Analysis Of Chinese Common GJB2 Mutation And Study Of Subcellular Localization Of Mutant Protein

Hearing impairment affects 1 in 1000 infants.Approximately 50%of congenital hearing loss is hereditary,and 80%of hereditary hearing loss is inherited in an autosomal recessiveway.Mutations in the connexin26(Cx26) gene(GJB2) are known as a major cause of autosomal recessive nonsyndromic sensorineural hearing loss(DFNB1).The most frequent mutation of GJB2 associated with deafness is 235delC,which is common in chinese populations.Some other relative high frenquency of GJB2 mutation are 299-300delAT and 176del16bp.The GJB2 235delC,299-300delAT,176del16bp mutation are all frameshift mutation.Along with the exciting development in genome medicine and medical molecular bioiogy,the genetic testing for deafness has been applied in clinic.Since 2008,the ENT department of our hosipital has begun the clinical diagnosis of common gennetic testing for deathess.Our study summarize and discuss the cases of GJB2 mutation,that were detected by common gennetic chip testing for deafness.In order to provide a solid foundation for futher experimental studies on the mechanism of hearing loss about chinese frenquent mutation,we constrcut eukaryotic expression vectors of these frameshift mutations.The aim of the present study was to identify that the expression and subcellucar of three mutations(235delC,299-300delAT and 176delbp) affect the function of GJB2 using molecular biological techniques.1 Clinical analysis of deafness genetic gene chip for GJB2 mutationAll the 120 deafness patients that were detected by deafness gene chip,there are 16 cases of GJB2 mutation.In 120 cases of deaf patients,the existence of GJB2 mutation accounted for 13.33%(16/120).Ther are 6 cases of 235delC/235delC;3 cases of 235delC/299-300delAT; 1 case of 235delC/176dell6bp;1 case of 235delC/35delG,5 cases of 235delC/WT.Our record accords with the research of molecular epidemiology of deafness in China.Although the most frequency mutation is 235delC,and next are 299-300delAT and 176del16bp,there are less reports on the mechanism of these three franmeshift mutations in China. 2 Subcellular localization of Chinese common GJB2 mutant proteinThe 235delC,299-300delAT and 176del16bp mutations are all the hot spots of GJB2 mutation in chinese population.They all result in frameshift mutation and premature termation.These mutations prouduces a short,defective protein of some amino acids,not the normal 226 amino acids.In the vitro study of deafness mechanism,genes containing the mutation are usually cloned into the definite expression vector,and the cells are tranfected with the recombinant DNA sanmples by some tranfection method.The protein that expessed by mutant gene are determined,using antibodies against amino acid sequences of the protein.Because of the limitation of prorein immunoblot analysis,the provided messages are limated.In order to study the deafness mechanism of common GJB2 gene mutations in the Chinese population,we construct the EGFP(Enhanced Green Fluorescent Protein)fusion protein vectors with the site -directed mutagenesis and the TA cloning technique.Then, HEK-293 cell were respectively tranfected by the 235delC,299-300delAT and 176del16bp EGFP fusion protein vectors.Our results obtained indicated that these three mutation proteins are deeply impaired in their intracelluar trafficking and fails to reach the plasma membrane.Gap junctions are membrane specializations containing intercellar for the passage of a wide variety of small molecules,including ions nucletides,siRNAs,and inositol phosphate.When the mutant protein fail to reach the plasma membrane,the physiological function of Cx26 would be impaired.Therefore,the mutant protein in my study may cause human deafness.In conclusion,our results are useful to understand the pathogenesis of the GJB2 mutation which is common in Chinese population.