Differential increases of specific FMR1 mRNA isoforms in premutation carriers.

BACKGROUND: Over 40% of male and ∼16% of female carriers of a premutation FMR1 allele (55-200 CGG repeats) will develop fragile X-associated tremor/ataxia syndrome, an adult onset neurodegenerative disorder, while about 20% of female carriers will develop fragile X-associated primary ovarian insufficiency. Marked elevation in FMR1 mRNA transcript levels has been observed with premutation alleles, and RNA toxicity due to increased mRNA levels is the leading molecular mechanism proposed for these disorders. However, although the FMR1 gene undergoes alternative splicing, it is unknown whether all or only some of the isoforms are overexpressed in premutation carriers and which isoforms may contribute to the premutation pathology. METHODS: To address this question, we have applied a long-read sequencing approach using single-molecule real-time (SMRT) sequencing and qRT-PCR. RESULTS: Our SMRT sequencing analysis performed on peripheral blood mononuclear cells, fibroblasts and brain tissue samples derived from premutation carriers and controls revealed the existence of 16 isoforms of 24 predicted variants. Although the relative abundance of all mRNA isoforms was significantly increased in the premutation group, as expected based on the bulk increase in mRNA levels, there was a disproportionate (fourfold to sixfold) increase, relative to the overall increase in mRNA, in the abundance of isoforms spliced at both exons 12 and 14, specifically Iso10 and Iso10b, containing the complete exon 15 and differing only in splicing in exon 17. CONCLUSIONS: These findings suggest that RNA toxicity may arise from a relative increase of all FMR1 mRNA isoforms. Interestingly, the Iso10 and Iso10b mRNA isoforms, lacking the C-terminal functional sites for fragile X mental retardation protein function, are the most increased in premutation carriers relative to normal, suggesting a functional relevance in the pathology of FMR1-associated disorders.

CGG allele size somatic mosaicism and methylation in FMR1 premutation alleles.

BACKGROUND: Greater than 200 CGG repeats in the 5'UTR of the FMR1 gene lead to epigenetic silencing and lack of the FMR1 protein, causing fragile X Syndrome. Individual carriers of a premutation (PM) allele with 55-200 CGG repeats are typically unmethylated and can present with clinical features defined as FMR1-associated conditions. METHODS: Blood samples from 17 male PM carriers were assessed clinically and molecularly by Southern blot, western blot, PCR and QRT-PCR. Blood and brain tissue from an additional 18 PM males were also similarly examined. Continuous outcomes were modelled using linear regression and binary outcomes were modelled using logistic regression. RESULTS: Methylated alleles were detected in different fractions of blood cells in all PM cases (n=17). CGG repeat numbers correlated with percent of methylation and mRNA levels and, especially in the upper PM range, with greater number of clinical involvements. Inter-tissue/intra-tissue somatic instability and differences in percent methylation were observed between blood and fibroblasts (n=4) and also observed between blood and different brain regions in three of the 18 PM cases examined. CGG repeat lengths in lymphocytes remained unchanged over a period of time ranging from 2 to 6 years, three cases for whom multiple samples were available. CONCLUSIONS: In addition to CGG size instability, individuals with a PM expanded allele can exhibit methylation and display more clinical features likely due to RNA toxicity and/or FMR1 silencing. The observed association between CGG repeat length and percent of methylation with the severity of the clinical phenotypes underscores the potential value of methylation in affected PM to further understand penetrance, inform diagnosis and expand treatment options.

CNS expression of murine fragile X protein (FMRP) as a function of CGG-repeat size.

Large expansions of a CGG-repeat element (>200 repeats; full mutation) in the fragile X mental retardation 1 (FMR1) gene cause fragile X syndrome (FXS), the leading single-gene form of intellectual disability and of autism spectrum disorder. Smaller expansions (55-200 CGG repeats; premutation) result in the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Whereas FXS is caused by gene silencing and insufficient FMR1 protein (FMRP), FXTAS is thought to be caused by 'toxicity' of expanded-CGG-repeat mRNA. However, as FMRP expression levels decrease with increasing CGG-repeat length, lowered protein may contribute to premutation-associated clinical involvement. To address this issue, we measured brain Fmr1 mRNA and FMRP levels as a function of CGG-repeat length in a congenic (CGG-repeat knock-in) mouse model using 57 wild-type and 97 expanded-CGG-repeat mice carrying up to ~250 CGG repeats. While Fmr1 message levels increased with repeat length, FMRP levels trended downward over the same range, subject to significant inter-subject variation. Human comparisons of protein levels in the frontal cortex of 7 normal and 17 FXTAS individuals revealed that the mild FMRP decrease in mice mirrored the more limited data for FMRP expression in the human samples. In addition, FMRP expression levels varied in a subset of mice across the cerebellum, frontal cortex, and hippocampus, as well as at different ages. These results provide a foundation for understanding both the CGG-repeat-dependence of FMRP expression and for interpreting clinical phenotypes in premutation carriers in terms of the balance between elevated mRNA and lowered FMRP expression levels.

Reduced excitatory amino acid transporter 1 and metabotropic glutamate receptor 5 expression in the cerebellum of fragile X mental retardation gene 1 premutation carriers with fragile X-associated tremor/ataxia syndrome.

A premutation (PM) expansion (55-200 CGG) in the fragile X mental retardation gene 1 causes elevated messenger RNA and reduced fragile X mental retardation gene 1 protein. Young PM carriers can develop characteristic physical features and mild cognitive disabilities. In addition, individuals with PM, particularly male carriers, are at high risk to develop fragile X-associated tremor/ataxia syndrome (FXTAS) with aging. Human postmortem FXTAS brains show extensive white matter disease in the cerebellum and the presence of intranuclear inclusions throughout the brain, although their etiologic significance is unknown. In the current work, expression levels of the metabotropic glutamate (Glu) receptor 5 and the Glu transporter excitatory amino acid transporter 1, examined by reverse transcription polymerase chain reaction and western blot analyses, were found to be reduced in the postmortem cerebellum of PM carriers with FXTAS compared with age matched controls, with higher CGG repeat number having greater reductions in both proteins. These data suggests a dysregulation of Glu signaling in PM carriers, which would likely contribute to the development and severity of FXTAS.

High MMP-9 activity levels in fragile X syndrome are lowered by minocycline.

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by lack of the FMR1 protein, FMRP, a translational repressor. Its absence leads to up-regulation of locally translated proteins involved in synaptic transmission and plasticity, including the matrix metalloproteinase-9 (MMP-9). In the Fmr1 knock-out (KO), a mouse model of FXS, an abnormal elevated expression of MMP-9 in the brain was pharmacologically down-regulated after treatment with the tetracycline derivative minocycline. Moreover, the rescue of immature dendritic spine morphology and a significant improvement of abnormal behavior were associated with down-regulation of MMP-9. Here, we report on high plasma activity of MMP-9 in individuals with FXS. In addition, we investigate MMP-9 changes in patients with FXS who have gone through a minocycline controlled clinical trial and correlate MMP-9 activity to clinical observations. The results of this study suggest that, in humans, activity levels of MMP-9 are lowered by minocycline and that, in some cases, changes in MMP-9 activity are positively associated with improvement based on clinical measures.

Intranuclear inclusions in a fragile X mosaic male.

Lack of the fragile X mental retardation protein leads to Fragile X syndrome (FXS) while increased levels of FMR1 mRNA, as those observed in premutation carriers can lead to Fragile X- associated tremor ataxia syndrome (FXTAS). Until recently, FXTAS had been observed only in carriers of an FMR1 premutation (55-200 CGG repeats); however the disorder has now been described in individuals carriers of an intermediate allele (45-54 CGG repeats) as well as in a subject with a full mutation with mosaicism.Here, we report on molecular and clinical data of a male FMR1 mosaic individual with full and premutation alleles. Molecular analysis of FMR1 and FMRP expression in this subject is consistent with a FXS phenotype. We observed reduced expression of FMRP in both peripheral blood and brain leading to the FXS diagnosis. In addition, a dramatic 90% depletion of both FMR1 mRNA and FMRP levels was observed in the blood, as normally observed in FXS cases, and an even greater depletion in the brain. A clinical report of this patient, at age 71, described neurodegenerative signs of parkinsonism that were likely, in retrospect, part of a FXTAS scenario as post-mortem examination shows the presence of intranuclear inclusions, the hallmark pathology of FXTAS.The findings presented in this study indicate co-morbidity for both FXS and FXTAS in this individual carrying both full and premutation FMR1 alleles. In addition, based on symptoms and pathological and molecular evidence, this report suggests the need to redefine the diagnostic criteria of FXTAS.

Structural dynamics and single-stranded DNA binding activity of the three N-terminal domains of the large subunit of replication protein A from small angle X-ray scattering.

Replication protein A (RPA) is the primary eukaryotic single-stranded DNA (ssDNA) binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial interdomain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments with two multidomain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high-affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA-bound state and therefore freely available to serve as a protein recruitment module.

NMR analysis of the architecture and functional remodeling of a modular multidomain protein, RPA.

Modular proteins with multiple domains tethered by flexible linkers have variable global architectures. Using the eukaryotic ssDNA binding protein, Replication Protein A (RPA), we demonstrate that NMR spectroscopy is a powerful tool to characterize the remodeling of architecture in different functional states. The first direct evidence is obtained for the remodeling of RPA upon binding ssDNA, including an alteration in the availability of the RPA32N domain that may help explain its damage-dependent phosphorylation.