A functional tetranucleotide (AAAT) polymorphism in an Alu element in the NF1 gene is associated with mental retardation.

Mental retardation (MR) is frequent in neurofibromatosis type 1 (NF1). Allele 5 of a tetranucleotide polymorphism in an Alu element (GXAlu) localized in intron 27b of the NF1 gene has previously been associated with autism. We considered that the microsatellite GXAlu could also represent a risk factor in MR without autism. We developed a rapid method for genotyping by non-denaturing HPLC and assayed the allelic variation of GXAlu marker on in vitro gene expression in Cos-7 cells. A French population of 157 individuals (68 non syndromic non familial MR (NS-MR) patients diagnosed in the University Hospital of Tours; 89 controls) was tested in a case-control assay. We observed a significant association (χ(2)=7.96; p=0.005) between alu4 carriers (7 AAAT repeats) and MR (OR: 7.86; 95% C.I.: 2.13-28.9). The relative in vitro expression of a reporter gene encoding chloramphenicol acetyl transferase (CAT) was higher for alu4 and alu5, suggesting a regulation effect for these alleles on gene expression in vivo. Our results showed an association with a polymorphism regulating the NF1 gene or other genes during brain development.

Study of the serotonin transporter (SLC6A4) and BDNF genes in French patients with non syndromic mental deficiency.

BACKGROUND: Mental deficiency has been linked to abnormalities in cortical neuronal network connectivity and plasticity. These mechanisms are in part under the control of two interacting signalling pathways, the serotonergic and the brain-derived neurotrophic (BDNF) pathways. The aim of the current paper is to determine whether particular alleles or genotypes of two crucial genes of these systems, the serotonin transporter gene (SLC6A4) and the brain-derived neurotrophic factor gene (BDNF), are associated with mental deficiency (MD). METHODS: We analyzed four functional polymorphisms (rs25531, 5-HTTLPR, VNTR, rs3813034) of the SLC6A4 gene and one functional polymorphism (Val66 Met) of the BDNF gene in 98 patients with non-syndromic mental deficiency (NS-MD) and in an ethnically matched control population of 251 individuals. RESULTS: We found no significant differences in allele and genotype frequencies in the five polymorphisms studied in the SLC6A4 and BDNF genes of NS-MD patients versus control patients. While the comparison of the patterns of linkage disequilibrium (D') in the control and NS-MD populations revealed a degree of variability it did not, however, reach significance. No significant differences in frequencies of haplotypes and genotypes for VNTR/rs3813034 and rs25531/5-HTTLPR were observed. CONCLUSION: Altogether, results from the present study do not support a role for any of the five functional polymorphisms of SLC6A4 and BDNF genes in the aetiology of NS-RM. Moreover, they suggest no epistatic interaction in NS-MD between polymorphisms in BDNF and SLC6A4. However, we suggest that further studies on these two pathways in NS-MD remain necessary.

Recurrent deletion of ZNF630 at Xp11.23 is not associated with mental retardation.

ZNF630 is a member of the primate-specific Xp11 zinc finger gene cluster that consists of six closely related genes, of which ZNF41, ZNF81, and ZNF674 have been shown to be involved in mental retardation. This suggests that mutations of ZNF630 might influence cognitive function. Here, we detected 12 ZNF630 deletions in a total of 1,562 male patients with mental retardation from Brazil, USA, Australia, and Europe. The breakpoints were analyzed in 10 families, and in all cases they were located within two segmental duplications that share more than 99% sequence identity, indicating that the deletions resulted from non-allelic homologous recombination. In 2,121 healthy male controls, 10 ZNF630 deletions were identified. In total, there was a 1.6-fold higher frequency of this deletion in males with mental retardation as compared to controls, but this increase was not statistically significant (P-value = 0.174). Conversely, a 1.9-fold lower frequency of ZNF630 duplications was observed in patients, which was not significant either (P-value = 0.163). These data do not show that ZNF630 deletions or duplications are associated with mental retardation.

A new chromosome x exon-specific microarray platform for screening of patients with X-linked disorders.

Recent studies and advances in high-density oligonucleotide arrays have shown that microdeletions and microduplications occur at a high frequency in the human genome, causing various genetic conditions including mental retardation. Thus far little is known about the pathways leading to this disease, and implementation of microarrays is hampered by their increasing cost and complexity, underlining the need for new diagnostic tools. The aim of this study was to introduce a new targeted platform called "chromosome X exon-specific array" and to apply this new platform to screening of 20 families (including one blind positive control) with suspected X-linked mental retardation, to identify new causative X-linked mental retardation genes. The new microarray contains of 21,939 oligonucleotides covering 92.9% of all exons of all genes on chromosome X. Patient screening resulted in successful identification of the blind positive control included in the sample of 20 families, and one of the remaining 19 families was found to carry a 1.78-kilobase deletion involving all exons of pseudogene BRAF2. The BRAF2 deletion segregated in the family and was not found in 200 normal male samples, and no copy number variations are reported in this region. Further studies and focused investigation of X-linked disorders have the potential to reveal the molecular basis of human genetic pathological conditions that are caused by copy-number changes in chromosome X genes.

Could autism with mental retardation result from digenism and frequent de novo mutations?

The high concordance for autism symptoms in monozygotic twin-pairs compared to di-zygotic twins and/or non-twin sib-ships suggests a high genetic determinism in autism. Those results have hypothesized multi-factorial determinism in accordance with family studies and mathematical models. However, linkage and association or candidate gene strategies have failed to-date to identify clearly involved mechanisms. Mental retardation (MR) is known as frequently associated to autism. Multiplex XLMR pedigrees have been reported with only one mutated patient having autism and MR: different X-located MR genes have been shown to be involved (NLGN4, MECP2, OPHN1, ZNF674 and FRAXA) which does not suggest that they could be "autism genes". Tuberous sclerosis studies and report of numerous autosomal domains shown deleted in MR-autistic subjects suggest that several autosomal dominant (AD) genes could be also involved in MR with autism. Whereas multiplex AD-MR families are rare, AD de novo mutations could explain numerous sporadic situations of non-specific MR and of autism with MR, in accordance with twin studies. Finally, we hypothesize that in those autistic subjects with mendelian MR, the XL-MR or AD-MR gene (G1) would pave the way for a second Mendelian factor (G2) responsible for autism symptoms.

MCT8 mutation analysis and identification of the first female with Allan-Herndon-Dudley syndrome due to loss of MCT8 expression.

Mutations in the thyroid monocarboxylate transporter 8 gene (MCT8/SLC16A2) have been reported to result in X-linked mental retardation (XLMR) in patients with clinical features of the Allan-Herndon-Dudley syndrome (AHDS). We performed MCT8 mutation analysis including 13 XLMR families with LOD scores >2.0, 401 male MR sibships and 47 sporadic male patients with AHDS-like clinical features. One nonsense mutation (c.629insA) and two missense changes (c.1A>T and c.1673G>A) were identified. Consistent with previous reports on MCT8 missense changes, the patient with c.1673G>A showed elevated serum T3 level. The c.1A>T change in another patient affects a putative translation start codon, but the same change was present in his healthy brother. In addition normal serum T3 levels were present, suggesting that the c.1A>T (NM_006517) variation is not responsible for the MR phenotype but indicates that MCT8 translation likely starts with a methionine at position p.75. Moreover, we characterized a de novo translocation t(X;9)(q13.2;p24) in a female patient with full blown AHDS clinical features including elevated serum T3 levels. The MCT8 gene was disrupted at the X-breakpoint. A complete loss of MCT8 expression was observed in a fibroblast cell-line derived from this patient because of unfavorable nonrandom X-inactivation. Taken together, these data indicate that MCT8 mutations are not common in non-AHDS MR patients yet they support that elevated serum T3 levels can be indicative for AHDS and that AHDS clinical features can be present in female MCT8 mutation carriers whenever there is unfavorable nonrandom X-inactivation.

Screening of 20 patients with X-linked mental retardation using chromosome X-specific array-MAPH.

The rapid advancement of high-resolution DNA copy number assessment methods revealed the significant contribution of submicroscopic genetic imbalances to abnormal phenotypes, including mental retardation. In order to detect submicroscopic genetic imbalances, we have screened 20 families with X-linked mental retardation (XLMR) using a chromosome X-specific array-MAPH platform with median resolution of 238kb. Among the 20 families, 18 were experimental, as they were not previously screened with any microarray method, and two were blind controls with known aberrations, as they were previously screened by array-CGH. This study presents the first clinical application of chromosome X-specific array-MAPH methodology. The screening of 20 affected males from 20 unrelated XLMR families resulted in the detection of an unknown deletion, spanning a region of 7-23kb. Family studies and population screening demonstrated that the detected deletion is an unknown rare copy number variant. One of the control samples, carrying approximately 6-Mb duplication was correctly identified, moreover it was found to be interrupted by a previously unknown 19kb region of normal copy number. The second control 50kb deletion was not identified, as this particular region was not covered by array-MAPH probes. This study demonstrates that the chromosome X-specific array-MAPH platform is a valuable tool for screening patients with XLMR, or other X-linked disorders, and emerges the need for introducing new high-resolution screening methods for the detection of genetic imbalances.

Pleiotropic effects of CEP290 (NPHP6) mutations extend to Meckel syndrome.

Meckel syndrome (MKS) is a rare autosomal recessive lethal condition characterized by central nervous system malformations, polydactyly, multicystic kidney dysplasia, and ductal changes of the liver. Three loci have been mapped (MKS1-MKS3), and two genes have been identified (MKS1/FLJ20345 and MKS3/TMEM67), whereas the gene at the MKS2 locus remains unknown. To identify new MKS loci, a genomewide linkage scan was performed using 10-cM-resolution microsatellite markers in eight families. The highest heterogeneity LOD score was obtained for chromosome 12, in an interval containing CEP290, a gene recently identified as causative of Joubert syndrome (JS) and isolated Leber congenital amaurosis. In view of our recent findings of allelism, at the MKS3 locus, between these two disorders, CEP290 was considered a candidate, and homozygous or compound heterozygous truncating mutations were identified in four families. Sequencing of additional cases identified CEP290 mutations in two fetuses with MKS and in four families presenting a cerebro-reno-digital syndrome, with a phenotype overlapping MKS and JS, further demonstrating that MKS and JS can be variable expressions of the same ciliopathy. These data identify a fourth locus for MKS (MKS4) and the CEP290 gene as responsible for MKS.

Loss of SLC38A5 and FTSJ1 at Xp11.23 in three brothers with non-syndromic mental retardation due to a microdeletion in an unstable genomic region.

Using high resolution X chromosome array-CGH we identified an interstitial microdeletion at Xp11.23 in three brothers with moderate to severe mental retardation (MR) without dysmorphic features. The extent of the deletion was subsequently delineated to about 50 kb by regular PCR and included only the SLC38A5 and FTSJ1 genes. The loss of the FTSJ1 MR gene in males is expected to result in the observed phenotype but the contribution of the deletion of the solute carrier SLC38A5 gene is less clear. Their mother also carries the deletion and completely inactivates the aberrant X chromosome. Interestingly, the distal breakpoint is situated within a 200 kb SSX repeat region that appears to stimulate recombination since subtle copy number changes often occur at this location and it is frequently involved in translocations in tumours. Since this apparent SSX unstable structure is flanked proximally by FTSJ1 and PQBP1, subtle deletions or duplications at this location would be expected to cause MR, as in our family. So far, we have screened a cohort of 300 patients but did not find additional aberrations at the FTSJ1 locus indicating that the frequency is likely to be low.

Mutation frequencies of X-linked mental retardation genes in families from the EuroMRX consortium.

The EuroMRX family cohort consists of about 400 families with non-syndromic and 200 families with syndromic X-linked mental retardation (XLMR). After exclusion of Fragile X (Fra X) syndrome, probands from these families were tested for mutations in the coding sequence of 90 known and candidate XLMR genes. In total, 73 causative mutations were identified in 21 genes. For 42% of the families with obligate female carriers, the mental retardation phenotype could be explained by a mutation. There was no difference between families with (lod score >2) or without (lod score <2) significant linkage to the X chromosome. For families with two to five affected brothers (brother pair=BP families) only 17% of the MR could be explained. This is significantly lower (P=0.0067) than in families with obligate carrier females and indicates that the MR in about 40% (17/42) of the BP families is due to a single genetic defect on the X chromosome. The mutation frequency of XLMR genes in BP families is lower than can be expected on basis of the male to female ratio of patients with MR or observed recurrence risks. This might be explained by genetic risk factors on the X chromosome, resulting in a more complex etiology in a substantial portion of XLMR patients. The EuroMRX effort is the first attempt to unravel the molecular basis of cognitive dysfunction by large-scale approaches in a large patient cohort. Our results show that it is now possible to identify 42% of the genetic defects in non-syndromic and syndromic XLMR families with obligate female carriers.

X-linked mental retardation: a comprehensive molecular screen of 47 candidate genes from a 7.4 Mb interval in Xp11.

About 30% of the mutations causing nonsyndromic X-linked mental retardation (MRX) are thought to be located in Xp11 and in the pericentromeric region, with a particular clustering of gene defects in a 7.4 Mb interval flanked by the genes ELK1 and ALAS2. To search for these mutations, 47 brain-expressed candidate genes located in this interval have been screened for mutations in up to 22 mental retardation (MR) families linked to this region. In total, we have identified 57 sequence variants in exons and splice sites of 27 genes. Based on these data, four novel MR genes were identified, but most of the sequence variants observed during this study have not yet been described. The purpose of this article is to present a comprehensive overview of this work and its outcome. It describes all sequence variants detected in 548 exons and their flanking sequences, including disease-causing mutations as well as possibly relevant polymorphic and silent sequence changes. We show that many of the studied genes are unlikely to play a major role in MRX. This information will help to avoid duplication of efforts in the ongoing endeavor to unravel the molecular causes of MRX.

Mutation screening of brain-expressed X-chromosomal miRNA genes in 464 patients with nonsyndromic X-linked mental retardation.

MiRNAs are small noncoding RNAs that control the expression of target genes at the post-transcriptional level and have been reported to modulate various biological processes. Their function as regulatory factors in gene expression renders them attractive candidates for harbouring genetic variants with subtle effects on IQ. In an attempt to investigate the potential role of miRNAs in the aetiology of X-linked mental retardation, we have examined all 13 known, brain-expressed X-chromosomal miRNAs in a cohort of 464 patients with non-syndromic X-linked MR and found four nucleotide changes in three different pre-miRNA hairpins. All the observed changes appear to be functionally neutral which, taken together with the rarity of detected nucleotide changes in miRNA genes, may reflect strong selection and thus underline the functional importance of miRNAs.

ZNF674: a new kruppel-associated box-containing zinc-finger gene involved in nonsyndromic X-linked mental retardation.

Array-based comparative genomic hybridization has proven to be successful in the identification of genetic defects in disorders involving mental retardation. Here, we studied a patient with learning disabilities, retinal dystrophy, and short stature. The family history was suggestive of an X-linked contiguous gene syndrome. Hybridization of full-coverage X-chromosomal bacterial artificial chromosome arrays revealed a deletion of ~1 Mb in Xp11.3, which harbors RP2, SLC9A7, CHST7, and two hypothetical zinc-finger genes, ZNF673 and ZNF674. These genes were analyzed in 28 families with nonsyndromic X-linked mental retardation (XLMR) that show linkage to Xp11.3; the analysis revealed a nonsense mutation, p.E118X, in the coding sequence of ZNF674 in one family. This mutation is predicted to result in a truncated protein containing the Kruppel-associated box domains but lacking the zinc-finger domains, which are crucial for DNA binding. We characterized the complete ZNF674 gene structure and subsequently tested an additional 306 patients with XLMR for mutations by direct sequencing. Two amino acid substitutions, p.T343M and p.P412L, were identified that were not found in unaffected individuals. The proline at position 412 is conserved between species and is predicted by molecular modeling to reduce the DNA-binding properties of ZNF674. The p.T343M transition is probably a polymorphism, because the homologous ZNF674 gene in chimpanzee has a methionine at that position. ZNF674 belongs to a cluster of seven highly related zinc-finger genes in Xp11, two of which (ZNF41 and ZNF81) were implicated previously in XLMR. Identification of ZNF674 as the third XLMR gene in this cluster may indicate a common role for these zinc-finger genes that is crucial to human cognitive functioning.

Disruptions of the novel KIAA1202 gene are associated with X-linked mental retardation.

The extensive heterogeneity underlying the genetic component of mental retardation (MR) is the main cause for our limited understanding of the aetiology of this highly prevalent condition. Hence we set out to identify genes involved in MR. We investigated the breakpoints of two balanced X;autosome translocations in two unrelated female patients with mild/moderate MR and found that the Xp11.2 breakpoints disrupt the novel human KIAA1202 (hKIAA1202) gene in both cases. We also identified a missense exchange in this gene, segregating with the Stocco dos Santos XLMR syndrome in a large four-generation pedigree but absent in >1,000 control X-chromosomes. Among other phenotypic characteristics, the affected males in this family present with severe MR, delayed or no speech, seizures and hyperactivity. Molecular studies of hKIAA1202 determined its genomic organisation, its expression throughout the brain and the regulation of expression of its mouse homologue during development. Transient expression of the wild-type KIAA1202 protein in HeLa cells showed partial colocalisation with the F-actin based cytoskeleton. On the basis of its domain structure, we argue that hKIAA1202 is a new member of the APX/Shroom protein family. Members of this family contain a PDZ and two ASD domains of unknown function and have been shown to localise at the cytoskeleton, and play a role in neurulation, cellular architecture, actin remodelling and ion channel function. Our results suggest that hKIAA1202 may be important in cognitive function and/or development.

Duplication of the MECP2 region is a frequent cause of severe mental retardation and progressive neurological symptoms in males.

Loss-of-function mutations of the MECP2 gene at Xq28 are associated with Rett syndrome in females and with syndromic and nonsyndromic forms of mental retardation (MR) in males. By array comparative genomic hybridization (array-CGH), we identified a small duplication at Xq28 in a large family with a severe form of MR associated with progressive spasticity. Screening by real-time quantitation of 17 additional patients with MR who have similar phenotypes revealed three more duplications. The duplications in the four patients vary in size from 0.4 to 0.8 Mb and harbor several genes, which, for each duplication, include the MR-related L1CAM and MECP2 genes. The proximal breakpoints are located within a 250-kb region centromeric of L1CAM, whereas the distal breakpoints are located in a 300-kb interval telomeric of MECP2. The precise size and location of each duplication is different in the four patients. The duplications segregate with the disease in the families, and asymptomatic carrier females show complete skewing of X inactivation. Comparison of the clinical features in these patients and in a previously reported patient enables refinement of the genotype-phenotype correlation and strongly suggests that increased dosage of MECP2 results in the MR phenotype. Our findings demonstrate that, in humans, not only impaired or abolished gene function but also increased MeCP2 dosage causes a distinct phenotype. Moreover, duplication of the MECP2 region occurs frequently in male patients with a severe form of MR, which justifies quantitative screening of MECP2 in this group of patients.

Mutations in the JARID1C gene, which is involved in transcriptional regulation and chromatin remodeling, cause X-linked mental retardation.

In families with nonsyndromic X-linked mental retardation (NS-XLMR), >30% of mutations seem to cluster on proximal Xp and in the pericentric region. In a systematic screen of brain-expressed genes from this region in 210 families with XLMR, we identified seven different mutations in JARID1C, including one frameshift mutation and two nonsense mutations that introduce premature stop codons, as well as four missense mutations that alter evolutionarily conserved amino acids. In two of these families, expression studies revealed the almost complete absence of the mutated JARID1C transcript, suggesting that the phenotype in these families results from functional loss of the JARID1C protein. JARID1C (Jumonji AT-rich interactive domain 1C), formerly known as "SMCX," is highly similar to the Y-chromosomal gene JARID1D/SMCY, which encodes the H-Y antigen. The JARID1C protein belongs to the highly conserved ARID protein family. It contains several DNA-binding motifs that link it to transcriptional regulation and chromatin remodeling, processes that are defective in various other forms of mental retardation. Our results suggest that JARID1C mutations are a relatively common cause of XLMR and that this gene might play an important role in human brain function.

Mild phenotypes in a series of patients with Opitz GBBB syndrome with MID1 mutations.

Opitz syndrome (OS; MIM 145410 and MIM 300000) is a congenital midline malformation syndrome characterized by hypertelorism, hypospadias, cleft lip/palate, laryngotracheoesophageal (LTE) abnormalities, imperforate anus, developmental delay, and cardiac defects. The X-linked form (XLOS) is caused by mutations in the MID1 gene, which encodes a microtubule-associated RBCC protein. In this study, phenotypic manifestations of patients with and without MID1 mutations were compared to determine genotype-phenotype correlations. We detected 10 novel mutations, 5 in familial cases, 2 in sporadic cases, and 3 in families for whom it was not clear if they were familial or sporadic. The genotype and phenotype was compared for these 10 families, clinically diagnosed OS patients found not to have MID1 mutations, and 4 families in whom we have previously reported MID1 mutations. This combined data set includes clinical and mutation data on 70 patients. The XLOS patients with MID1 mutations were less severely affected than patients with MID1 mutations reported in previous studies, particularly in functionally significant neurologic, LTE, anal, and cardiac abnormalities. Minor anomalies were more prevalent in patients with MID1 mutations compared to those without mutations in this study. Female MID1 mutation carriers had milder phenotypes compared to male MID1 mutation carriers, with the most common manifestation being hypertelorism in both sexes. Most of the anomalies found in the patients of the present study do not correlate with the MID1 mutation type, with the possible exception of LTE malformations. This study demonstrates the wide spectrum of severity and manifestations of OS. It also shows that XLOS patients with MID1 mutations may be less severely affected than indicated in prior reports.

Möbius sequence, Robin complex, and hypotonia: severe expression of brainstem disruption spectrum versus Carey-Fineman-Ziter syndrome.

We report on nine unrelated children fitting a diagnosis of Carey-Fineman-Ziter syndrome (CFZS). All children presented with Möbius sequence, Pierre Robin complex (6/9) or micrognathia, and hypotonia. Some had primary hypoventilation, delayed development, and acral anomalies. The neuropathological investigations performed in two patients showed a combination of dysplastic lesions (neuronal heterotopias) and encephaloclastic changes consisting of small foci of necrosis with microcalcifications. The mother of a third child had severe trauma during her 2nd month of pregnancy. Based on a review of the literature on MS and CFZS, we suggest designating as "Robin-Möbius phenotype" a distinct clinical variant of MS with extensive brainstem involvement, Robin complex, hypotonia without specific muscle disorder, clubfeet and variable acral anomalies. This condition appears to bear a higher risk of mental handicap and perhaps a higher recurrence risk than "common" MS. Neuropathology and neuroimaging are suggestive, at least in some cases, of a vascular disruption, which could be exogenous, or secondary to a genetic predisposition. Etiologic heterogeneity seems likely and, in that respect, the original CFZS family could represent a private syndrome fitting on the "Robin-Möbius" spectrum. Despite the existence of two familial reports, recurrence risk is probably much lower than 25%, although exact figures cannot be extracted from the available literature.

High prevalence of SLC6A8 deficiency in X-linked mental retardation.

A novel X-linked mental retardation (XLMR) syndrome was recently identified, resulting from creatine deficiency in the brain caused by mutations in the creatine transporter gene, SLC6A8. We have studied the prevalence of SLC6A8 mutations in a panel of 290 patients with nonsyndromic XLMR archived by the European XLMR Consortium. The full-length open reading frame and splice sites of the SLC6A8 gene were investigated by DNA sequence analysis. Six pathogenic mutations, of which five were novel, were identified in a total of 288 patients with XLMR, showing a prevalence of at least 2.1% (6/288). The novel pathogenic mutations are a nonsense mutation (p.Y317X) and four missense mutations. Three missense mutations (p.G87R, p.P390L, and p.P554L) were concluded to be pathogenic on the basis of conservation, segregation, chemical properties of the residues involved, as well as the absence of these and any other missense mutation in 276 controls. For the p.C337W mutation, additional material was available to biochemically prove (i.e., by increased urinary creatine : creatinine ratio) pathogenicity. In addition, we found nine novel polymorphisms (IVS1+26G-->A, IVS7+37G-->A, IVS7+87A-->G, IVS7-35G-->A, IVS12-3C-->T, IVS2+88G-->C, IVS9-36G-->A, IVS12-82G-->C, and p.Y498) that were present in the XLMR panel and/or in the control panel. Two missense variants (p.V629I and p.M560V) that were not highly conserved and were not associated with increased creatine : creatinine ratio, one translational silent variant (p.L472), and 10 intervening sequence variants or untranslated region variants (IVS6+9C-->T, IVS7-151_152delGA, IVS7-99C-->A, IVS8-35G-->A, IVS8+28C-->T, IVS10-18C-->T, IVS11+21G-->A, IVS12+15C-->T, *207G-->C, IVS12+32C-->A) were found only in the XLMR panel but should be considered as unclassified variants or as a polymorphism (p.M560V). Our data indicate that the frequency of SLC6A8 mutations in the XLMR population is close to that of CGG expansions in FMR1, the gene responsible for fragile-X syndrome.