Method for the molecular cytogenetic visualization of fragile site FRAXA

Fragile X syndrome is one of the most common reasons for human hereditary mental retardation. It is associated with the expansion of CGG repeats in the 5'-untranslated region of the FMR1 gene, which results in the suppression of its expression and the development of the disease. At present, methods based on PCR and Southern blot analysis are used for diagnostics of the fragile X syndrome. The presence of a fragile site FRAXA on the X chromosome is typical for patients with this pathology. We developed a method of visualizing this site in cell cultures obtained from patients using the fluorescent in situ hybridization (FISH) and the combination of two probes. The method allows one to detect five types of signals on the X chromosome, three of which are normal, while two are associated with the emergence of fragile site FRAXA. An analysis of the distribution of all signal types in cell lines from healthy individuals and patients with fragile X syndrome demonstrated that the method allows one to determine differences between lines with a high statistical significance and that it is applicable to detecting cells that are carriers of the syndrome.     

A survey of fragile X syndrome in a sample from Spanish Basque country

Fragile X syndrome is the most common inherited form of mental retardation. The syndrome is associated with a CGG repeat expansion in the 5'UTR of the first exon of the FMR1 gene. This gene maps to Xq27.3 and coincides with the cytogenetic fragile site (FRAXA). The present study deals with the prevalence of fragile X syndrome among individuals with mental retardation of unknown cause from institutions and special schools from the Spanish Basque Country. Results of cytogenetic and molecular studies, performed in a group of 134 unrelated individuals (92 males and 42 females) are presented. The cytogenetic marker at Xq27.3 was identified in 12 patients. Other chromosomal abnormalities were found in two cases that this and previous studies confirmed as Angelman and Prader-Willi syndromes. Two males, in whom the cytogenetic marker was identified, were found negative for FRAXA and FRAXE expansion at the molecular level. The present study shows that the frequency of the FRAXA full mutation in individuals of Spanish non-Basque origin is in the range of other Spanish populations. In the sample of Spanish Basque origin we have not found cytogenetic FRAXA site expression, and the CGG repeat size of FMR1 gene is in the normal range. The significance of these results are discussed.     

The fragile X syndromes

Fragile X syndrome is a leading cause of mental retardation worldwide, with an incidence of approximately one case in 2000 live births. It is amongst the most common of human genetic diseases, and was the first to be associated with an unstable trinucleotide (CGG) repeat sequence. It is also characterized by a chromosomal fragile site which was the first of (now) four such sites to be identified at the molecular level. Each shows very similar features suggesting that other representatives of this type of fragile site will likely involve similar sequences. As with the other unstable trinucleotide repeats, the sequence at the fragile X locus is found to be remarkably unstable upon genetic transmission, however many features differ from the other repeats. As repeat expansion at the fragile X locus results in loss of expression of the co-resident FMR1 gene, the basis for clinical features is best understood in this disorder. Two additional fragile sites in the vicinity have been identified, and at least one of these is associated with mental retardation.     

An origin of DNA replication in the promoter region of the human fragile X mental retardation (FMR1) gene

Fragile X syndrome, the most common form of inherited mental retardation in males, arises when the normally stable 5 to 50 CGG repeats in the 5' untranslated region of the fragile X mental retardation protein 1 (FMR1) gene expand to over 200, leading to DNA methylation and silencing of the FMR1 promoter. Although the events that trigger local CGG expansion remain unknown, the stability of trinucleotide repeat tracts is affected by their position relative to an origin of DNA replication in model systems. Origins of DNA replication in the FMR1 locus have not yet been described. Here, we report an origin of replication adjacent to the FMR1 promoter and CGG repeats that was identified by scanning a 35-kb region. Prereplication proteins Orc3p and Mcm4p bind to chromatin in the FMR1 initiation region in vivo. The position of the FMR1 origin relative to the CGG repeats is consistent with a role in repeat maintenance. The FMR1 origin is active in transformed cell lines, fibroblasts from healthy individuals, fibroblasts from patients with fragile X syndrome, and fetal cells as early as 8 weeks old. The potential role of the FMR1 origin in CGG tract instability is discussed.     

Instability of the CGG repeat and expression of the FMR1 protein in a male fragile X patient with a lung tumor.

The molecular mechanism of the fragile X syndrome is based on the expansion of an CGG repeat in the 5' UTR of the FMR1 gene in the majority of fragile X patients. This repeat displays instability both between individuals and within an individual. We studied the instability of the CGG repeat and the expression of the FMR1 protein (FMRP) in several different tissues derived from a male fragile X patient. Using Southern blot analysis, only a full mutation is detected in 9 of the 11 tissues tested. The lung tumor contains a methylated premutation of 160 repeats, whereas in the testis, besides the full mutation, a premutation of 60 CGG repeats is detected. Immunohistochemistry of the testis revealed expression of FMR1 in the spermatogonia only, confirming the previous finding that, in the sperm cells of fragile X patients with a full mutation in their blood cells, only a premutation is present. Immunohistochemistry of brain and lung tissue revealed that 1% of the cells are expressing the FMRP. PCR analysis demonstrated the presence of a premutation of 160 repeats in these FMR1-expressing cells. This indicates that the tumor was derived from a lung cell containing a premutation. Remarkably, despite the methylation of the EagI and BssHII sites, FMRP expression is detected in the tumor. Methylation of both restriction sites has thus far resulted in a 100% correlation with the lack of FMR1 expression, but the results found in the tumor suggest that the CpGs in these restriction sites are not essential for regulation of FMR1 expression. This indicates a need for a more accurate study of the exact promoter of FMR1.     

Screening for FXTAS in 95 Spanish patients negative for Huntington disease

Fragile X syndrome is the most common form of hereditary mental retardation. The molecular basis of this syndrome is mainly a CGG expansion in the 5' untranslated region of the FMR1 gene. Expansions with more than 200 CGG repeats abolish gene expression causing the classical fragile X phenotype. Premutation carriers (55-200 CGG) have normal cognitive function with increased risk of developing premature ovarian failure and fragile X-associated tremor-ataxia syndrome (FXTAS). Some clinical features associated with FXTAS, such as tremor, gait ataxia, cognitive decline, and generalized brain atrophy, are also seen in other movement disorders. Ninety-five patients referred for HD, who tested negative for the expansion in the IT15 gene, were screened for FMR1 CGG-repeat expansion. One FMR1 premutation male carrier was detected, giving an FXTAS frequency of 1.6%. Our results highlight that FXTAS is still not well diagnosed; therefore, we recommend FMR1 premutation screenings in all patients with late-onset tremor, ataxia, and cognitive dysfunction.     

A fragile X case with an amplification/deletion mosaic pattern

Fragile X syndrome is the most common cause of hereditary mental retardation. The FMR1 gene, which is involved in fragile X syndrome, contains a polymorphic CGG repeat, which expands in affected patients. Expanding triplet repeats have been shown to be a new type of mutation, termed "dynamic mutation", responsible for more than 12 genetic diseases. These mutations occur as multiple steps rather than as a single event. The first step leads to an unstable allele that then becomes increasingly unstable generally achieving further increases in copy or occasionally contraction. In this report, we describe a fragile X boy with both a hypermethylated full mutation and a deletion of 905 bp encompassing the CGG repeat. The upstream breakpoint is 438 bp 5' to the CGG repeat and the downstream breakpoint is 420 bp 3' of the triplet repeats. The deletion includes the ATG starting codon for translation of the FMR1 gene. This was confirmed by using FMRP immunocytochemistry both on blood smears and hair roots. The deleted region is flanked by a ccgg direct repeat next to the breakpoints; this may have had a critical role in the formation of a secondary DNA structure leading to the deletion.     

Screening for FMR1 and FMR2 mutations in 222 individuals from Spanish special schools: identification of a case of FRAXE-associated mental retardation

Fragile X syndrome is the most common inherited form of familial mental retardation. It results from a (CGG) _n trinucleotide expansion in the FMR1 gene leading to the typical Martin-Bell phenotype. Clinical features vary depending on age and sex. Expansion of a (CCG) _n repeat in the FMR2 gene corresponds to the FRAXE fragile site which lies distal to FRAXA and is also associated with mental retardation, but it is less frequent and lacks a consistent phenotype. Analysis of repeat expansions in these two genes allows the molecular diagnosis of these different entities. We report here the screening of the FRAXA and FRAXE mutations in 222 unrelated mentally retarded individuals attending Spanish special schools. PCR and/or Southern blotting methods were used. We detected full mutations in the FMR1 gene in 11 boys (4.9%) and 1 boy (0.5%) with a CCG repeat expansion in the FMR2 gene. The latter shows mild mental retardation with psychotic behaviour and no remarkable physical traits. Molecular studies revealed a mosaicism for methylation in the FMR2 gene. This case supports the observation that expansions greater than 100 repeats can be partially methylated and cause the phenotype. Received: 11 February 1997 / Accepted: 9 June 1997     

Methylation mosaicism of 5'-(CGG)(n)-3' repeats in fragile X, premutation and normal individuals

Fragile X syndrome (FRAXA) is characterized at the molecular level by an expansion of a naturally occurring 5′-(CGG)_n-3′ repeat in the promoter and 5′-untranslated region (5′-UTR) of the fragile X mental retardation (FMR1) gene on human chromosome Xq27.3. When expanded, this region is usually hypermethylated. Inactivation of the FMR1 promoter and absence of the FMR1 protein are the likely cause of the syndrome. By using the bisulfite protocol of the genomic sequencing method, we have determined the methylation patterns in this region on single chromosomes of healthy individuals and of selected premutation carriers and FRAXA patients. In control experiments with unmethylated or M-SssI-premethylated DNAs, this protocol has been ascertained to reliably detect all cytidines or 5-methylcytidines as unmethylated or methylated nucleotides, respectively. Analyses of the DNA from FRAXA patients reveal considerable variability in the lengths of the 5′-(CGG)_n-3′ repeats and in the levels of methylation in the repeat and the 5′-UTR. In one patient (OEl) with high repeat length heterogeneity (n = 15 to >200), shorter repeats (n = 20–80) were methylated or unmethylated, longer repeats (n = 100–150) were often completely methylated, but one repeat with n = 160 proved to be completely unmethylated. This type of methylation mosaicism was observed in several FRAXA patients. In healthy females, methylated 5′-CG-3′ sequences were found in some repeats and 5′-UTRs, as expected for the sequences from one of the X chromosomes. The natural FMR1 promoter is methylation sensitive, as demonstrated by the loss of activity in transfection experiments using the unmethylated or M-SssI-premethylated FMR1 promoter fused to the luciferase gene as an activity indicator.     

Analysis of the Fragile X Trinucleotide Repeat in Basques: Association of Premutation and Intermediate Sizes,Anchoring AGGs and Linked Microsatellites with Unstable Alleles

Fragile X Syndrome (FXS) is associated with an unstable CGG repeat sequence in the 5’ untranslated region in the first exon of the FMR1 gene which resides at chromosome position Xq27.3 and is coincident with the fragile site FRAXA. The CGG sequence is polymorphic with respect to size and purity of the repeat. Interpopulation variation in the polymorphism of the FMR1 gene and consequently, in the predisposition to FXS due to the prevalence of certain unstable alleles has been observed. Spanish Basque population is distributed among narrow valleys in northeastern Spain with little migration between them until recently. This characteristic may have had an effect on allelic frequency distributions. We had previously reported preliminary data on the existence of FMR1 allele differences between two Basque valleys (Markina and Arratia). In the present work we extended the study to Uribe, Gernika, Durango, Goierri and Larraun, another five isolated valleys enclosing the whole area within the Spanish Basque region. We analyzed the prevalence of FMR1 premutated and intermediate/grey zone alleles. With the aim to complete the previous investigation about the stability of the Fragile X CGG repeat in Basque valleys, we also analyzed the existence of potentially unstable alleles, not only in relation with size and purity of CGG repeat but also in relation with DXS548 and FRAXAC1 haplotypes implicated in repeat instability. The data show that differences in allele frequencies as well as in the distribution of the mutational pathways previously identified are present among Basques. The data also suggest that compared with the analyzed Basque valleys, Gernika had increased frequency of susceptibility to instability alleles, although the prevalence of premutation and intermediate/grey zone alleles in all the analyzed valleys was lower than that reported in Caucasian populations.Key Words: Fragile X syndrome, FMR1 gene, CGG repeat, FRAXAC1, DXS548, basque country.     

Haplotype study of intermediate-length alleles at the fragile X (FMR1) gene: ATL1, FMRb, and microsatellite haplotypes differ from those found in common-size FMR1 alleles

The CGG repeat within the X-chromosome-linked FMR1 gene, which in hyperexpansion (> 200 copies) results in fragile X syndrome, is highly polymorphic. The mechanism of expansion is not well understood, but CGG repeats called intermediate-length or gray zone alleles (approximately equal 35-60 repeats) are thought to make up the FMR1 alleles showing initial steps in this expansion process. It has been hypothesized that the background haplotype of these alleles plays a role in their susceptibility to expansion. In this study we investigate whether or not the frequencies of alleles and haplotypes at four marker loci in the FMR1 gene region (microsatellites DXS548 and FRAXAC1 and SNPs ATL1 and FMRb) in 84 intermediate-length male chromosomes differ from those in 94 common-size male alleles. The ATL1*G and FMRB*A alleles were more frequent among intermediate-length alleles than among common alleles. In addition, the DXS548-FRAXAC1 T50-T42 and T40-T42 haplotypes were strongly associated with intermediate-length alleles between 41 and 60 CGG repeats (p < 0.001). Two extended haplotypes, DXS548-FRAXAC1-ATL1-FMRb T50-T42-G-A and T40-T42-G-A, are strongly associated (p < 0.001) with intermediate-length alleles between 41 and 60 CGG repeats, and these haplotypes have also been reported as fragile X associated haplotypes in European populations. These data suggest that these haplotypes are among the most susceptible to further expansion among the intermediate-length alleles. T50-T42-G-A was also much more prevalent in males with 35-40 CGG repeats than in males with common-size alleles. ATL1 did not increase discrimination among intermediate-length alleles beyond that detected by DXS548-FRAXAC1 haplotypes, but the FMRb locus did, particularly for the DXS548-FRAXAC1-ATL1 T50-T42-G and T40-T42-G haplotypes. Comparison with fragile X associated haplotypes, from the literature, suggests that repeat hyperexpansion occurs most frequently on chromosomes carrying FMRB*A. Within the intermediate-length allele category, however, there were some significant differences in haplotype frequencies between smaller and larger alleles, and this finding has implications for future studies.