title: Differential increases of specific FMR1 mRNA isoforms in premutation carriers.
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.
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, California, USA.
authors.forename: Dalyir I
authors.initials: DI
authors.lastname: Pretto
authors.affiliation: Pacific Biosciences, Menlo Park, California, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Eid
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, California, USA.
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Yrigollen
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, California, USA.
authors.forename: Hiu-Tung
authors.initials: HT
authors.lastname: Tang
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, California, USA.
authors.forename: Erick W
authors.initials: EW
authors.lastname: Loomis
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, California, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Raske
authors.affiliation: Department of Public Health Sciences, University of California Davis, School of Medicine, Davis, California, USA.
authors.forename: Blythe
authors.initials: B
authors.lastname: Durbin-Johnson
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, California, USA MIND Institute, University of California Davis Medical Center, Sacramento, California, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hagerman
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, California, USA MIND Institute, University of California Davis Medical Center, Sacramento, California, USA.
authors.forename: Flora
authors.initials: F
authors.lastname: Tassone
mesh_terms: D000328:Adult; D001259:Ataxia / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics* / Q000378:metabolism*; D005600:Fragile X Syndrome / Q000235:genetics*; D040461:Gene Components; D020869:Gene Expression Profiling; D015723:Gene Library; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016649:Primary Ovarian Insufficiency / Q000235:genetics*; D059368:RNA Isoforms / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D014202:Tremor / Q000235:genetics*
chemical_list: D017931:DNA Primers; D051860:Fragile X Messenger Ribonucleoprotein 1; D059368:RNA Isoforms; D012333:RNA, Messenger; C497683:FMR1 protein, human
keywords: Genetics