Gene Mutation Detection Of PMP22, CX32, MFN2in CMT And The Functional Research Of CX32Protein

Background and purpose:Charcot-Marie-Tooth disease (CMT) is the most common form of Hereditary neuropathies are a genetically heterogeneous group of diseases that affect the peripheral nervous system.And it’s also called the hereditary motor and sensory neuropathy (HMSN).CMT1is the most commo type of CMT, account for50%. And CMT1A account70%of CMT1, which results from the duplication of a genomic fragment that encompasses the PMP22gene. CMT2is Autosomal-Dominant axonal neuropathies, account20%～40%of CMT, And CMT2A account8%-%of CMT2, which results from the mutation of MFN2gene. CMTX account10%～15%of CMT,as the second most type of CMT.CMTX1account90%of CMTX, which results from CX32gene mutation. Up to date, about50loci and30genes have been identified. However, it should be noted that there is not a good genotype-phenotype correlation and that great variability exists, both within and between families, regarding the degree of clinical expression. There is insufficient evidence to determine the usefulness of routine genetic testing in patients with system,convenient and effective way.In this research,we screening the mutation of the most common CMT disease-causing of PMP22, CX32and MFN2. And build CX32(c.65G>A) plasmid to validation of point mutations on the expression of CX32function.Methods:1. Deteced70proband patients with PCR-RFLP analysis based on PCR-based test that amplified a3.6kb region including the1.7kb hotspot from specific CMT1A-repetitions (CMT1A-REPs).2. Thirty-four CMT proband patients were selected for CX32mutation screening after the exclusion of the PMP22duplication and male-to-male transmission.Mutation analysis was carried out by using PCR combined with direct sequencing.3. Twenty-three CMT proband patients were selected for MFN2mutation screening with clinical features, electrophysiological or pathology prompt CMT2type.Mutation analysis was carried out by using PCR combined with direct sequencing.4. Cloning CX32(c.65G>A) pEGFP-N1-wtCX32and pEGFP-N1-mtCX32plasmid,then transfectign into Hela cells to study CX32function expression.Results:1. Find one case of PMP22large fragment duplication mutation positive family and one case of PMP22deletion mutation positive family.2. Five CX32gene mutations were detected in6CMT families: c.37G>A, c.65G>A,c.246C>G,c.256A>G and c.533A>G, with c.246C>G and c.533A>G firstly reported. CMT1X is the second most common form of CMT, with a frequency of about9%in our study.3. Two MFN2gene mutations were detected:c.280C>T and c.395G>A.The mutation frequency of MFN2in CMT2is8%.4. Successful cloning of CX32(c.65G>A) plasmid, and transiently transfected into Hela cells. The mutation c.65G>A deffect the formation of the gap junction plaques on the cell membrane, wich underlie the pathogenesis of CMT1X disease.Conclusion:1. PCR-RFLP analysis as the first line gene screening method of PMP22large fragment mutation is still uncertainty.2. CX32and MFN2gene point mutation could tested by PCR combined with direct sequencing. The mutation frequency of these two genes is consistent with international reports. With c.246C>G and c.533A>G of CX32gene and c.395G>A of MFN2gene firstly reported.3. Successful cloning of CX32(c.65G>A) pEGFP-N1-wtCX32and pEGFP-N1-mtCX32plasmid, and transiently transfected into Hela cells. The next steps are building stable transfected strain, cloning other mutation point especially c.246C>G and c.533A>G and then do more further protein function research.