Expansion of the fragile X CGG repeat in females with premutation or intermediate alleles

The CGG repeat in the 5' untranslated region of the fragile X mental retardation 1 gene (FMR1) exhibits remarkable instability upon transmission from mothers with premutation alleles. A collaboration of 13 laboratories in eight countries was established to examine four issues concerning FMR1 CGG-repeat instability among females with premutation (approximately 55-200 repeats) and intermediate (approximately 46-60 repeats) alleles. Our central findings were as follows: (1) The smallest premutation alleles that expanded to a full mutation (>200 repeats) in one generation contained 59 repeats; sequence analysis of the 59-repeat alleles from these two females revealed no AGG interruptions within the FMR1 CGG repeat. (2) When we corrected for ascertainment and recalculated the risks of expansion to a full mutation, we found that the risks for premutation alleles with <100 repeats were lower than those previously published. (3) When we examined the possible influence of sex of offspring on transmission of a full mutation-by analysis of 567 prenatal fragile X studies of 448 mothers with premutation and full-mutation alleles-we found no significant differences in the proportion of full-mutation alleles in male or female fetuses. (4) When we examined 136 transmissions of intermediate alleles from 92 mothers with no family history of fragile X, we found that, in contrast to the instability observed in families with fragile X, most (99/136 [72.8%]) transmissions of intermediate alleles were stable. The unstable transmissions (37/136 [27.2%]) in these families included both expansions and contractions in repeat size. The instability increased with the larger intermediate alleles (19% for 49-54 repeats, 30.9% for 55-59, and 80% for 60-65 repeats). These studies should allow improved risk assessments for genetic counseling of women with premutation or intermediate-size alleles.     

The fragile X premutation: into the phenotypic fold

Premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 gene (FMR1) are known to contribute to the fragile X phenotype through genetic instability and transmission of full mutation alleles (>200 repeats). There is now mounting evidence that the premutation alleles themselves contribute to clinical involvement, including premature ovarian failure among female carriers and a new tremor/ataxia syndrome among older male carriers. Recent observations also provide direct evidence of dysregulation of the FMR1 gene in the premutation range, which may explain many of the clinical observations.     

Decrease in the CGGn trinucleotide repeat mutation of the fragile X syndrome to normal size range during paternal transmission.

The fragile X syndrome, the most common inherited form of mental retardation, is caused by the expansion of a CGGn trinucleotide repeat in the FMR-1 gene. Although the repeat number usually increases during transmission, few cases with reduction of an expanded CGGn repeat back to the normal size range have been reported. We describe for the first time a family in which such reduction has occurred in the paternal transmission. The paternal premutation (delta = 300 bp) was not detected in one of the five daughters or in the son of this daughter, although he had the grandpaternal RFLP haplotype. Instead, fragments indicating the normal CGGn repeat size were seen on a Southern blot probed with StB12.3. PCR analysis of the CGGn repeat confirmed this; in addition to a maternal allele of 30 repeats, an allele of 34 repeats was detected in the daughter and, further, in her son. Sequencing of this new allele revealed a pure CGGn repeat configuration without AGG interruptions. No evidence for a somatic mosaicism of a premutation allele in the daughter or a normal allele in her father was detected when investigating DNA derived from blood lymphocytes and skin fibroblasts. Another unusual finding in this family was lack of the PCR product of the microsatellite marker RS46 (DXS548) in one of the grandmaternal X chromosomes, detected as incompatible inheritance of RS46 alleles. The results suggest an intergenerational reduction in the CGGn repeat from premutation size to the normal size range and stable transmission of the contracted repeat to the next generation. However, paternal germ-line mosaicism could not be excluded as an alternative explanation for the reverse mutation.     

Prevalence of carriers of premutation-size alleles of the FMRI gene--and implications for the population genetics of the fragile X syndrome.

The fragile X syndrome is the second leading cause of mental retardation after Down syndrome. Fragile X premutations are not associated with any clinical phenotype but are at high risk of expanding to full mutations causing the disease when they are transmitted by a carrier woman. There is no reliable estimate of the prevalence of women who are carriers of fragile X premutations. We have screened 10,624 unselected women by Southern blot for the presence of FMR1 premutation alleles and have confirmed their size by PCR analysis. We found 41 carriers of alleles with 55-101 CGG repeats, a prevalence of 1/259 women (95% confidence interval 1/373-1/198). Thirty percent of these alleles carry an inferred haplotype that corresponds to the most frequent haplotype found in fragile X males and may indeed constitute premutations associated with a significant risk of expansion on transmission by carrier women. We identified another inferred haplotype that is rare in both normal and fragile X chromosomes but that is present on 13 (57%) of 23 chromosomes carrying FMR1 alleles with 53-64 CGG repeats. This suggests either (1) that this haplotype may be stable or (2) that the associated premutation-size alleles have not yet reached equilibrium in this population and that the incidence of fragile X syndrome may increase in the future.     

Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.

Fragile X syndrome results from mutations in a (CGG)n repeat found in the coding sequence of the FMR-1 gene. Analysis of length variation in this region in normal individuals shows a range of allele sizes varying from a low of 6 to a high of 54 repeats. Premutations showing no phenotypic effect in fragile X families range in size from 52 to over 200 repeats. All alleles with greater than 52 repeats, including those identified in a normal family, are meiotically unstable with a mutation frequency of one, while 75 meioses of alleles of 46 repeats and below have shown no mutation. Premutation alleles are also mitotically unstable as mosaicism is observed. The risk of expansion during oogenesis to the full mutation associated with mental retardation increases with the number of repeats, and this variation in risk accounts for the Sherman paradox.     

CTG trinucleotide repeat "big jumps": large expansions, small mice

Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including fragile X mental retardation, Huntington disease, and myotonic dystrophy type 1. Disease severity and age of onset are critically linked to expansion size. Previous mouse models of repeat instability have not recreated large intergenerational expansions ("big jumps"), observed when the repeat is transmitted from one generation to the next, and have never attained the very large tract lengths possible in humans. Here, we describe dramatic intergenerational CTG*CAG repeat expansions of several hundred repeats in a transgenic mouse model of myotonic dystrophy type 1, resulting in increasingly severe phenotypic and molecular abnormalities. Homozygous mice carrying over 700 trinucleotide repeats on both alleles display severely reduced body size and splicing abnormalities, notably in the central nervous system. Our findings demonstrate that large intergenerational trinucleotide repeat expansions can be recreated in mice, and endorse the use of transgenic mouse models to refine our understanding of triplet repeat expansion and the resulting pathogenesis.     

Molecular genetics of the fragile-X syndrome: a novel type of unstable mutation.

Fragile-X syndrome, the most common inherited form of mental retardation, has a very unusual mode of inheritance. The disease is caused by a multistep expansion, in successive generations, of a polymorphic CGG repeat localized in a 5' exon of FMR-1, a gene of unknown function. Two main mutation types have been categorized. Premutations are moderate expansions of the repeat and do not cause mental retardation. Full mutations are found in affected individuals and involve larger expansions of the repeat, with abnormal methylation of the neighboring CpG island. The full mutations demonstrate striking somatic instability and extinguish expression of FMR-1. Premutations are changed to full mutation only when transmitted by a female with a frequency that increases up to 100% as a function of the initial size of the premutation. Direct detection of the mutations provides an accurate test for pre- and postnatal diagnosis of the disease, and for carrier detection. A similar unstable expansion of a trinucleotide repeat occurs in myotonic dystrophy.     

Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation

The human FMR1 gene contains a CGG repeat in its 5' untranslated region. The repeat length in the normal population is polymorphic (5-55 CGG repeats). Lengths beyond 200 CGGs (full mutation) result in the absence of the FMR1 gene product, FMRP, through abnormal methylation and gene silencing. This causes Fragile X syndrome, the most common inherited form of mental retardation. Elderly carriers of the premutation, defined as a repeat length between 55 and 200 CGGs, can develop a progressive neurodegenerative syndrome: Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS, FMR1 mRNA levels are elevated and it has been hypothesised that FXTAS is caused by a pathogenic RNA gain-of-function mechanism. We have developed a knock in mouse model carrying an expanded CGG repeat (98 repeats), which shows repeat instability and displays biochemical, phenotypic and neuropathological characteristics of FXTAS. Here, we report further repeat instability, up to 230 CGGs. An expansion bias was observed, with the largest expansion being 43 CGG units and the largest contraction 80 CGG repeats. In humans, this length would be considered a full mutation and would be expected to result in gene silencing. Mice carrying long repeats ( approximately 230 CGGs) display elevated mRNA levels and decreased FMRP levels, but absence of abnormal methylation, suggesting that modelling the Fragile X full mutation in mice requires additional repeats or other genetic manipulation.     

CTG Trinucleotide Repeat “Big Jumps”: Large Expansions, Small Mice

Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including fragile X mental retardation, Huntington disease, and myotonic dystrophy type 1. Disease severity and age of onset are critically linked to expansion size. Previous mouse models of repeat instability have not recreated large intergenerational expansions (“big jumps”), observed when the repeat is transmitted from one generation to the next, and have never attained the very large tract lengths possible in humans. Here, we describe dramatic intergenerational CTG•CAG repeat expansions of several hundred repeats in a transgenic mouse model of myotonic dystrophy type 1, resulting in increasingly severe phenotypic and molecular abnormalities. Homozygous mice carrying over 700 trinucleotide repeats on both alleles display severely reduced body size and splicing abnormalities, notably in the central nervous system. Our findings demonstrate that large intergenerational trinucleotide repeat expansions can be recreated in mice, and endorse the use of transgenic mouse models to refine our understanding of triplet repeat expansion and the resulting pathogenesis.     

Apparently unstable normal FMR1 alleles in nine developmentally delayed patients: implications for molecular diagnosis of the fragile X syndrome

The Fragile X syndrome is a common form of X-linked mental retardation, affecting approximately 1 in 4,000 males. Since the discovery of the FMR1 gene responsible for the syndrome, molecular, rather than cytogenetic, diagnosis of Fragile X syndrome has become the gold standard. Numerous molecular diagnostic centers worldwide use PCR and Southern blotting to characterize the size of the CGG repeats within the gene, expansion of which has been shown to be associated with the vast majority of cases of Fragile X syndrome. Instability of this repeat through successive generations has been demonstrated in many patients and has been associated with numerous factors, including repeat length and molecular structure of the repeat. Nine males with normal-size alleles that exhibit repeat length instability by the presence of a second normal length distinct band by repeated PCR analysis from peripheral lymphocytes are reported. Many hypotheses addressing the reason for this apparent instability were tested without elucidating the underlying molecular causes, including cytogenetic analysis, sequence analysis of the repeat locus, and analysis of flanking dinucleotide repeat loci. All patients exhibited a normal complement of sex chromosomes by cytogenetic and molecular analysis. These results from the widely used PCR analysis illustrate an interesting molecular phenomenon and raise many questions relating to the factors and mechanisms involved in trinucleotide instability as well as having implications for the diagnostic testing of the Fragile X syndrome.     

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.     

The risk of fragile X premutation expansion is lower in carriers detected by general prenatal screening than in carriers from known fragile X families

The Fragile X syndrome is the most common cause of inherited mental retardation. For a female premutation carrier, the risk of having a child with a full mutation is positively correlated with the size of the premutation. The current study was performed to evaluate the risk of premutation expansion in the offspring of average-risk carriers detected by general prenatal screening. Over a 4-year period, 9,660 women underwent DNA screening for FMR1 mutation/premutation at the Tel Aviv Sourasky Medical Center. A premutation was defined as a CGG repeat number >50 in the 5' untranslated region (UTR) of exon 1 in the FMR1 gene. The study included only individuals with no family history of X-linked mental retardation or known FMR1 mutations. A premutation was found in 85 women (1 in 114), 68 of whom consented to have prenatal diagnoses in 74 pregnancies. The abnormal allele was transmitted to the offspring in 44 pregnancies. Of these, no change in allele size was noted in 35 pregnancies (79.6%), and expansion within premutation range was evident in 4 pregnancies (9%). In 5 pregnancies (11.4%), expansion to the full mutation was noted. This occurred only in carriers having more than 90 repeats. We conclude that the likelihood of Fragile X premutation expansion to full mutation is significantly lower in individuals ascertained by general prenatal carrier testing than in those from known Fragile X families.     

Familial transmission of the FMR1 CGG repeat.

To better define the nature of FMR1 CGG-repeat expansions, changes in allele sizes for 191 families with fragile X and for 33 families with gray-zone repeats (40-60) were analyzed. Expansion of the fragile X chromosome to the full mutation was seen in 13.4% of offspring from premutation mothers with 56-59 repeats, 20.6% of those with 60-69 repeats, 57.8% of those with 70-79 repeats, 72.9% of those with 80-89 repeats, and 97.3% of those with 90-199 repeats. For premutation fathers, the majority (62%) of their daughters had a larger repeat number, while a few had either a smaller (22%) or the same (16%) repeat number, compared with their fathers' sizes. However, daughters with a smaller repeat number were observed only if their fathers had > or = 80 repeats. Fifteen (39.5%) of 38 such daughters carried a smaller repeat than did their fathers. We observed that a similar repeat number was inherited more often than expected by chance, among the members of a sibship segregating fragile X. This familial clustering, observed in the offspring of both males and females with a premutation, implies there may be an additional factor, independent of parental repeat size, that influences CGG-repeat instability. Instability in gray-zone allele transmissions was observed in 25% of alleles with 50-60 CGGs but in <8% of those with 40-49 CGGs. Examination of gray-zone allele organization revealed that long tracts of pure CGGs (>34) are not always unstably transmitted. These results raise new questions regarding the familial factors that may determine transmission expansions.     

Recurrent and unexpected segregation of the FMR1 CGG repeat in a family with fragile X syndrome

Fragile X syndrome, the most common cause of hereditary mental retardation, results from amplification of a CGG trinucleotide repeat in the FMR1 gene. The transmission of the CGG repeat from premutated individuals to their premutated descendants is usually unstable, showing an increase in the size of the repeat. We report here a family which exhibits recurrent and unexpected transmission of the maternal premutation to three daughters. The first daughter exhibited mosaicism with two premutated alleles, one contracted and the other expanded. The second daughter showed a reversion from the maternal premutation to the normal range, and the third carried an expanded premutated allele associated with an expanded paternal allele within the normal range. These variations in the size of the CGG repeat may result from many different mechanisms such as DNA polymerase slippage on the leading or lagging strand during replication, large contractions of repeats on the parental strand during replication, or recombination through unequal crossover between sister chromatids. Our results suggest that the variation of the CGG premutated alleles in this family may be the result of intrinsic instability associated with a trans-acting factor such as a mismatch repair gene product. Received: 21 August 1995 / Revised: 21 September 1995     

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.     

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.     

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%.     

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.     

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.