A female compound heterozygote (pre- and full mutation) for the CGG FMR1 expansion

Fragile X syndrome is the most common cause of inherited mental retardation. The incidence has been estimated to be 1 in 1250 males and 1 in 2000 females. Molecular studies have shown that 95% of fragile X syndrome cases are caused by the expansion of a CGG triplet in the FMR1 gene with hypermethylation of the adjacent CpG island. In spite of the high incidence of this syndrome, a female with both FMR1 genes in the expanded form has never been reported. We present here a female from the Canary Islands presenting mental retardation and attention problems. Molecular analysis has revealed that both of her FMR1 genes have the CGG expansion, one with a premutation and the other with a full mutation. We have studied the CGG repeat in the FMR1 gene in 64 members of her family and detected 33 normal individuals, 14 carriers with the premutation (1 male and 13 females), and 18 individuals with full mutations (8 males and 10 females). The index case illustrates that the possibility of both parents being carriers of the fragile X syndrome premutation should be considered in consanguineous families or in small communities. Received: 4 April 1996 / Revised: 3 May 1996     

Understanding fragile X syndrome: insights from animal models

The fragile X mental retardation syndrome is caused by large methylated expansions of a CGG repeat in the FMR1 gene leading to the loss of expression of FMRP, an RNA-binding protein. FMRP is proposed to act as a regulator of mRNA transport or translation that plays a role in synaptic maturation and function. To study the physiological function of the FMR1 protein, mouse and Drosophila models have been developed. The loss-of-function mouse model shows slightly enlarged testes, a subtle behavioral phenotype, and discrete anomalies of dendrite spines similar to those observed in brains of patients. Studies in Drosophila indicate that FXMR plays an important role in synaptogenesis and axonal arborization, which may underlie the observed deficits in flight ability and circadian behavior of FXR mutant flies. The relevance of these studies to our understanding of fragile X syndrome is discussed.     

A fragile gene

Fragile X syndrome is the most common cause of inherited mental retardation in humans. The fragile X gene (FMR1) has been cloned and the mutation causing the disease is known. The molecular basis of the disease is an expansion of a trinucleotide repeat sequence (CGG) present in the first exon within the 5′ untranslated region of the FMR1 gene. Affected individuals have repeat CGG sequences of above 200. As a result the gene is not producing protein. It has been shown that the FMR1 protein has RNA binding activity, but the function of this RNA binding activity is not known. The timing and mechanism of repeat amplification are not yet understood. An animal model for fragile X syndrome has been generated, which can be used to study the clinical and biochemical abnormalities caused by absence of FMR1 protein product.     

FRAXA screening in Brazilian institutionalized individuals with nonspecific severe mental retardation

Individuals with mental disabilities are a heterogeneous group, mainly when we consider the etiology of mental retardation (MR). Recent advances in molecular genetics techniques have enabled us to unveil more about the molecular basis of several genetic syndromes associated with MR. In this study, we surveyed 85 institutionalized individuals with severe MR, 38 males and 47 females, by two molecular techniques, to detect CGG amplifications in the FMR1 gene. No FRAXA mutations were found in the FMR1 gene, reinforcing the low prevalence of Fragile X syndrome among institutionalized individuals with severe MR. We considered the PCR protocol used adequate for screening males with mental retardation of unknown etiology. The use of the Southern blot is still necessary for the decisive diagnosis of the Fragile X syndrome. To exclude chromosomal abnormalities associated with MR as a possible cause of the phenotype in these individuals, G-banded chromosome analysis was performed in all patients and 7.3% of chromosomal aberrations were found. Our results are similar to those reported previously and point to the necessity of expanding the molecular investigation toward other causes of MR, such as subtle chromosomal rearrangements, as suggested recent by a combination of fluorescence in situ hybridization (FISH) and PCR studies.     

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.     

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     

The fragile X syndrome: exploring its molecular basis and seeking a treatment

Fragile X syndrome (FXS) - the leading cause of inherited mental retardation - is an X-linked disease caused by loss of expression of the FMR1 (fragile X mental retardation 1) gene. In addition to impairment of higher-cognitive functions, FXS patients show a variety of physical and other mental abnormalities. FMRP, the protein encoded by the FMR1 gene, is thought to play a key role in translation, trafficking and targeting of mRNA in neurons. To better understand FMRP's functions, the protein partners and mRNA targets that interact with FMRP have been sought. These and functional studies have revealed links with processes such as cytoskeleton remodelling via the RhoGTPase pathway and mRNA processing via the RNA interference pathway. In this review, we focus on recent insights into the function of FMRP and speculate on how the absence of FMRP might cause the clinical phenotypes seen in FXS patients. Finally, we explore potential therapies for FXS.     

Understanding fragile X syndrome: insights from retarded flies

Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss-of-function mutations in the fragile X mental retardation 1 (fmr1) gene. FMR1 is an RNA binding protein that is highly expressed in neurons of the central nervous system. Recent studies in Drosophila indicate that FMR1 plays an important role in synaptogenesis and axonal arborization, which may underlie the observed deficits in flight ability and circadian behavior of fmr1 mutant flies. The relevance of these studies to our understanding of fragile X syndrome is discussed.     

A fragile X mental retardation-like gene in a cnidarian

The fragile X mental retardation syndrome in humans is caused by a mutational loss of function of the fragile X mental retardation gene 1 (FMR1). FMR1 is an RNA-binding protein, involved in the development and function of the nervous system. Despite of its medical significance, the evolutionary origin of FMR1 has been unclear. Here, we report the molecular characterization of HyFMR1, an FMR1 orthologue, from the cnidarian hydroid Hydractinia echinata. Cnidarians are the most basal metazoans possessing neurons. HyFMR1 is expressed throughout the life cycle of Hydractinia. Its expression pattern correlates to the position of neurons and their precursor stem cells in the animal. Our data indicate that the origin of the fraxile X related (FXR) protein family dates back at least to the common ancestor of cnidarians and bilaterians. The lack of FXR proteins in other invertebrates may have been due to gene loss in particular lineages.     

Detection of the fragile X syndrome protein for the evaluation of FMR1 intermediate alleles

Molecular screening programs in mentally retarded individuals have been performed in several populations worldwide. One finding has been an excess of FMR1 intermediate alleles in a population with learning difficulties. However, other published reports with similar characteristics did not corroborate those previous results. In order to contribute additional data from our population, we studied 563 patients affected with nonspecific mental retardation (MRX) that did not present a CGG expansion in the FMR1 gene and 208 individuals as a control population. Forty MRX patients presented alleles within the intermediate range. Among them, one case showed a pattern of expression of the FMR1 protein (FMRP) concordant with a fragile X syndrome case with an intermediate allele/full mutation mosaicism, although it was not detected by Southern blot analysis. Statistical analysis was performed again showing no statistically significant difference regarding the intermediate allele frequency in the MRX and control populations. This finding is in agreement with the hypothesis that the incidence of intermediate FMR1 alleles in MRX populations does not seem to be higher than in control populations, and it emphasizes the importance of FMRP detection as a diagnostic tool for fragile X syndrome.     

Screening and diagnosis for the fragile X syndrome among the mentally retarded: an epidemiological and psychological survey. Collaborative Fragile X Study Group.

The fragile X syndrome is an X-linked mental retardation disorder caused by an expanded CGG repeat in the first exon of the fragile X mental retardation (FMR1) gene. Its frequency, X-linked inheritance, and consequences for relatives all prompt for diagnosis of this disorder on a large scale in all affected individuals. A screening for the fragile X syndrome has been conducted in a representative sample of 3,352 individuals in schools and institutes for the mentally retarded in the southwestern Netherlands, by use of a brief physical examination and the DNA test. The attitudes and reactions of (non)consenting parents/guardians were studied by (pre- and posttest) questionnaires. A total of 2,189 individuals (65%) were eligible for testing, since they had no valid diagnosis, cerebral palsy, or a previous test for the FMR1 gene mutation. Seventy percent (1,531/2,189) of the parents/guardians consented to testing. Besides 32 previously diagnosed fragile X patients, 11 new patients (9 males and 2 females) were diagnosed. Scoring of physical features was effective in preselection, especially for males (sensitivity .91 and specificity .92). Major motives to participate in the screening were the wish to obtain a diagnosis (82%), the hereditary implications (80%), and the support of research into mental retardation (81%). Thirty-four percent of the parents/guardians will seek additional diagnostic workup after exclusion of the fragile X syndrome. The prevalence of the fragile X syndrome was estimated at 1/ 6,045 for males (95% confidence interval 1/9,981-1/ 3,851). On the basis of the actual number of diagnosed cases in the Netherlands, it is estimated that >50% of the fragile X cases are undiagnosed at present.     

RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppress fragile X CGG premutation repeat-induced neurodegeneration in a Drosophila model of FXTAS

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a recently described neurodegenerative disorder of older adult carriers of premutation alleles (60-200 CGG repeats) in the fragile X mental retardation gene (FMR1). It has been proposed that FXTAS is an RNA-mediated neurodegenerative disease caused by the titration of RNA-binding proteins by the CGG repeats. To test this hypothesis, we utilize a transgenic Drosophila model of FXTAS that expresses a premutation-length repeat (90 CGG repeats) from the 5' UTR of the human FMR1 gene and displays neuronal degeneration. Here, we show that overexpression of RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppresses the phenotype of the CGG transgenic fly. Furthermore, we show that hnRNP A2/B1 directly interacts with riboCGG repeats and that the CUGBP1 protein interacts with the riboCGG repeats via hnRNP A2/B1.     

Drosophila fragile X protein,DFXR, regulates neuronal morphology and function in the brain

Mental retardation is a pervasive societal problem, 25 times more common than blindness for example. Fragile X syndrome, the most common form of inherited mental retardation, is caused by mutations in the FMR1 gene. Fragile X patients display neurite morphology defects in the brain, suggesting that this may be the basis of their mental retardation. Drosophila contains a single homolog of FMR1, dfxr (also called dfmr1). We analyzed the role of dfxr in neurite development in three distinct neuronal classes. We find that DFXR is required for normal neurite extension, guidance, and branching. dfxr mutants also display strong eclosion failure and circadian rhythm defects. Interestingly, distinct neuronal cell types show different phenotypes, suggesting that dfxr differentially regulates diverse targets in the brain.     

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.     

CYFIP/Sra-1 controls neuronal connectivity in Drosophila and links the Rac1 GTPase pathway to the fragile X protein

Neuronal plasticity requires actin cytoskeleton remodeling and local protein translation in response to extracellular signals. Rho GTPase pathways control actin reorganization, while the fragile X mental retardation protein (FMRP) regulates the synthesis of specific proteins. Mutations affecting either pathway produce neuronal connectivity defects in model organisms and mental retardation in humans. We show that CYFIP, the fly ortholog of vertebrate FMRP interactors CYFIP1 and CYFIP2, is specifically expressed in the nervous system. CYFIP mutations affect axons and synapses, much like mutations in dFMR1 (the Drosophila FMR1 ortholog) and in Rho GTPase dRac1. CYFIP interacts biochemically and genetically with dFMR1 and dRac1. Finally, CYFIP acts as a dRac1 effector that antagonizes FMR1 function, providing a bridge between signal-dependent cytoskeleton remodeling and translation.     

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.