Study Of Novel Disease-causing Gene And Molecular Mechanisms For Charcot-marie-tooth Disease

BACKGROUND AND PURPOSEThe Charcot-Marie-Tooth disease(CMT)is a group of inherited neurodegenerative disorders,which encompasses the peripheral motor and sensory neuropathies,exhibiting highly clinical and genetic heterogeneity.Here,we investigated an autosomal dominant CMT pedigree whose causative gene was unknown and further explored the role of the novel CMT causative gene in the peripheral nervous system by in vitro and in vivo functional studies.METHOD AND MATERIALCMT patients were diagnosed by clinical phenotype,electrophysiological and pathological examinations.After exclusion of common CMT causative gene with short tandem repeats(STR),we used whole exome sequencing(WES)to map the causative gene.Then,we carried out PCR and Sanger sequencing to screen the mutations in C1ORF194 in 107 patients with CMT.Next,we studied the mRNA and protein expression levels of wild type and mutant C10RF194 gene.We also detected the degradation rate and degradation pathway of C10RF194 protein.Then,we observed the cellular localization of C1ORF194 in neuroblastoma cell lines.Subsequently,we produced knock-in mice bearing the p.I121N mutation in Clorf194 to assess the in vivo consequences of the missense mutation.We analyzezd the physiology,behavioral and morphological of 4-month and 8-month-old mice.In addition,we also performed ITRAQ proteomics analysis on the sciatic nerves of wild type and heterozygous mice,and explored the molecular mechanisms of C10RF194-related CMT by immunoblotting and immunofluorescence.RESULTWe mapped a candidate locus of CMT to 1p21.2-p13.2 and identified two heterozygous missense mutations c.83A>T(p.K28I)and c.365T>A(p.I122N)and a splicing mutation(c.404-2_c.404-3delCA)in C1ORF194 from three unrelated Chinese families.In vitro functional assays demonstrated that variable clinical phenotypes observed from three families are due to different genetic mechanisms for these three mutations.The transgenic mouse model associated with a pathogenic CMT condition on peripheral neuropathy and recapitulates abnormal motor and sensory behaviors.Furthermore,we show that the mutant p.I121N was able to alter extracellular matrix interaction and calcium signaling pathway,which are required as previously identified in priming the cell for demyelination and axon degeneration,CONCLUSIONOur results collectively show that C1ORF194 is a novel CMT causative gene and these findings also link mutations in C1ORF194 to alter both ECM molecules and calcium homeostasis which leads to peripheral neuropathy.Our study broaden the new avenues for diagnosis and treatment of CMT.