title: Clinical, in silico, and experimental evidence for pathogenicity of two novel splice site mutations in the SH3TC2 gene.
abstract: Charcot-Marie-Tooth (CMT) neuropathy is the most common inherited neuromuscular disorder. CMT is genetically very heterogeneous. Mutations in the SH3TC2 gene cause Charcot-Marie-Tooth neuropathy type 4C (CMT4C), a demyelinating form with autosomal recessive inheritance. In this study, two novel splice site mutations in the SH3TC2 gene have been studied (c.279G → A, c.3676-8G → A). Mutation c.279G → A was detected on one allele in two unrelated families with CMT4C in combination with a known pathogenic mutation (c.2860 C →T in one family, c.505T → C in the other) on the second allele of SH3TC2 gene. Variant c.3676-8G → A was detected in two patients from unrelated families on one allele of the SH3TC2 gene in combination with c.2860C →T mutation on the other allele. Several in silico tests were performed and exon trap experiments were undertaken in order to prove the effect of both mutations on proper splicing of SH3TC2. Fragments of SH3TC2 were subcloned into pET01 exon trap vector (Mobitec) and transfected into COS-7 cells. Aberrant splicing was predicted in silico for both mutations, which was confirmed by exon trap analysis. For c.279G → A mutation, 19 bases from intron 3 are retained in cDNA. The mutation c.3676-8G→ A produces a novel splice acceptor site for exon 17 and complex changes in splicing were observed. We present evidence that mutations c.279G → A and c.3676-8G →A in the SH3TC2 gene cause aberrant splicing and are therefore pathogenic and causal for CMT4C.
authors.affiliation: DNA laboratory, Department of Paediatric Neurology, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech Republic. petra.lassuthova@gmail.com
authors.forename: Petra
authors.initials: P
authors.lastname: Laššuthová
authors.forename: Martin
authors.initials: M
authors.lastname: Gregor
authors.forename: Lenka
authors.initials: L
authors.lastname: Sarnová
authors.forename: Eliška
authors.initials: E
authors.lastname: Machalová
authors.forename: Radek
authors.initials: R
authors.lastname: Sedláček
authors.forename: Pavel
authors.initials: P
authors.lastname: Seeman
mesh_terms: D000328:Adult; D000818:Animals; D019556:COS Cells; D002607:Charcot-Marie-Tooth Disease / Q000235:genetics* / Q000473:pathology / Q000503:physiopathology; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D005192:Family Health; D005260:Female; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D009431:Neural Conduction / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D012016:Reference Values; D014162:Transfection
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C488620:SH3TC2 protein, human