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.     

Noninvasive test for fragile X syndrome, using hair root analysis.

Identification of the FMR1 gene and the repeat-amplification mechanism causing fragile X syndrome led to development of reliable DNA-based diagnostic methods, including Southern blot hybridization and PCR. Both methods are performed on DNA isolated from peripheral blood cells and measure the repeat size in FMR1. Using an immunocytochemical technique on blood smears, we recently developed a novel test for identification of patients with fragile X syndrome. This method, also called "antibody test," uses monoclonal antibodies against the FMR1 gene product (FMRP) and is based on absence of FMRP in patients' cells. Here we describe a new diagnostic test to identify male patients with fragile X syndrome, on the basis of lack of FMRP in their hair roots. Expression of FMRP in hair roots was studied by use of an FMRP-specific antibody test, and the percentage of FMRP-expressing hair roots in controls and in male fragile X patients was determined. Control individuals showed clear expression of FMRP in nearly every hair root, whereas male fragile X patients lacked expression of FMRP in almost all their hair roots. Mentally retarded female patients with a full mutation showed FMRP expression in only some of their hair roots (<55%), and no overlap with normal female controls was observed. The advantages of this test are (1) plucking of hair follicles does no appreciable harm to the mentally retarded patient, (2) hairs can be sent in a simple envelope to a diagnostic center, and (3) the result of the test is available within 5 h of plucking. In addition, this test enabled us to identify two fragile X patients who did not show the full mutation by analysis of DNA isolated from blood cells.     

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.     

FISH-detected delay in replication timing of mutated FMR1 alleles on both active and inactive X-chromosomes

X-chromosome inactivation and the size of the CGG repeat number are assumed to play a role in the clinical, physical, and behavioral phenotype of female carriers of a mutated FMR1 allele. In view of the tight relationship between replication timing and the expression of a given DNA sequence, we have examined the replication timing of FMR1 alleles on active and inactive X-chromosomes in cell samples (lymphocytes or amniocytes) of 25 females: 17 heterozygous for a mutated FMR1 allele with a trinucleotide repeat number varying from 58 to a few hundred, and eight homozygous for a wild-type allele. We have applied two-color fluorescence in situ hybridization (FISH) with FMR1 and X-chromosome α-satellite probes to interphase cells of the various genotypes: the α-satellite probe was used to distinguish between early replicating (active) and late replicating (inactive) X-chromosomes, and the FMR1 probe revealed the replication pattern of this locus. All samples, except one with a large trinucleotide expansion, showed an early replicating FMR1 allele on the active X-chromosome and a late replicating allele on the inactive X-chromosome. In samples of mutation carriers, both the early and the late alleles showed delayed replication compared with normal alleles, regardless of repeat size. We conclude therefore that: (1) the FMR1 locus is subjected to X-inactivation; (2) mutated FMR1 alleles, regardless of repeat size, replicate later than wild-type alleles on both the active and inactive X-chromosomes; and (3) the delaying effect of the trinucleotide expansion, even with a low repeat size, is superimposed on the delay in replication associated with X-inactivation. Electronic Publication     

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.     

Characterisation of Fmrp in zebrafish: evolutionary dynamics of the <Emphasis Type="Italic">fmr1</Emphasis> gene

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by the lack of the Fragile X Mental Retardation Protein (FMRP), which is encoded by the FMR1 gene. Although Fmr1 knockout mice display some characteristics also found in fragile X patients, it is a complex animal model to study brain abnormalities, especially during early embryonic development. Interestingly, the ortholog of the FMR1 gene has been identified not only in mouse, but also in zebrafish (Danio rerio). In this study, an amino acid sequence comparison of FMRP orthologs was performed to determine the similar regions of FMRP between several species, including human, mouse, frog, fruitfly and zebrafish. Further characterisation of Fmrp has been performed in both adults and embryos of zebrafish using immunohistochemistry and western blotting with specific antibodies raised against zebrafish Fmrp. We have demonstrated a strong Fmrp expression in neurons of the brain and only a very weak expression in the testis. In brain tissue, a different distribution of the isoforms of Fmrp, compared to human and mouse brain tissue, was shown using western blot analysis. Due to the high similarity between zebrafish Fmrp and human FMRP and their similar expression pattern, the zebrafish has great potential as a complementary animal model to study the pathogenesis of the fragile X syndrome, especially during embryonic development.Edited by D. Tautz     

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.     

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.     

Programming social behavior by the maternal fragile X protein

The developing fetus and neonate are highly sensitive to maternal environment. Besides the well‐documented effects of maternal stress, nutrition and infections, maternal mutations, by altering the fetal, perinatal and/or early postnatal environment, can impact the behavior of genetically normal offspring. Mutation/premutation in the X‐linked FMR1 (encoding the translational regulator FMRP) in females, although primarily responsible for causing fragile X syndrome (FXS) in their children, may also elicit such maternal effects. We showed that a deficit in maternal FMRP in mice results in hyperactivity in the genetically normal offspring. To test if maternal FMRP has a broader intergenerational effect, we measured social behavior, a core dimension of neurodevelopmental disorders, in offspring of FMRP‐deficient dams. We found that male offspring of Fmr1^+/? mothers, independent of their own Fmr1 genotype, exhibit increased approach and reduced avoidance toward conspecific strangers, reminiscent of ‘indiscriminate friendliness’ or the lack of stranger anxiety, diagnosed in neglected children and in patients with Asperger's and Williams syndrome. Furthermore, social interaction failed to activate mesolimbic/amygdala regions, encoding social aversion, in these mice, providing a neurobiological basis for the behavioral abnormality. This work identifies a novel role for FMRP that extends its function beyond the well‐established genetic function into intergenerational non‐genetic inheritance/programming of social behavior and the corresponding neuronal circuit. As FXS premutation and some psychiatric conditions that can be associated with reduced FMRP expression are more prevalent in mothers than full FMR1 mutation, our findings potentially broaden the significance of FMRP‐dependent programming of social behavior beyond the FXS population.     

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.     

Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. In addition to cognitive deficits, FXS patients exhibit hyperactivity, attention deficits, social difficulties, anxiety, and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 5' untranslated region of the Fragile X Mental Retardation (FMR1) gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP). Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations, we report on the generation of induced pluripotent stem cell (iPSC) lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons, these iPSC models will aid in the discovery of novel therapeutics for FXS and other autism-spectrum disorders sharing common pathophysiology.     

The X chromosome and fragile X mental retardation

Fragile X syndrome represents the most common inherited cause of mental retardation. It is caused by a stretch of CGG repeats within the fragile X gene, which increases in length as it is transmitted from generation to generation. Once the repeat exceeds a threshold length, no protein is produced, resulting in the fragile X phenotype. Both X chromosome inactivation and inactivation of the FMR1 gene are the result of methylation. X inactivation occurs earlier than inactivation of the FMR1 gene. The instability to a full mutation is dependent on the sex of the transmitting parent and occurs only from mother to child. For most X-chromosomal diseases, female carriers do not express the phenotype. A clear exception is fragile X syndrome. It is clear that more than 50% of the neurons have to express the protein to ensure a normal phenotype in females. This means that a normal phenotype in female carriers of a full mutation is accompanied by a distortion of the normal distribution of X inactivation.     

Restoring the phenotype of fragile X syndrome: insight from the mouse model

A mouse model for the fragile X syndrome, the most common form of inherited mental retardation, was generated a number of years ago. It shows characteristics compatible with the clinical symptoms of human patients. These include pathological changes such as macroorchidism, behavioral problems, and diminished visuo-spatial abilities. To investigate whether the fragile X syndrome is a potentially correctable disorder, several groups attempted to 'rescue' the knockout mutation by introduction of an intact copy of the FMR1 gene in the knockout mouse. Two different types of rescue mice have been created by injection of constructs based on FMR1 cDNA or on FMR1 genomic DNA. Several pathological, behavioral and cognitive function tests were performed on these two different rescue mouse lines to compare their characteristics with those of the knockout and control littermates. Each rescue line resembled the control in some aspects though neither of the 2 lines was a full 'rescue', e.g. resemble the control in all aspects investigated. Thus, rescue of some aspects of the phenotype has been achieved by introduction of FMR1 constructs in the fragile X knockout mice. The results implicate that, even if FMR1 production is cell type specific, the quantity of the FMRP expression is highly critical as overproduction may have a harmful effect.