral data were assessed andcollected for each affected individual and a pedigree was drawn.Blood samples were obtained after obtaining informed consent.

2.2. Molecular studies

Genomic DNA was isolated from peripheral blood leucocytes bya standard salting out method [24] for all mentally retarded indi-viduals of these families.

The initial analysis of the CGG repeat region of the FMR1 genewas performed by PCR amplification using the primers FR1 and FR3[5]. Amplification products were resolved by 8% polyacrylamide gelelectrophoresis (PAGE). The gels were silver-stained according toBassam’s protocol [1]. PCR analysis was used to exclude males withnormal repeat lengths or females carrying two distinguishablealleles of normal size. To detect and confirm the presence ofa trinucleotide expansion in males or females, Southern blot anal-ysis was performed.

Genomic DNA (10 mg) was digested with a combination ofHindIII and the methylation-sensitive restriction enzymes EclXI(isoschizomer of EagI). The digested DNA samples were size-sepa-rated by electrophoresis on a 0.8% agarose gel with using a DIG-labeled molecular marker (M-VII) and transferred to a positivelycharged nylon membrane. The StB 12.3 probe (kindly provided by JLMandel) specific for fragments containing the CGG repeat waslabeled using a non-radioactive label (digoxigenin). After hybrid-ization, the membrane was washed and the digoxigenin labeledprobe was detected by exposure to an X-ray film.

3. Results

In total, we have collected 508 families with 1940 affectedindividuals, 1178 males (60.7%) of which 428 were selected asproband and 762 females (39.3%) of which 80 of them wereselected as proband. Sex distribution among the studied population

has been shown in Table 1. In 384 (75.6%) of these families, theparents were related, and in 124 (24.4%) they were not. 22 of theconsanguineous and 19 of the unrelated parents had only oneaffected child.

279 of the consanguineous parents were first cousin, 76 weresecond cousin and 29 were more distantly related. Among the 124non-consanguineous families, 26 had at least two affected boys indifferent sibships that were related through their mothers,(established X-linked MR), and 79 families had a minimum of twoaffected sons in one generation, (putative X-linked MR).

Testing for the FMR1 gene mutation by using PCR and Southernblot analysis were performed for all families’ probands andrevealed 32 (6.3%) families with the full mutation that leads to FXS.Of these, 13 families had consanguineous parents, and 4 of these 32mentally retarded patients (12.5%) were sporadic cases (Table 2). Inthe course of this study, a single female with related parents wasfound to carry a FMR1 pre-mutation.

13 of the (male or female) index patients from 384 consan-guineous families (or 3.4%) were positive for fragile X syndrome. Incontrast, 19 of the affected index patients from 124 non-consan-guineous families (15.3%) had full FMR1 mutations. This proportionis much higher than reported for other populations, e.g., Chineseand Indian [9,37].

4. Discussion

Fragile X syndrome is the most common inherited form ofmental retardation currently known and its effects are seen morefrequently, and with greater severity, in males than females[12,28,34].

Since the cloning of the FMR1 gene, there have been variousattempts at determining the frequency of full FMR1 mutations inpatients with idiopathic mental retardation by using moleculartechniques, including PCR, Southern blot, and immunodetection ofthe protein product of the FMR1 gene FMRP [27,32,33].

Most frequency estimates vary between less than 0.5% and 5%,and the larger, more reliable studies converge on frequenciesbetween 1 and 3% [2,9,12,37]. The present study is the firstsystematic attempt to assess the relative frequency of FXS in pop-ulations with a high degree of parental consanguinity.

In the context of a large-scale project aiming to determine themolecular causes of autosomal recessive and other familial forms ofMR in Iran. We have employed PCR and Southern blotting to screenindex patients from 508 families with idiopathic MR for expansionof the CGG trinucleotide stretch in the FMR1 gene, which is themolecular hallmark of FXS. Families had been referred to theGenetics Research Center in Teheran from various regions of Iran fordiagnostic purposes. Many of the families came from consanguinityfamily since most of our families follow autosomal recessivepattern of inheritance. This may explain in part why 75.6%(384/508) of the collected families had consanguineous parents,which is almost twice as high as previously reported [30]. Since thefocus of our study was on familial MR, families with a single

Table 2Correlation of the FXS screening among MR families with parental consanguinity status.

Consanguineous marriage Non-consanguineous marriage Total families

Male probands(n¼ 318)

Female probands(n¼ 66)

% in 384families

Male probands(n¼ 110)

Female probands(n¼ 14)

% in 124families

Male probands(n¼ 428)

Female probands(n¼ 80)

% in 508families

Sporadic cases with FXS 1 – 0.3% (1) 3 – 2.4% (3) 4 – 0.8% (4)Familial cases with FXS 11 1 3.1% (12) 16 – 12.9% (16) 27 1 5.5% (28)

Total FXS families 12 1 3.4% (13) 19 – 15.3% (19) 31 1 6.3% (32)

A.R. Pouya et al. / European Journal of Medical Genetics 52 (2009) 170–173172

affected child may be underrepresented, but otherwise, the familiesstudied were not pre-selected, and certainly not for FXS or otherforms of X-linked MR.

In this study, the frequency of full FMR1 mutations is 6.3% whichis significantly higher than reported previously for large cohorts[2,7–9,12,34,37]. This excess is largely due to the very highproportion of FXS (15.3%) in the affected offspring of non-consan-guineous parents; in families with consanguineous parents, thefrequency of FXS was only moderately elevated. Still, the presencefull FMR1 mutations in 3.4% of the families emphasizes the need toevaluate all mentally retarded individuals for fragile X syndrome,regardless of parental consanguinity, and particularly in countrieswhere it is a common practice to marry within families.

The most striking result of this study is the unexpectedly highfrequency of FXS in the mentally retarded offspring of unrelatedparents. Given the fact that in a previous studies, FXS accounted foronly 10–12% of the families with XLMR [17,21], this observationeven raises the possibility that in non-consanguineous Iranianfamilies, X-linked forms are the predominant genetic cause of MR.On the other hand, founder effects might be a plausible explanationfor our findings, particularly because most of the families’ studieswere referred from the same region in the South-West of Iran. Aswell as it also could be due to the fact that we selected significantportion of families with putative X-linked inheritance pattern (79/124) in the studied population.

Unlike other severe forms of XLMR, where mutations are rapidlyeliminated from the population because most affected males do notreproduce, founder effects are possible in FXS, because there is no(or at least very little) selection against pre-mutations whichincludes individuals with POI, tremor/ataxia. Thus, the frequency ofpre-mutations might differ considerably between Iranian subpop-ulations due to random sampling from small founder populationsor to other factors. Indeed, apparently unrelated Dutch patientswith FXS have been shown to share the same allele of a micro-satellite marker close to the FXS locus [27], suggesting that new FXSmutations are rare. It is tempting to speculate that this might alsoexplain the exceptionally high frequency of FXS in non-consan-guineous families of our cohort. Studies to test this hypothesis arecurrently underway.

Acknowledgments

We thank the families for their kind help and cooperation andthe physicians who supported our recruitment efforts.

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