CGG repeat in the FMR1 gene: size matters

The FMR1 gene contains a CGG repeat present in the 5'-untranslated region which can be unstable upon transmission to the next generation. The repeat is up to 55 CGGs long in the normal population. In patients with fragile X syndrome (FXS), a repeat length exceeding 200 CGGs (full mutation: FM) generally leads to methylation of the repeat and the promoter region, which is accompanied by silencing of the FMR1 gene. The absence of FMR1 protein, FMRP, seen in FM is the cause of the mental retardation in patients with FXS. The premutation (PM) is defined as 55-200 CGGs. Female PM carriers are at risk of developing primary ovarian insufficiency. Elderly PM carriers might develop a progressive neurodegenerative disorder called fragile X-associated tremor/ataxia syndrome (FXTAS). Although arising from the mutations in the same gene, distinct mechanisms lead to FXS (absence of FMRP), FXTAS (toxic RNA gain-of-function) and FXPOI. The pathogenic mechanisms thought to underlie these disorders are discussed. This review gives insight on the implications of all possible repeat length categories seen in fragile X families.     

Fragile X syndrome: the FMR1 CGG repeat distribution among world populations

Fragile X syndrome (FXS) is characterized by moderate to severe intellectual disability, which is accompanied by macroorchidism and distinct facial morphology. FXS is caused by the expansion of the CGG trinucleotide repeat in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. The syndrome has been studied in ethnically diverse populations around the world and has been extensively characterized in several populations. Similar to other trinucleotide expansion disorders, the gene-specific instability of FMR1 is not accompanied by genomic instability. Currently we do not have a comprehensive understanding of the molecular underpinnings of gene-specific instability associated with tandem repeats. Molecular evidence from in vitro experiments and animal models supports several pathways for gene-specific trinucleotide repeat expansion. However, whether the mechanisms reported from other systems contribute to trinucleotide repeat expansion in humans is not clear. To understand how repeat instability in humans could occur, the CGG repeat expansion is explored through molecular analysis and population studies which characterized CGG repeat alleles of FMR1. Finally, the review discusses the relevance of these studies in understanding the mechanism of trinucleotide repeat expansion in FXS.     

Small CGG repeat expansion alleles of FMR1 gene are associated with parkinsonism

Fragile X-associated tremor/ataxia syndrome (FXTAS) affects older males carrying premutation, that is, expansions of the CGG repeat (in the 55–200 range), in the FMR1 gene. The neurological changes are linked to the excessive FMR1 messenger RNA (mRNA), becoming toxic through a 'gain-of-function'. Because elevated levels of this mRNA are also found in carriers of the smaller expansion (grey zone) alleles, ranging from 40 to 54 CGGs, we tested for a possible role of these alleles in the origin of movement disorders associated with tremor.
We screened 228 Australian males affected with idiopathic Parkinson's disease and other causes of parkinsonism recruited from Victoria and Tasmania for premutation and grey zone alleles. The frequencies of either of these alleles were compared with the frequencies in a population-based sample of 578 Guthrie spots from consecutive Tasmanian male newborns (controls). There was a significant excess of premutation carriers (Fisher's exact test p = 0.006). There was also a more than twofold increase in grey zone carriers in the combined sample of the Victorian and Tasmanian cases, with odds ratio (OR ) = 2.36, and 95% confidence intervals (CI): 1.20–4.63, as well as in Tasmanian cases only (OR = 2.33, 95% CI: 1.06–5.13), compared with controls. The results suggest that the FMR1 grey zone alleles, as well as premutation alleles, might contribute to the aetiology of disorders associated with parkinsonism.     

Expansions and contractions of the FMR1 CGG repeat in 5,508 transmissions of normal,intermediate, and premutation alleles

Instability of the FMR1 repeat, commonly observed in transmissions of premutation alleles (55–200 repeats), is influenced by the size of the repeat, its internal structure and the sex of the transmitting parent. We assessed these three factors in unstable transmissions of 14/3,335 normal (~5 to 44 repeats), 54/293 intermediate (45–54 repeats), and 1561/1,880 premutation alleles. While most unstable transmissions led to expansions, contractions to smaller repeats were observed in all size classes. For normal alleles, instability was more frequent in paternal transmissions and in alleles with long 3′ uninterrupted repeat lengths. For premutation alleles, contractions also occurred more often in paternal than maternal transmissions and the frequency of paternal contractions increased linearly with repeat size. All paternal premutation allele contractions were transmitted as premutation alleles, but maternal premutation allele contractions were transmitted as premutation, intermediate, or normal alleles. The eight losses of AGG interruptions in the FMR1 repeat occurred exclusively in contractions of maternal premutation alleles. We propose a refined model of FMR1 repeat progression from normal to premutation size and suggest that most normal alleles without AGG interruptions are derived from contractions of maternal premutation alleles.     

Molecular-intelligence correlations in young fragile X males with a mild CGG repeat expansion in the FMR1 gene

Several mechanisms can explain the occurrence of full-mutation fragile X males with an IQ level above −2 SD below mean, also called “high-functioning fragile X males.” Incomplete methylation of the CpG island at the 5′ end of the FMR1 gene is one of these mechanisms. The present study describes the physical and behavior phenotypes in 7 fragile X boys with CGG repeat insertions in the FMR1 gene between 600–2,400 base pairs. The degree of methylation at the FMR1-associated CpG island ranges in peripheral blood lymphocytes from 0–95%. Subjects with a low degree of methylation at this site have mild or absent physical characteristics of the fragile X syndrome, while subjects with a high degree of methylation at this site have more severe physical characteristics. In this range of CGG repeat insertion (600–2,400 base pairs), the degree of methylation at the FMR1-associated CpG island is a good predictor of intelligence, while CGG repeat insertion length is not. © 1996 Wiley-Liss, Inc.     

Instability of the FMR2 trinucleotide repeat region associated with expanded FMR1 alleles

The fragile sites FRAXA and FRAXE, located ∼600 kb apart on Xq27.3 and Xq28, respectively, are due to a CGG trinucleotide repeat expansion. Although the expansion mechanism for these and other trinucleotide repeat disorders remains unknown, the similarities between the FRAXA and FRAXE regions suggest a possible association between the 2 sites. DNA from 953 individuals was analyzed to determine the distribution of FRAXE repeat sizes in this population and to ascertain potential association between FRAXA and FRAXE repeat sizes. Thirty-four FMR2 alleles ranging from 3–42 repeats were identified. No FRAXE expansions were found in this population, supporting previous findings that FRAXE expansions are rare. However, in the fragile X syndrome affected group, a FMR2 delection, 2 cases of FRAXE repeat instability and a FRAXE mosaic male were identified. Also, a previously identified, rare FMR2 polymorphism was observed. Statistical analyis showed no correlation between normal FRAXA and FRAXE repeat sizes studied, although there was a significant size difference in larger FMR2 alleles that segregated with expanded FMR1 alleles. These findings support the idea of an association between repeat expansion in the FMR1 gene and instability or deletions in the FMR2 gene. Am. J. Med. Genet. 73:447–455, 1997. © 1997 Wiley-Liss, Inc.     

Association of FMR1 repeat size with ovarian dysfunction

BACKGROUND: Women who carry the FMR1 premutation allele have a significantly increased risk for ovarian dysfunction. We hypothesize that molecular characteristics of the FMR1 gene may explain this increased risk. METHODS: Thus, we examined the effect of FMR1 CGG repeat size and related factors on measures of ovarian dysfunction using data from 507 women with a wide range of repeat sizes. RESULTS AND CONCLUSIONS: We found a significant positive association of repeat size with ovarian dysfunction, but have preliminary evidence that this relationship is non-linear. We suggest that FMR1 repeat size in the lower range (<80 repeats) contributes to the variation in age at menopause; thus, FMR1 could be considered a quantitative trait locus. More importantly, when repeat size exceeds this threshold, the increase in risk for ovarian dysfunction is clinically significant. Intriguingly, this risk appears to plateau, or perhaps decrease, among women with very high repeats (> or =100 repeats).     

New evidence for,and challenges in,linking small CGG repeat expansion FMR1 alleles with Parkinson's disease

We recently reported a significant increase in the frequency of carriers of grey zone (GZ) alleles of FMR1 gene in Australian males with Parkinson's disease (PD) from Victoria and Tasmania. Here, we report data comparing an independent sample of 817 PD patients from Queensland to 1078 consecutive Australian male newborns from Victoria. We confirmed the earlier finding by observing a significant excess of GZ alleles in PD (4.8%) compared to controls (1.5%). Although both studies provided evidence in support of an association between GZ‐carrier status and increased risk for parkinsonism, the existing evidence in the literature from screening studies remains equivocal and we discuss the need for alternative approaches to resolve the issue.     

The FMR1 CGG repeat test is not a candidate prescreening tool for identifying women with a high probability of being carriers of BRCA mutations

The identification of women with a high probability of being carriers of pathogenic BRCA mutation is not straightforward and a major improvement would be the availability of markers of mutations that could be directly evaluated in individuals asking for genetic testing. The FMR1 gene testing was recently proposed as a candidate prescreening tool because an association between BRCA pathogenic mutations and FMR1 genotypes with ‘low alleles'' (CGG repeat number <26) was observed. To confirm this hypothesis, we evaluated the distribution of FMR1 alleles and genotypes between BRCA mutation carriers and non-carriers in a cohort of 147 Italian women, free of cancer or affected by breast and/or ovarian cancer, who were tested for the presence of BRCA mutation in a clinical setting. The distribution of FMR1 CGG repeat numbers in the two groups was similar (lower allele median/mean were 30/27.4 and 30/27.9, respectively; Mann–Whitney test P=0.997) and no difference in the FMR1 genotype distribution was present (χ²=0.503, d.f.=2, P=0.78). This result is in contrast with literature data and suggests that FMR1 genetic testing is not a candidate BRCA prescreening tool.     

Influence of intermediate and uninterrupted FMR1 CGG expansions in premature ovarian failure manifestation

BACKGROUND: Studies attempting to precisely define the range of fragile mental retardation 1 (FMR1) expansions and its inf luence in premature ovarian failure (POF) manifestation are partially lacking. To this aim, we evaluated a large cohort of POF patients for the size and, in selected cases, for the sequence of the CGG expansion. Furthermore, the correlation between POF and X-inactivation was investigated in FRAXA families. METHODS: By fluorescent PCR, 190 POF and 200 control women were sized for the CGG tract; some subjects were also characterized by sequencing and for the FMR1 activation ratio. RESULTS AND CONCLUSION: We found a significant association (19/190, 10%, P < 1 x 10(-6)) between POF and FMR1 premutation (range 63-163 repeats) and a significant enrichment (9/190, 4.7%, P = 0.021) of POF carriers of intermediate expansions (range 41-58 repeats). Interestingly, intermediate alleles were entirely composed of CGG repeats. Furthermore, the analysis of three pairs of siblings with similar FMR1 expansions and discordant for the POF phenotype showed a direct correlation between the expression of the intermediate/premutated allele and POF manifestation. The results obtained strengthen the correlation between FMR1 expansion and POF and suggest that the manifestation of the ovarian dysfunction could be influenced both by the pattern of interruption of the CGG repeat and by X-inactivation.     

不同民族人群FMR1基因CGG重复序列检测与分析

目的 检测和分析脆性X综合征致病基因FMR1 CGG重复序列在汉族和壮族人群中的多态性分布。方法 采用PCR扩增技术和聚丙烯酰胺凝胶电泳技术对1060例汉族人（男280人，女780人）和283例壮族人（男85人，女198人）FMR1基因CGG重复序列进行分析，并用Southern blot技术对结果进行了验证。结果 汉族人群中共检测到33种等位基因，其中CGG重复序列范围为6～43，壮族人群共检测到27种等位基因，CGG重复序列范围为6～57，两类人群中最大频率等位基因分别为28和29。结论 应用PCR扩增技术进行了脆性X综合征大面积筛查，中国汉族和壮族人群中FMR1基因CGG重复序列变异分布略有差异。     

Fragile X syndrome with FMR1 and FMR2 deletion

We report a 13 year old boy with fragile X syndrome resulting from a de novo deletion of the FMR1 and FMR2 genes extending from (and including) DXS7536 proximally to FMR2 distally. The patient has severe developmental delay, epilepsy, and behavioural difficulties, including autistic features. He has epicanthic folds, in addition to facial features typical of fragile X syndrome, and marked joint hypermobility. We compare our patient to the three other cases reported in which both FMR1 and FMR2 are deleted. This case has the smallest deletion reported to date. All four patients have epilepsy and a more severe degree of mental retardation than is usual in fragile X syndrome resulting from FMR1 triplet repeat expansion. Three of the patients have joint laxity and two have epicanthic folds. We suggest that these features, in particular severe developmental delay and epilepsy, may form part of the characteristic phenotype resulting from deletion of both FMR1 and FMR2 genes. The diagnosis in this case was delayed because routine cytogenetics showed no abnormality and standard molecular tests for FMR1 triplet repeat expansion (PCR and Southern blotting) failed. Further DNA studies should be undertaken to investigate for a deletion where clinical suspicion of fragile X syndrome is strong and routine laboratory tests fail.     

卵巢储备功能不全患者 FMR1基因CGG重复数的研究

目的:探讨 FMR1基因(CGG)n重复数与卵巢储备功能不全(diminished ovarian reserve,DOR)发病的相关性,为患者提供遗传咨询和生育指导。 方法:采集214例DOR患者的外周血样,提取DNA。用PCR扩增结合毛细管电泳测定患者及部分家系成员 FMR1基因的...     

A novel duplication in the FMR1 gene: implications for molecular analysis in fragile X syndrome and repeat instability

We have observed a 49 bp tandem duplication adjacent to the triplet repeat of the FMR1 gene and have shown it to occur as a variant in Finland. It affects the primers commonly used in molecular analysis of fragile X syndrome by polymerase chain reaction (PCR) methods. One concern is that females with the full mutation and variant alleles might be missed because of the two PCR products generated by the variant. We suggest that the duplication has arisen by a misalignment of the proximal end of the repeat tract and the non-adjacent GGCGGCGGCGG-sequence located 37 bp upstream and may indicate a mutation hot spot. The discovery of this duplication and the previous observations on deletions associated with full mutations in FMR1 indicate that realignment between the repeat tract and dispersed non-adjacent homologous repetitive sequences may also play a role in repeat instability in fragile X.     

Single nucleotide polymorphism hunting in cyberspace

Large-scale sequencing of human cDNA and genomic DNA libraries has produced a large collection of sequence data in public databases. To date, >900,000 human expressed sequence tag (EST) sequences and >80,000,000 bases of genomic DNA sequence have been deposited in Genbank. This ever-expanding data set is a rich source of gene-associated and anonymous single nucleotide polymorphisms (SNPs). DNA sequence variations can be found by comparing the sequences of redundant ESTs and by comparing sequences from overlapping genomic clones. Initial studies have shown that, with proper computer screening, informative SNP markers can be developed from these DNA databases in an efficient and cost-effective manner. Complete public access to these databases will allow individual investigators to add biological value to the human sequence data generated by large-scale sequencing centers. Hum Mutat 12:221–225, 1998. © 1998 Wiley-Liss, Inc.     

FMR1 CGG Repeat Patterns and Flanking Haplotypes in Three Asian Populations and Their Relationship With Repeat Instability

Hyper-expansion of a CGG repeat in the 5' untranslated region of the FMR1 gene followed by methylation and silencing is the predominant cause of Fragile X syndrome, the most common inherited mental retardation disorder. Most detailed studies of the FMR1 gene have focused on Caucasian populations and patients. We performed a detailed haplotype and linkage disequilibrium analysis of the FMR1 gene in a total of 454 unselected normal X chromosomes from three Asian populations, Chinese, Malay and Indian. Compared to Caucasians and African Americans, the diversity of normal FMR1 CGG repeat lengths, patterns and flanking haplotypes were lower in Asians. Strong linkage disequilibrium was observed between the CGG repeat and flanking FMR1 markers in all three Asian populations, with strong association between specific CGG repeat alleles and flanking marker alleles observed only in the Chinese and Malays. A test for randomness of distribution between FRAXA CGG repeat patterns and flanking FMR1 marker haplotypes also revealed a highly significant non-random distribution between CGG repeat patterns and flanking haplotypes in all three ethnic groups (P < 0.001). Extending previous findings in Caucasians and African Americans we present a novel statistical approach, using data from unselected population samples alone, to show an association between absence of at least one AGG interruption in any position (5', 3', or middle) and increased CGG repeat instability.     

The association of FMR1 gene (CGG)n variation with idiopathic female infertility

IntroductionThe FMR1 gene plays an important role in brain development and in the regulation of ovarian function. The FMR1 gene contains CGG repeat variation and the expansion of the repeats is associated with various phenotypes e.g. fragile X syndrome, premature ovarian failure, etc. Repeats ranging < 55 CGG are considered normal, however recent studies suggest that high-normal (35–54 CGG) and low-normal (< 26 CGG) alleles may also have an impact on female reproductive function.Material and methodsWe have performed a case-control study to assess the impact of FMR1 gene CGG repeats on female infertility. The study comprised 161 women with primary and secondary idiopathic infertility and 12 females with diminished ovarian reserve. The control group consisted of 129 healthy women with children. The FMR1 gene trinucleotide CGG repeat variation was detected using a triplet repeat primed polymerase chain reaction with capillary electrophoresis.ResultsThe analysis of CGG repeats revealed that high-normal alleles are statistically significantly more common in the secondary infertility group than in controls (12% vs. 4.3%, p = 0.03, OR = 3.1, 95% CI: 1.1–8.3). The distribution of high-normal alleles and genotypes did not differ between patients with primary infertility and controls (p > 0.05). In addition, the analysis of low-normal allele and genotype frequencies did not present a difference between primary, secondary infertility and the control group (p > 0.05).ConclusionsIn our study, the FMR1 gene high-normal alleles were associated with secondary infertility. However, to address the controversies related to the role of FMR1 genes in the development of diminished ovarian reserve, further studies on the subject are required.