1. Differential increases of specificFMR1mRNA isoforms in premutation carriers
- Author
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Hiu Tung Tang, Blythe Durbin-Johnson, Carolyn M. Yrigollen, John Eid, Paul J. Hagerman, Chris Raske, Erick Loomis, Dalyir I. Pretto, and Flora Tassone
- Subjects
Adult ,Male ,Gene isoform ,Molecular Sequence Data ,Primary Ovarian Insufficiency ,Biology ,Real-Time Polymerase Chain Reaction ,Article ,Fragile X Mental Retardation Protein ,Exon ,Tremor ,RNA Isoforms ,Genetics ,Humans ,RNA, Messenger ,Allele ,Genetics (clinical) ,DNA Primers ,Gene Library ,Messenger RNA ,Base Sequence ,Gene Expression Profiling ,Alternative splicing ,RNA ,Sequence Analysis, DNA ,FMR1 ,Molecular biology ,Gene Components ,Fragile X Syndrome ,RNA splicing ,Ataxia ,Female - Abstract
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.
- Published
- 2014