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.     

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.     

Fragile X-premutation tremor/ataxia syndrome (FXTAS) in a young woman: clinical, genetics, MRI and 1H-MR spectroscopy correlates

It is generally thought that fragile X-associated tremor/ataxia syndrome (FXTAS) represents a late-onset neurodegenerative disorder occuring in male carriers of a premutation expansion (55-200 CGG repeats) in the fragile X mental retardation 1 (FMR 1) gene. However, several female patients with FXTAS have also been reported recently. Here, we describe a 23-year old woman with positive family history of mental retardation and autism who presented clinically with action tremor, ataxia, emotional disturbances and cognitive dysfunction. Magnetic resonance imaging (MRI) of the brain showed diffuse cortical atrophy, while 1H-MR spectroscopy (MRS) revealed decreased levels of N-acetylaspartate (NAA) in the cerebellum, basal ganglia, and pons. Genetic testing confirmed heterozygous FMR 1 gene premutation of 100 CGG repeats in the abnormal allele and 29 CGG repeats in the normal allele. We concluded that FXTAS may be an under-recognized disorder, particularly in women.     

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.     

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.     

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.     

Preliminary evidence of an effect of cerebellar volume on postural sway in FMR1 premutation males

Recent evidence suggests that early changes in postural control may be discernible among females with premutation expansions (55–200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene at risk of developing fragile X‐associated tremor ataxia syndrome (FXTAS). Cerebellar dysfunction is well described in males and females with FXTAS, yet the interrelationships between cerebellar volume, CGG repeat length, FMR1 messenger RNA (mRNA) levels and changes in postural control remain unknown. This study examined postural sway during standing in a cohort of 22 males with the FMR1 premutation (ages 26–80) and 24 matched controls (ages 26–77). The influence of cerebellar volume, CGG repeat length and FMR1 mRNA levels on postural sway was explored using multiple linear regression. The results provide preliminary evidence that increasing CGG repeat length and decreasing cerebellar volume were associated with greater postural sway among premutation males. The relationship between CGG repeat length and postural sway was mediated by a negative association between CGG repeat size and cerebellar volume. While FMR1 mRNA levels were significantly elevated in the premutation group and correlated with CGG repeat length, FMR1 mRNA levels were not significantly associated with postural sway scores. These findings show for the first time that greater postural sway among males with the FMR1 premutation may reflect CGG repeat‐mediated disruption in vulnerable cerebellar circuits implicated in postural control. However, longitudinal studies in larger samples are required to confirm whether the relationships between cerebellar volume, CGG repeat length and postural sway indicate greater risk for neurological decline.     

Cerebral Protein Synthesis in a Knockin Mouse Model of the Fragile X Premutation

The (CGG)n-repeat in the 5′-untranslated region of the fragile X mental retardation gene (FMR1) gene is polymorphic and may become unstable on transmission to the next generation. In fragile X syndrome, CGG repeat lengths exceed 200, resulting in silencing of FMR1 and absence of its protein product, fragile X mental retardation protein (FMRP). CGG repeat lengths between 55 and 200 occur in fragile X premutation (FXPM) carriers and have a high risk of expansion to a full mutation on maternal transmission. FXPM carriers have an increased risk for developing progressive neurodegenerative syndromes and neuropsychological symptoms. FMR1 mRNA levels are elevated in FXPM, and it is thought that clinical symptoms might be caused by a toxic gain of function due to elevated FMR1 mRNA. Paradoxically, FMRP levels decrease moderately with increasing CGG repeat length in FXPM. Lowered FMRP levels may also contribute to the appearance of clinical problems. We previously reported increases in regional rates of cerebral protein synthesis (rCPS) in the absence of FMRP in an Fmr1 knockout mouse model and in a FXPM knockin (KI) mouse model with 120 to 140 CGG repeats in which FMRP levels are profoundly reduced (80%–90%). To explore whether the concentration of FMRP contributes to the rCPS changes, we measured rCPS in another FXPM KI model with a similar CGG repeat length and a 50% reduction in FMRP. In all 24 brain regions examined, rCPS were unaffected. These results suggest that even with 50% reductions in FMRP, normal protein synthesis rates are maintained.     

Reduced FMR1 mRNA translation efficiency in fragile X patients with premutations

The Fragile X mental retardation gene (FMR1) contains a polymorphic trinucleotide CGG repeat in the 5' untranslated region (UTR) of the FMR1 messenger. We have characterized three lymphoblastoid cell lines derived from unrelated male carriers of a premutation that overexpress FMR1 mRNA and show reduced FMRP level compared to normal cells. The analysis of polysomes/mRNPs distribution of mRNA in the cell lines with a premutation shows that the polysomal association of FMR1 mRNA, which is high in normal cells, becomes progressively lower with increasing CGG repeat expansion. In addition, we could detect a very low level of FMR1 mRNA in a lymphoblastoid cell line from a patient with a full mutation. In this case, FMR1 mRNA is not at all associated with polysomes, in agreement with the complete absence of FMRP. The impairment of FMR1 mRNA translation in patients with the Fragile X syndrome with FMR1 premutation is the cause of the lower FMRP levels that leads to the clinical involvement.     

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.     

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.     

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.     

Evaluation of the fragile X (FRAXA) syndrome with methylation-sensitive PCR

The fragile X (FRAXA) syndrome is the most common form of inherited mental retardation in males. Its peculiar pattern of inheritance results from the parent of origin-specific expansion of a CGG-repeat within the FMR1 gene on the X chromosome. In patients, gene function is abolished by hypermethylation of the promoter and the massively expanded repeat. We have developed a methylation-sensitive polymerase chain reaction (MS-PCR) strategy that combines repeat-length and methylation analysis of the CGG-repeat and the promoters of the FMR1 and XIST genes. The allelic methylation of the latter opposes that of the FMR promoter and serves as an internal control and standard for semiquantitative analyses. This system enables the delineation of 11 distinct patterns encountered in nonaffected, carrier, and affected males and females. We have evaluated our system on well-defined samples with different FMR1 mutations and have used it for the diagnostic evaluation of 253 male and 80 female probands. In the male group, we have identified five full mutations, and three gray-zone and premutation alleles with 54, 55, and 62 repeats, respectively. The female group consists of 33 normal homozygote and 41 heterozygote individuals, two of whom harbor a gray-zone allele with 47 repeats, none with a premutation, and six with a full mutation. Our MS-PCR approach allows the currently most comprehensive diagnostic evaluation of the FRAXA syndrome in a cost- and time-efficient fashion. In addition, it is a valuable tool for the analysis of clonality and skewing phenomena in females.     

Fragiles-X Syndrom

Fragile X syndrome is an X-linked neurodevelopmental disorder affecting both males and females. Phenotypical characteristics include intellectual deficits, somatic symptoms and behavioural abnormalities caused by loss of the FMRP protein, which leads to destruction of synapses with metabotropic glutamate receptors. The FMR1 gene harbours a CGG repeat in the 5’-untranslated region. The vast majority of fragile-X syndrome patients have a largely expanded CGG repeat (220 or more triplets, designated “full mutation”) and an inactive gene. Full mutation alleles originate upon proliferation of oogonia in the fetal ovary of females who carry a mitotically unstable premutation (59–200 repeats). Premutation carriers have no symptoms of fragile X syndrome; they may, however, experience premature ovarian insufficiency and/or fragile X-associated tremor/ataxia syndrome. The diagnosis of both syndromes requires genetic testing to measure the number of CGG repeats. Prenatal diagnostics of fragile X syndrome is offered to females carrying a pre- or full mutation.