Clinical and molecular implications of mosaicism in FMR1 full mutations

Expansions of more than 200 CGG repeats (full mutation) in the FMR1 gene give rise to fragile X syndrome (FXS) through a process that generally involves hypermethylation of the FMR1 promoter region and gene silencing, resulting in absence of expression of the encoded protein, FMRP. However, mosaicism with alleles differing in size and extent of methylation often exist within or between tissues of individuals with FXS. In the current work, CGG-repeat lengths and methylation status were assessed for eighteen individuals with FXS, including 13 mosaics, for which peripheral blood cells (PBMCs) and primary fibroblast cells were available. Our results show that for both PBMCs and fibroblasts, FMR1 mRNA and FMRP expression are directly correlated with the percent of methylation of the FMR1 allele. In addition, Full Scale IQ (FSIQ) scores were inversely correlated with the percent methylation and positively correlated with higher FMRP expression. These latter results point toward a positive impact on cognition for full mutation, methylation mosaics with lower methylation compared to individuals with fully methylated, full mutation alleles. However, we did not observe a significant reduction in the number of seizures, nor in the severity of hyperactivity or autism spectrum disorder, among individuals with mosaic genotypes in the presentation of FXS.These observations suggest that low, but non-zero expression of FMRP may be sufficient to positively impact cognitive function in individuals with FXS, with methylation mosaicism (lowered methylation fraction) contributing to a more positive clinical outcome

The Role of AGG Interruptions in the Transcription of FMR1 Premutation Alleles

Fragile X associated disorders are caused by a premutation allele in the fragile X mental retardation 1 gene (FMR1) and are hypothesized to result from the toxic effect of elevated levels of expanded FMR1 transcripts. Increased levels of FMR1 mRNA have indeed been reported in premutation carriers; however the mechanism by which expanded alleles lead to elevated levels of FMR1 mRNA in premutation carriers is unknown. Within the CGG repeat tract AGG interruptions are found, generally 1–3 present in normal/intermediate alleles (6–54 CGG repeats) and usually 0–1 in premutation alleles (55–200 CGG repeats). They are present at specific locations, generally occurring after 9 or 10 uninterrupted CGG repeats [(CGG)9AGG(CGG)9AGG(CGG)n]. We evaluated both the number of AGG interruptions and the resulting length of the uninterrupted 3′ CGG repeat pure tract in premutation alleles derived from two large cohorts of male and female carriers to determine whether the presence of AGG interruptions or the length of a pure stretch of CGG repeats influence the levels of FMR1 mRNA in blood. Our findings indicate that neither the number of AGG interruptions, nor their position along the CGG tract have a significant affect on mRNA levels in premutation carriers. We also, as expected based on previous findings, observed a highly significant correlation between CGG repeat number (as both total length and length of pure CGG stretch) and FMR1 mRNA expression levels, in both males and females. Importantly, we did not observe any significant difference in FMR1 mRNA levels in premutation carriers based on age

CRISPR to the Rescue: Advances in Gene Editing for the <i>FMR1</i> Gene

Gene-editing using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is promising as a potential therapeutic strategy for many genetic disorders. CRISPR-based therapies are already being assessed in clinical trials, and evaluation of this technology in Fragile X syndrome has been performed by a number of groups. The findings from these studies and the advancement of CRISPR-based technologies are insightful as the field continues towards treatments and cures of Fragile X-Associated Disorders (FXADs). In this review, we summarize reports using CRISPR-editing strategies to target Fragile X syndrome (FXS) molecular dysregulation, and highlight how differences in FXS and Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) might alter treatment strategies for each syndrome. We discuss the various modifications and evolutions of the CRISPR toolkit that expand its therapeutic potential, and other considerations for moving these strategies from bench to bedside. The rapidly growing field of CRISPR therapeutics is providing a myriad of approaches to target a gene, pathway, or transcript for modification. As cures for FXADs have remained elusive, CRISPR opens new avenues to pursue

Molecular biomarkers predictive of sertraline treatment response in young children with fragile X syndrome

ObjectivesSeveral neurotransmitters involved in brain development are altered in fragile X syndrome (FXS), the most common monogenic cause of autism spectrum disorder (ASD). Serotonin plays a vital role in synaptogenesis and postnatal brain development. Deficits in serotonin synthesis and abnormal neurogenesis were shown in young children with autism, suggesting that treating within the first years of life with a selective serotonin reuptake inhibitor might be the most effective time. In this study we aimed to identify molecular biomarkers involved in the serotonergic pathway that could predict the response to sertraline treatment in young children with FXS.MethodsGenotypes were determined for several genes involved in serotonergic pathway in 51 children with FXS, ages 24-72months. Correlations between genotypes and deviations from baseline in primary and secondary outcome measures were modeled using linear regression models.ResultsA significant association was observed between a BDNF polymorphism and improvements for several clinical measures, including the Clinical Global Impression scale (P=0.008) and the cognitive T score (P=0.017) in those treated with sertraline compared to those in the placebo group. Additionally, polymorphisms in the MAOA, Cytochrome P450 2C19 and 2D6, and in the 5-HTTLPR gene showed a significant correlation with some of the secondary measures included in this study.ConclusionThis study shows that polymorphisms of genes involved in the serotonergic pathway could play a potential role in predicting response to sertraline treatment in young children with FXS. Larger studies are warranted to confirm these initial findings

Genes Controlling Affiliative Behavior as Candidate Genes for Autism

Photograph of a scene at the ruins of Fort Nichols, along the Santa Fe Trail

Differential increases of specific FMR1 mRNA isoforms in premutation carriers

BackgroundOver 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&nbsp;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.MethodsTo address this question, we have applied a long-read sequencing approach using single-molecule real-time (SMRT) sequencing and qRT-PCR.ResultsOur 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.ConclusionsThese 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

Macrophage migration inhibitory factor and autism spectrum disorders

BACKGROUND: Autism-spectrum disorders (ASD) are childhood neurodevelopmental disorders characterized by social and communicative impairment and repetitive and stereotypical behavior. Macrophage migration inhibitory factor (MIF) is an upstream regulator of innate immunity that promotes monocyte/macrophage activation responses by increasing the expression of Toll-like receptors and inhibiting activation-induced apoptosis. Based on results of prior genetic linkage studies and reported altered innate immune response in ASD, we hypothesized that MIF could represent a candidate gene for ASD or its diagnostic components. METHODS: Genetic association between ASD and MIF was investigated in two independent sets of families of probands with ASD, from USA (527 participants from 152 families) and Holland (532 participants from 183 families). Probands and their siblings, when available, were evaluated with clinical instruments used for ASD diagnoses. Genotyping was performed for two polymorphisms in the promoter region of the MIF gene in both samples sequentially. In addition, MIF plasma analyses were carried out in a subset of Dutch patients from whom plasma was available. RESULTS: There were genetic associations between known functional polymorphisms in the promoter for MIF and ASD-related behaviors. Also, probands with ASD exhibited higher circulating MIF levels than did their unaffected siblings; the amount of MIF in the plasma correlated with the severity of multiple ASD symptoms. CONCLUSIONS: These results identify MIF as a susceptibility gene for ASD. Further research is warranted on the precise relationship between MIF and the behavioral components of ASD, the mechanism by which MIF contributes to ASD pathogenesis, and the clinical utility of MIF genotyping