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.     

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.     

Fragile X “gray zone” alleles: AGG patterns,expansion risks,and associated haplotypes

The risk for fragile X “gray-zone” alleles to expand appears to depend on the absence of stabilizing AGGs, which interrupt the CGG repeat region. To characterize such alleles better, we analyzed a series of 101 chromosomes with triplet repeat lengths ranging from 35 to 59 for variations in their AGG interspersion patterns. Among these, 11.9% had 3 AGGs, 59.3% had 2, 24.8% had 1, and 4.0% had 0. An inverse relationship between FMR1 repeat length and the number of interrupting AGGs was observed. Within the range of 35–44 repeats, 98.7% of alleles were found to have a pure CGG repeat length (P_CGG) of less than 33. However, among alleles with 45–59 repeats, 50% were found to have 0 or 1 AGG and a P_CGG of more than 33. Thus, gray-zone alleles with 45–59 repeats frequently have a long stretch of pure CGGs and thus are more likely to be unstably inherited than alleles with 35–44 repeats. We found length associations of P_CGG with 2 flanking microsatellites, DXS548 and FRAXAC1: a P_CGG ≤20 was strongly associated with haplotype 20−19, whereas a P_CGG > 20 was more strongly associated with the haplotype 25−21. This result could reflect a founder effect or a generalized instability of CGGs and microsatellites. © 1996 Wiley-Liss, Inc.     

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.     

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.     

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.     

The fragile X gene and its function

The fragile X syndrome represents the most common inherited cause of mental retardation worldwide. It is caused by a stretch of CGG repeats within the fragile X gene, which increases in length as it is transmitted from generation to generation. Once the repeat exceeds a threshold length, no protein is produced resulting in the fragile X phenotype. Ten years after the discovery of the gene, much has been learned about the function of the fragile X protein. Knowledge has been collected about the mutation mechanism, although still not all players that allow the destabilization of the CGG repeat are known.     

Haplotype and AGG‐interspersion analysis of FMR1 (CGG)n alleles in the Danish population: Implications for multiple mutational pathways towards fragile X alleles

The AGG interspersion pattern and flanking microsatellite markers and their association with instability of the FMR1 (CGG)_n repeat, involved in the fragile X syndrome, were analyzed in DNA from filter‐paper blood spots randomly collected from the Danish newborn population. Comparison of DXS548‐FRAXAC1 haplotype frequencies in the normal population and among fragile X patients suggested strong linkage disequilibrium between normal alleles and haplotype 7–3 and between fragile X alleles and haplotype 2–1 and 6–4. Comparison of the AGG interspersion pattern in 143 alleles, ranging in size from 34–62 CGG, and their associated haplotypes indicates the existence of at least three mutational pathways from normal alleles toward fragile X alleles in the Danish population. Two subgroups of normal alleles, with internal sequences of (CGG)₁₀AGG(CGG)₁₉ and (CGG)₉AGG(CGG)₁₂ AGG(CGG)₉, possibly predisposed for expansion, were identified in the data set. When alleles larger than 34 CGG were investigated, comparing the length of 3' uninterrupted CGG triplets (uCGG), we found that alleles associated with haplotype 2–1 and 6–4 contain significantly longer stretches of uCGG than alleles associated with haplotype 7–3. Thus, the data support that (CGG)_n instability is correlated to the length of uCGG. Am. J. Med. Genet. 93:99–106, 2000. © 2000 Wiley‐Liss, Inc.     

Genetic Diversity of the Fragile X Syndrome Gene (FMR1) in a Large Sub‐Saharan West African Population

Fragile X syndrome (OMIM #300624) is caused by the expansion of a CGG trinucleotide repeat found in the 5′ untranslated region of the X‐linked FMR1 gene. Although examinations of characteristics associated with repeat instability and expansion of the CGG repeat upon transmission from parent to offspring has occurred in various world populations, none has been conducted in large Sub‐Saharan African populations. We have examined the FMR1 CGG repeat structure in a sample of 350 males drawn from the general population of Ghana. We found that Ghanaians and African Americans have similar allele frequency distributions of CGG repeat and its flanking STR markers, DXS548 and FRAXAC1. However, the distribution of the more complex marker, FRAXAC2, is significantly different. The haplotype structure of the FMR1 locus indicated that Ghanaians share several haplotypes with African Americans and Caucasians that are associated with the expanded full mutation. In Ghanaians, the majority of repeat structures contained two AGG interruptions, however, the majority of intermediate alleles (35–49) lacked AGG interruptions. Overall, we demonstrate that allelic diversity of the FMR1 locus among Ghanaians is comparable to African Americans, but includes a minority of CGG array structures not found in other populations.     

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.     

Fragile X syndrome: clinical,cytogenetic and molecular screening among autism spectrum disorder children in Indonesia

Fragile X testing is a priority in the evaluation of autism spectrum disorders (ASD) cases because identification of the FMR1 mutation leads to new treatment options. This study is focused on determining the prevalence of the FMR1 gene mutation among ASD cases in Indonesia. DSM‐IV‐TR criteria were administered to diagnose ASD; symptom severity was classified using the Childhood Autism Rating Scale. Cytogenetic analysis, polymerase chain reaction, and Southern blot for FMR1 gene analysis were carried out to confirm the diagnosis of fragile X syndrome. The fragile X site and FMR1 full mutation allele were identified in 3 out of 65 (4.6%) and 4 out of 65 (6.15%) children aged 3–17 years (57 boys and 8 girls), respectively. The Fragile X laboratory workup is essential in the evaluation of patients with ASD. Molecular analysis is most accurate, while cytogenetic documentation of the fragile X site can also be useful if molecular testing is not available.     

Expand Long PCR for fragile X mutation detection

Fragile X mutation detection by DNA analysis enables accurate diagnosis of the fragile X syndrome. The mutation involves the expansion of CGG repeats in the FMR1 gene and has been primarily detected by the Southern blotting method. In this study we present a novel, efficient and reliable PCR protocol that is more convenient for routine diagnosis of the fragile X syndrome. This method is based on the use of the Expand Long PCR System, which enables the amplification of normal, premutated and full-mutated alleles, and therefore provides complete CGG repeat analysis of the FMR1 gene. Normal alleles were easily detected by ethidium bromide staining of the agarose gels, suggesting that this assay could be used as a screening test for a large number of referrals. The amplified premutations and full mutations were identified by hybridization with a digoxigenin-labeled 5'-(CGG)_5–3' probe, followed by chemiluminescent detection. The accuracy of our Expand Long PCR protocol was confirmed by Southern blot analysis, illustrating that the Expand Long PCR results concur with those of Southern blotting. In this paper we propose a new strategy for molecular diagnosis of the fragile X syndrome in which our Expand Long PCR assay is used as the first screening test for fragile X mutation detection.     

一种脆性X综合征新检测方法的应用及探讨

目的建立一种更快捷、准确、简便的检测脆性X综合征的方法。方法用传统的基因组酶切-PCR法与TP-PCR法同时对26例可疑患儿进行基因检测。结果基因组酶切PCR法共检测4例男性患者,其余均未见异常;TP-PCR法不仅检测出4例男性患者,且检测出1例女性携带者。结论 TP-PCR法是一种准确、快速、简便和相对价廉的方法,可以快速筛查脆性X综合征的基因诊断方法。     

Fragile X syndrome: An overview and update of the FMR1 gene

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability and the leading form of the monogenic cause of autism. Fragile X mental retardation type 1 (FMR1) gene premutation is the first single‐gene cause of primary ovarian failure (Fragile X‐associated primary ovarian insufficiency [FXPOI]) and one of the most common causes of ataxia (fragile X‐associated tremor/ataxia syndrome [FXTAS]), multiple additional phenotypes such as fibromyalgia, hypothyroidism, migraine headaches, sleep disturbances, sleep apnea, restless legs syndrome, central pain syndrome, neuropathy and neuropsychiatric alterations has been described. Clinical involvement in men and women carrying the FMR1 premutation currently constitutes a real health problem in the society that should be taken into account. It is important to highlight that while in FXS there is a loss‐of‐function of the FMR1 gene, in premutation associated disorders there is a gain of FMR1 mRNA function. To date, the tremendous progress achieved in the understanding of the pathophysiology of FXS, has led to the development of several targeted therapies aimed at preventing or improving the neurological manifestations of the disease. This review is an update of the diseases associated with the FMR1 gene.     

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.     

Fragile X syndrome due to a missense mutation

Fragile X syndrome is a common inherited form of intellectual disability and autism spectrum disorder. Most patients exhibit a massive CGG-repeat expansion mutation in the FMR1 gene that silences the locus. In over two decades since the discovery of FMR1, only a single missense mutation (p.(Ile304Asn)) has been reported as causing fragile X syndrome. Here we describe a 16-year-old male presenting with fragile X syndrome but without the repeat expansion mutation. Rather, we find a missense mutation, c.797G>A, that replaces glycine 266 with glutamic acid (p.(Gly266Glu)). The Gly266Glu FMR protein abolished many functional properties of the protein. This patient highlights the diagnostic utility of FMR1 sequencing.     

Standardization of PCR amplification for fragile X trinucleotide repeat measurements

To provide the clinical diagnostics community with accurate protocols and measurements for the detection of genetic disorders, we have established a quantitative measurement program for trinucleotide repeats associated with human disease. In this study, we have focused on the triplet repeat associated with fragile X syndrome. Five cell lines obtained from the Coriell Cell Repository were analyzed after polymerase chain reaction (PCR) amplification and size separation. These cell lines were reported to contain CGG repeat elements (ranging from 29 to 110 repeats). Our initial measurements focused on measurement variability: (a) between slab-PAGE and capillary (CE) separation systems (b) interlane variability (slab-PAGE) (c) intergel variability, and (d) variability associated with amplification. Samples were run in triplicate for all measurements, and the analysis performed using Gene Scan analysis software. The repeat sizes were verified by DNA sequence analyzes. The standard deviations for interlane measurements in slab-gels ranged from 0.05 to 0.35. There was also little variation in size measurements performed on different gels and among PCR amplifications. The CGG repeat measurements performed by capillary electrophoresis were more precise, with standard deviations ranging from 0.02 to 0.29. The slab-PAGE and CE size measurements were in agreement except for the pre-mutation alleles, which yielded significantly smaller sizes by CE.