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.     

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.     

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.     

Prevalence of fragile X syndrome among patients with mental retardation in the west of Iran

Background

Fragile X syndrome (FXS), an X-linked disorder, is the most common cause of inherited mental retardation. This is caused by a trinucleotide CGG repeat expansion (>200) on the fragile X mental retardation 1 gene (FMR1) becoming methylated leading to a deficiency or absence of the FMR1 protein. Determining FXS prevalence in the mentally retarded individuals in the west of Iran was the aim of this study.

Methods

200 patients with moderate mental retardation who were clinically suspicious to FXS were screened using cytogenetic and molecular methods. Blood samples were collected and cultured in the specific culture media. The G-Banding method was used for karyotyping and DNA sequencing performed for verifying the results of the cytogenetic tests.

Results

16 patients (8%) were found to have fragile X syndrome. The results showed that there is no significant association between the fragile X syndrome and economic status and place of residence, however, the relationship between fragile X syndrome and mental retardation in the family history is significant.

Conclusion

The frequency of FXS was similar to other reports in the preselected patients. For diagnosis of FXS, chromosome analysis must be accompanied by molecular studies.

     

Fragile x syndrome

Recent data from a national survey highlighted a significant difference in obesity rates in young fragile X males (31%) compared to age matched controls (18%). Fragile X syndrome (FXS) is the most common cause of intellectual disability in males and the most common single gene cause of autism. This X-linked disorder is caused by an expansion of a trinucleotide CGG repeat (>200) on the promotor region of the fragile X mental retardation 1 gene (FMR1). As a result, the promotor region often becomes methylated which leads to a deficiency or absence of the FMR1 protein (FMRP). Common characteristics of FXS include mild to severe cognitive impairments in males but less severe cognitive impairment in females. Physical features of FXS include an elongated face, prominent ears, and post-pubertal macroorchidism. Severe obesity in full mutation males is often associated with the Prader-Willi phenotype (PWP) which includes hyperphagia, lack of satiation after meals, and hypogonadism or delayed puberty; however, there is no deletion at 15q11-q13 nor uniparental maternal disomy. Herein, we discuss the molecular mechanisms leading to FXS and the Prader-Willi phenotype with an emphasis on mouse FMR1 knockout studies that have shown the reversal of weight increase through mGluR antagonists. Finally, we review the current medications used in treatment of FXS including the atypical antipsychotics that can lead to weight gain and the research regarding the use of targeted treatments in FXS that will hopefully have a significantly beneficial effect on cognition and behavior without weight gain.     

Establishment of FXS-A9 panel with a single human X chromosome from fragile X syndrome-associated individual

Fragile X syndrome (FXS) is the most common inheritable form of intellectual disability. FMR1, the gene responsible for FXS, is located on human chromosome Xq27.3 and contains a stretch of CGG trinucleotide repeats in its 5′ untranslated region. FXS is caused by CGG repeats that expand beyond 200, resulting in FMR1 silencing via promoter hypermethylation. The molecular mechanism underlying CGG repeat expansion, a fundamental cause of FXS, remains poorly understood, partly due to a lack of experimental systems. Accumulated evidence indicates that the large chromosomal region flanking a CGG repeat is critical for repeat dynamics. In the present study, we isolated and introduced whole human X chromosomes from healthy, FXS premutation carriers, or FXS patients who carried disease condition-associated CGG repeat lengths, into mouse A9 cells via microcell-mediated chromosome transfer. The CGG repeat length-associated methylation status and human FMR1 expression in these monochromosomal hybrid cells mimicked those in humans. Thus, this set of A9 cells containing CGG repeats from three different origins (FXS-A9 panel) may provide a valuable resource for investigating a series of genetic and epigenetic CGG repeat dynamics during FXS pathogenesis.     

脆性X综合征致病机理与治疗

脆性X综合征(fragile X syndrome, FXS)是最常见的遗传性智力障碍疾病,主要是由于X染色体上脆性X智力低下基因1(fragile X-mental retardation gene 1, FMR1)5’端非翻译区CGG三核苷酸的重复扩增及其相邻部位CpG岛的异常甲基化而导致其编码产物脆性X智力低下蛋白(fragile X mental retardation protein, FMRP)的缺失引起。目前,基因诊断已成为FXS诊断的金标准,但临床治疗仍缺乏特异性。本文首先介绍了FMRP的结构与功能,剖析了FXS的致病机制,然后阐述了FXS中与FMRP表达相关的信号转导途径,深入探讨并总结了靶向干预FXS中信号通路、基因编辑逆转FMR1沉默以及靶向降解FXS异常表达蛋白的治疗策略。     

From FMRP Function to Potential Therapies for Fragile X Syndrome

Fragile X syndrome (FXS) is caused by mutations in the fragile X mental retardation 1 (FMR1) gene. Most FXS cases occur due to the expansion of the CGG trinucleotide repeats in the 5′ un-translated region of FMR1, which leads to hypermethylation and in turn silences the expression of FMRP (fragile X mental retardation protein). Numerous studies have demonstrated that FMRP interacts with both coding and non-coding RNAs and represses protein synthesis at dendritic and synaptic locations. In the absence of FMRP, the basal protein translation is enhanced and not responsive to neuronal stimulation. The altered protein translation may contribute to functional abnormalities in certain aspects of synaptic plasticity and intracellular signaling triggered by Gq-coupled receptors. This review focuses on the current understanding of FMRP function and potential therapeutic strategies that are mainly based on the manipulation of FMRP targets and knowledge gained from FXS pathophysiology.     

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.     

Transmission of double <Emphasis Type="Italic">FMR1</Emphasis> allelic premutations in a family

The fragile X mental retardation 1 (FMR1) gene is the only gene known responsible for fragile X related disorders, including fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency, and fragile X-associated tremor/ataxia. Although FMR1 premutation carriers are common, double mutations of the FMR1 gene is very rare. To our knowledge, only twelve such reports including twenty-three cases from fourteen families have been documented. We report here another family with a FXS family history in which the proband’s maternal grandmother had compound FMR1 gene premutations and we review twelve published papers associated with double allelic mutations. Our study and literature review indicated that compound premutations may have influences regarding the early onset of fragile X-associated primary ovarian insufficiency and severity of psychiatric issues, and less likely aggravate the cognitive deficits compared with one allele mutant patients. Further detailed studies of similar cases are needed to clarify the profile of double FMR1 premutaions.     

Crystal structures of CGG RNA repeats with implications for fragile X-associated tremor ataxia syndrome

The CGG repeats are present in the 5'-untranslated region (5'-UTR) of the fragile X mental retardation gene FMR1 and are associated with two diseases: fragile X-associated tremor ataxia syndrome (FXTAS) and fragile X syndrome (FXS). FXTAS occurs when the number of repeats is 55-200 and FXS develops when the number exceeds 200. FXTAS is an RNA-mediated disease in which the expanded CGG tracts form stable structures and sequester important RNA binding proteins. We obtained and analysed three crystal structures of double-helical CGG repeats involving unmodified and 8-Br modified guanosine residues. Despite the presence of the non-canonical base pairs, the helices retain an A-form. In the G-G pairs one guanosine is always in the syn conformation, the other is anti. There are two hydrogen bonds between the Watson-Crick edge of G(anti) and the Hoogsteen edge of G(syn): O6·N1H and N7·N2H. The G(syn)-G(anti) pair shows affinity for binding ions in the major groove. G(syn) causes local unwinding of the helix, compensated elsewhere along the duplex. CGG helical structures appear relatively stable compared with CAG and CUG tracts. This could be an important factor in the RNA's ligand binding affinity and specificity.     

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.     

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.