Fragile X syndrome and the (CGG)n mutation: two families with discordant MZ twins.

The fragile X phenotype has been found, in the majority of cases, to be due to the expansion of a CGG repeat in the 5'-UTR region of the FMR-1 gene, accompanied by methylation of the adjacent CpG island and inactivation of the FMR-1 gene. Although several important aspects of the genetics of fragile X have been resolved, it remains to be elucidated at which stage in development the transition from the premutation to the full mutation occurs. We present two families in which discordance between two sets of MZ twins illustrates two important genetic points. In one family, two affected MZ brothers differed in the number of CGG repeats, demonstrating in vivo mitotic instability of this CGG repeat and suggesting that the transition to the full mutation occurred postzygotically. In the second family, two MZ sisters had the same number of repeats, but only one was mentally retarded. When the methylation status of the FMR-1 CpG island was studied, we found that the majority of normal chromosomes had been inactivated in the affected twin, thus leading to the expression of the fragile X phenotype.     

Diagnosis of fragile X syndrome by direct mutation analysis

A total of 27 fragile X pedigrees consisting of over 100 nuclear families were analyzed by Southern blotting methods and probes StB12.3 and StB12.3xx to detect the expansion of the (CGG) _n repeat within the FMR-1 gene and the abnormal methylation pattern of the adjacent DNA region responsible for the fragile X syndrome. Clinical expression was found to be associated with the presence of a full mutation ( > 500 bp, associated with abnormal methylation) in all the males and 50% of the females studied, whereas individuals carrying a premutation ( = 100–700 bp) were normal. A preferential size increase in the enlarged (CGG) _n repeat was detected in successive generations, the instability being stronger when transmitted from a female than from a male. No expansion of the premutation to the full mutation occurred in the paternal transmissions, and the size increase was significantly smaller than in the maternal transmissions. This could partly explain the stability of the premutation through several generations in families with transmitting males. In the maternal transmissions, the risk of expansion of a premutation to a full mutation appeared to depend on its size. The critical maternal premutation size leading invariably to the full mutation was between = 175–200 bp. This is important for genetic counseling and also explains the commonly observed clustering of affected individuals in fragile X families.     

Data on the CGG repeat at the fragile X site in the non-retarded Japanese population and family suggest the presence of a subgroup of normal alleles predisposing to mutate

The fragile X mutation is the result of amplification in the repeat number of p(CGG) _n in FMR-1; alleles with more than 52 repeats have been shown to be so unstable as to mutate in the repeat number in almost every transmission. To improve our understanding of mutations in normal alleles of FMR-1, the following studies were carried out in the Japanese population: a study on length variation in the repeat to determine the allele distribution of the repeat length in a non-retarded population, family studies to observe new mutations in normal allele, and haplotype analyses with microsatellite markers flanking the repeat to confirm estimated mutation rates and founder chromosomes in the fragile X syndrome. Analysis of the p(CGG) _n in 370 unrelated males detected 24 distinct alleles with repeats of 18–44. A comparison with previously reported data suggests the presence of racial/ethnic differences in the allele distribution. No premutation allele was found in 824 unrelated X chromosomes examined by the polymerase chain reaction and Southern blot analysis. Family studies detected one new mutation in a total of 303 meioses. However, the mutation rate was not in accordance with the expected or observed heterozygosities in the population or with linkage disequilibrium observed between the repeat numbers and the haplotypes of the markers flanking the CGG. The haplotype in the chromosome in which the new mutation was found was the same as that frequently found in the Japanese fragile X chromosomes, and the variance in the CGG repeat number was wider in chromosomes with the haplotypes frequently found in the fragile X chromosome than in those with the other haplotypes. These observations suggest that a subgroup is present in normal alleles and that this subgroup is more liable to mutate than others.     

Polymerase chain reaction analysis of fragile X mutations

Summary The mutation that underlies the fragile X syndrome is presumed to be a large expansion in the number of CGG repeats within the gene FMR-1. The unusually GC-rich composition of the expanded region has impeded attempts to amplify it by the polymerase chain reaction (PCR). We have developed a PCR protocol that successfully amplifies the (CGG)_n region in normal, carrier and affected individuals. The PCR analysis of several large fragile X families is presented. The PCR results agree with those obtained by direct genomic Southern blot analyses. These favorable comparisons suggest that the PCR assay may be suitable for rapid testing for fragile X mutations and premutations and genetic screening of at-risk individuals.     

Distribution of CGG repeat sizes within the fragile X mental retardation 1 (<Emphasis Type="Italic">FMR1</Emphasis>) homologue in a non-human primate population

Fragile X syndrome, the most common inherited form of mental retardation, arises in individuals with more than 200 CGG repeats in the 5 untranslated region of the fragile X mental retardation 1 (FMR1) gene. Although CGG repeat numbers comparable to those found in the normal human population are found in various non-human primates, neither the within-species size variation nor the propensity for expansion of the CGG repeat has been described for any non-human primate species. The allele distribution has now been determined for FMR1 (homologue) CGG repeats of 265 unrelated founder females of Macaca mulatta monkeys. Among 530 X chromosomes, at least 26 distinct repeat lengths were identified, ranging from 16 to 54 CGG repeats. Of these alleles 79% have between 25 and 33 CGG repeats. Detailed examination of the CGG region revealed a conserved G (CGG)₂ G interruption, although in no case was an AGG trinucleotide detected. Two animals carried borderline premutation alleles with 54 CGG repeats, within the region of marginal instability for humans. Thus, M. mulatta may be useful as an animal model for the study of fragile X syndrome.     

Stability of triplet repeats of myotonic dystrophy and fragile X loci in human mutator mismatch repair cell lines

At least nine human genetic diseases, including myotonic dystrophy (DM) and fragile X syndrome have been associated with the expansion of CTG or CGG trinucleotide repeats within the disease loci. Little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. Mutations in human mismatch repair genes are associated with the increased polymorphism of many microsatellites, including dinucleotide repeats. The effect of mutations in two mismatch repair genes on the size of trinucleotide repeats in the DM and FRAXA loci has been analyzed. PCR and Southern analysis of the triplet repeat regions of the DM and fragile X mental retardation (FRAXA) loci in cell lines HTC116 and LoVo, which contain mutations in both alleles of the hMLH1 and hMSH2 genes, respectively, indicated that the size of the endogenous (CTG)_n and (CGG)_n tracts fall within the range observed in the normal population. This suggests that mutations in hMLH1 or hMSH2 do not result in the instability of CTG or CGG tracts to the levels observed in individuals with myotonic dystrophy or fragile X syndrome. Received: 4 December 1995 / Revised: 29 January 1996, 7 March 1996     

Molecular diagnosis of the fragile X and FRAXE syndromes in patients with mental retardation of unknown cause in Mexico

The fragile X syndrome (Fra-X) is the most common cause of inherited mental retardation with X-linked semi-dominant inheritance. The prevalence of Fra-X in the Mexican population is unknown. The aim of this population screening study was to determine if Fra-X or FRAXE mutations are the cause of a number of cases of mental retardation in a sample of Mexican children with mental retardation of unknown cause (MRUC) and to stress the importance of performing molecular analysis of the FMR-1 gene in all patients with MRUC. We report here the direct analysis of CGG and GCC repeats within the FMR-1 and FMR-2 genes, respectively, in 62 unrelated patients with MRUC. Two male index cases had the CGG expansion, although they did not express the Xq27.3 fragile site cytogenetically. Fra-X diagnosis was highly suspected on a clinical basis in one of the patients, but not in the other. Both mothers were found to be premutation carriers. The molecular studies of FMR-1 showed that the proportion of MRUC patients with Fra-X is 3.2%. This frequency was not significantly different to that reported in most populations. As reported in other series, no patients with FRAXE were found in our sample. Our findings confirm that the molecular analysis of the FMR-1 gene is necessary in MRUC patients to achieve unequivocal diagnosis of fragile X syndrome, carrier premutation detection and for accurate genetic counseling.     

Apparent regression of the CGG repeat in FMR1 to an allele of normal size

The fragile X syndrome is the result of amplification of a CGG trinucleotide repeat in the FMR1 gene and anticipation in this disease is caused by an intergenerational expansion of this repeat. Although regression of a CGG repeat in the premutation range is not uncommon, regression from a full premutation (>200 repeats) or premutation range (50–200 repeats) to a repeat of normal size (<50 repeats) has not yet been documented. We present here a family in which the number of repeats apparently regressed from approximately 110 in the mother to 44 in her daughter. Although the CGG repeat of the daughter is in the normal range, she is a carrier of the fragile X mutation based upon the segregation pattern of Xq27 markers flanking FMR1. It is unclear, however, whether this allele of 44 repeats will be stably transmitted, as the daughter has as yet no progeny. Nevertheless, the size range between normal alleles and premutation alleles overlap, a factor that complicates genetic counseling.     

The fragile X syndrome in Finland: demonstration of a founder effect by analysis of microsatellite haplotypes

Microsatellite markers RS46 (DXS548) and FRAXAC2 flanking the fragile X mutation, an expansion of a (CGG)_n repeat within the FMR-1 gene, were typed in 60 unrelated northern and eastern Finnish fragile X families and in a control population from the same geographical region. A significant difference was found in allelic and haplotypic distributions between the normal X and fragile X chromosomes. Evidence for a strong founder effect was detected, with the haplotype 196-153 being present on 80% of the fragile X chromosomes, but on only 8% of the normal X chromosomes. In addition to this major haplotype, four minor haplotypes were found on the fragile X chromosomes. These results suggest that the majority of present-day fragile X mutations in Finland may have a common initial ancestor, probably from the 16th century.     

Molecular analysis of mutations in the gene FMR-1 segregating in fragile X families

Molecular genetic analysis of the transmission of mutations in 73 families with fragile X (one of the largest samples evaluated so far) has confirmed previous hypotheses that the fragile X syndrome results from two consecutive mutational steps, designated premutation and full fragile X mutation. These mutations give rise to expansions of restriction fragments, most probably by amplification of the FMR-1 CGG repeat. Premutations are identified by small expansions that apparently have no effect on either the clinical or the cellular phenotype. Full mutations are reflected by large expansions and hypermethylation of the expanded gene region. All males showing large expansions were affected. Individuals with full mutations also expressed the fragile X, with only one exception. An affected mosaic male, showing a predominance of premutated fragments in his leukocytes, was shown to be fragile-X-negative on different occasions. About 50% of heterozygotes with full mutations were reported by clinicians to be mentally retarded. Conversion of the premutation to the full mutation may occur at oogenesis, as previously suggested, or after formation of a zygote at an early transitional stage in development when the CGG repeat behaves as a mitotically unstable element on maternally derived/imprinted X chromosomes carrying a premutation of sufficient repeat length.     

Analysis of a CGG sequence at the FMR-1 locus in fragile X families and in the general population.

In this study, we have characterized a CGG repeat at the FMR-1 locus in more than 100 families (more than 500 individuals) presenting for fragile X testing and in 247 individuals from the general population. Both Southern blot and PCR-based assays were evaluated for their ability to detect premutations, full mutations, and variability in normal allele sizes. Among the Southern blot assays, the probes Ox1.9 or StB12.3 with a double restriction-enzyme digest were the most sensitive in detecting both small and large amplifications and, in addition, provided information on methylation of an adjacent CpG island. In the PCR-based assays, analysis of PCR products on denaturing DNA sequencing gels allowed the most accurate determination of CGG repeat number up to approximately 130 repeats. A combination of a Southern blot assay with a double digest and the PCR-sequencing-gel assay detected the spectrum of amplification-type mutations at the FMR-1 locus. In the patient population, a CGG repeat of 51 was the largest to be stably inherited, and a repeat of 57 was the smallest size of premutation to be unstably inherited. When premutations were transmitted by females, the size of repeat correlated with risk of expansion to a full mutation in the next generation. Full mutations (large repeats typically associated with an abnormal methylation pattern and mitotic instability) were associated with clinical and cytogenetic manifestations in males but not necessarily in females. In the control population, the CGG repeat ranged from 13 to 61, but 94% of alleles had fewer than 40 repeats. The most frequent allele (34%) was a repeat of 30. One female had an allele (61 repeats) within a range consistent with fragile X premutations, while two other individuals each had a repeat of 52. This suggests that the frequency of unstable alleles in the general population may be approximately 1%.     

Molecular Analysis and Test of Linkage between the FMR-1 Gene and Infantile Autism in Multiplex Families

Approximately 2%–5% of autistic children show cytogenetic evidence of the fragile X syndrome. This report tests whether infantile autism in multiplex autism families arises from an unusual manifestion of the fragile X syndrome. This could arise either by expansion of the (CGG)n trinucleotide repeat in FMR-1 or from a mutation elsewhere in the gene. We studied 35 families that met stringent criteria for multiplex autism. Amplification of the trinucleotide repeat and analysis of methylation status were performed in 79 autistic children and in 31 of their unaffected siblings, by Southern blot analysis. No examples of amplified repeats were seen in the autistic or control children or in their parents or grandparents. We next examined the hypothesis that there was a mutation elsewhere in the FMR-1 gene, by linkage analysis in 32 of these families. We tested four different dominant models and a recessive model. Linkage to FMR-1 could be excluded (lod score between −24 and −62) in all models by using probes DXS548, FRAXAC1, and FRAXAC2 and the CGG repeat itself. Tests for heterogeneity in this sample were negative, and the occurrence of positive lod scores in this data set could be attributed to chance. Analysis of the data by the affected-sib method also did not show evidence for linkage of any marker to autism. These results enable us to reject the hypothesis that multiplex autism arises from expansion of the (CGG)n trinucleotide repeat in FMR-1. Further, because the overall lod scores for all probes in all models tested were highly negative, linkage to FMR-1 can also be ruled out in multiplex autistic families.     

应用多聚酶链式反应快速分析FMR-1基因突变

为了建立一种在先天性智力低下患儿中快速分析脆性X综合征智力低下基因1(Fragile X mental retardation gene 1.FMR-1)突变的方法,对先天性智力低下儿童进行脆性X综合征的大面积筛查和诊断,应用复式多聚酶链式反应一次性扩增FMR-1基因的(CGG)n的重复区,分析CGG重复序列的大小,判断FMR-1基因状态(正常、突变前、突变后),对脆性X综合征可疑患儿快速筛查,在113倒不明原因的先天性智力低下患儿中,分析有脆性X综合征携带者(FMR-1基因前突变者)7例(2男5女),脆性X综合征患者(FMR-1基因突变者)5例,应用多聚酶链式反应可以对脆性X综合征可疑患儿进行大面积初筛,确定携带者和患者。     

Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection

Molecular genetic analysis of individuals from 6 Egyptian and 33 German families with fragile X syndrome and 240 further patients with mental retardation was performed applying a completely non-radioactive system. The aim of our study was the development of a non-radioactive detection method and its implementation in molecular diagnosis of the fragile X syndrome. Furthermore, we wanted to assess differences in the mutation sizes between Egyptian and German patients and between Egyptian and German carriers of a premutation. Using non-radioactive polymerase chain reaction (PCR), agarose gel electrophoresis and blotting of the PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)₅ and chemiluminescent detection, we identified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of the CGG repeat between the Egyptian and German patients and carriers, respectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whereas Southern blot analysis with the digoxigenin labelled probe StB 12.3 revealed presence of a full mutation. Our newly established nonradioactive genomic blotting method is based on the conventional radioactive Southern blot analysis. Labelling of the probe StB 12.3 with digoxigenin via PCR allowed the detection of normal, premutated and fully mutated alleles. For exact sizing of small premutated or large normal alleles, we separated digoxigenin labelled PCR products through denaturing poly-acrylamide gelelectrophoresis (PAGE) and transfered them to a nylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody. The number of trinucleotide repeat units can be determined by scoring the detected bands against a digoxigenated M13 sequencing ladder. Our newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detection of full fragile X mutations and premutations.     

Interruption of the fragile X syndrome expanded sequence d(CGG)(n) by interspersed d(AGG) trinucleotides diminishes the formation and stability of d(CGG)(n) tetrahelical structures

Fragile X syndrome is caused by expansion of a d(CGG) trinucleotide repeat sequence in the 5′ untranslated region of the first exon of the FMR1 gene. Repeat expansion is thought to be instigated by formation of d(CGG)_n secondary structures. Stable FMR1 d(CGG)_n runs in normal individuals consist of 6–52 d(CGG) repeats that are interrupted every 9–11 triplets by a single d(AGG) trinucleotide. By contrast, individuals having fragile X syndrome premutation or full mutation present >54–200 or >200–2000 monotonous d(CGG) repeats, respectively. Here we show that the presence of interspersed d(AGG) triplets diminished in vitro formation of bimolecular tetrahelical structures of d(CGG)₁₈ oligomers. Tetraplex structures formed by d(CGG)_n oligomers containing d(AGG) interspersions had lower thermal stability. In addition, tetraplex structures of d(CGG)₁₈ oligomers interspersed by d(AGG) triplets were unwound by human Werner syndrome DNA helicase at rates and to an extent that exceeded the unwinding of tetraplex form consisting of monotonous d(CGG)₁₈. Diminished formation and stability of tetraplex structures of d(AGG)-containing FMR1 d(CGG)_2–50 tracts might restrict their expansion in normal individuals.     

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     

Population-based carrier screening and prenatal diagnosis of fragile X syndrome in East Asian populations

Identification of carriers of fragile X syndrome (FXS) with the subsequent prenatal diagnosis and knowledge of FXS-associated genetic profiles are essential for intervention in specific populations. We report the results of carrier screening of 39,458 East Asian adult women and prenatal diagnosis from 87 FXS carriers. The prevalence of FXS carriers and full mutation fetuses was estimated to be 1/581 and 1/3124 in East Asian populations, respectively. We confirmed the validity of the current threshold of CGG trinucleotide repeats for FMR1 categorization; the integral risks of full mutation expansion were approximately 6.0%, 43.8%, and 100% for premutation alleles with 55–74, 75–89, and ≥ 90 CGG repeats, respectively. The protective effect of AGG (adenine-guanine-guanine nucleotides) interruption in East Asian populations was validated, which is important in protecting premutation alleles with 75–89 CGG repeats from full mutation expansion. Finally, family history was shown not an effective indicator for FXS carrier screening in East Asian populations, and population-based screening was more cost-effective. This study provides an insight into the largest carrier screening and prenatal diagnosis for FXS in East Asian populations to date. The FXS-associated genetic profiles of East Asian populations are delineated, and population-based carrier screening is shown to be promising for FXS intervention.     

The guanine-rich fragile X chromosome repeats are reluctant to form tetraplexes

Using circular dichroism spectroscopy, UV absorption spectroscopy and polyacrylamide gel electrophoresis, we studied conformational properties of guanine-rich DNA strands of the fragile X chromosome repeats d(GGC)_n, d(GCG)_n and d(CGG)_n, with n = 2, 4, 8 and 16. These strands are generally considered in the literature to form guanine tetraplexes responsible for the repeat expansion. However, we show in this paper that the repeats are reluctant to form tetraplexes. At physiological concentrations of either Na⁺ or K⁺ ions, the hexamers and dodecamers associate to form homoduplexes and the longer repeats generate homoduplexes and hairpins. The tetraplexes are rarely observed being relatively most stable with d(GGC)_n and least stable with d(GCG)_n. The tetraplexes are exclusively formed in the presence of K⁺ ions, at salt concentrations higher than physiological, more easily at higher than physiological temperatures, and they arise with extremely long kinetics (even days). Moreover, the capability to form tetraplexes sharply diminishes with the oligonucleotide length. These facts make the concept of the tetraplex appearance in this motif in vivo very improbable. Rather, a hairpin of the fragile X repeats, whose stability increases with the repeat length, is the probable structure responsible for the repeat expansion in genomes.