Mutation Analysis Of TPRN Gene And Construction Of TPRN SiRNA Plasmid Expression Vector

Hereditary deafness is the most common hereditary disease. The incidence of congenital hearing loss is estimated at1in1,000births in the world, and about30,000deaf children with hearing loss are born in China every year. Sixty per cent of the hearing-loss disorders attributable to genetic causes,70%are classified as non-syndromic and the remaining.30%as syndromic. Autosomal recessive deafness is the most common form of genetic hearing loss, which accounts for nearly77%in non-syndromic hearing loss. To date,73loci and40genes have been reported for autosomal recessive non-syndromic hearing loss.The TPRN gene encodes taperin which is prominently expressed in stereocilia of hair cell. Mutations in TPRN were firstly proved to be responsible for autosomal recessive deafness-79(DFNB79) in2010.To identify the frequency and characteristic of mutations in TPRN gene in Chinese with prelingual nonsyndromic hearing impairment, sporadic congenital deaf patients and patients from hereditary prelingual deafness families in China were investigated in this study. Genomic DNA extracted from the cases was screened with DNA microarrav and sequenced to detect mutation in the exons of TPRN gene.To investigate the mechanism of Taperin on hearing fuction, we constructed TPRN siRNA plasmid expression vector to knockdown TPRN gene to discover the effect of Taperin protein on the morphology and function of the hair cells.Chapter1:Screening of the TPRN gene in Chinese hearing impairment patients with non-syndromic hearing lossObjective:To investigate the prevalence of TPRN gene mutation in Chinese hearing impairment patients with non-syndromic hearing loss.Methods:81non-syndromic hearing impairment patients and52normal controls were tested for TPRN gene screening with direct sequencing of DNA. Genomic DNA was taken from formal consent were obtained. The patients, who were detected with mutant alleles of9hot mutation spots in4deafness genes with microarray, were excluded. The other patients were subjected to further DNA sequencing of DNA samples to detect mutation in TPRN gene.Results:Two patients were detected to carry the same heterozygous mutations559G>T in exon1, which lead to the187amino acid substitute from Serine to Alanine (A187S). Two synonymous mutations157C>T and318C>T were found in the patients and controls. As the A187S were located in conservative amino acid sequence by analysis of homology, and the mutations found in the patients was not detected in the control subjects, it may be related to hearing loss.Conclusion:Mutation of TPRN gene is not common in Chinese patients with nonsyndromic hearing loss. Further studies for the gene should be made in our laboratory. Chapter2:Construction and identification of TPRN siRNA plasmid expression vectorObjective:To prepare for knockdown of TPRN gene, TPRN siRNA plasmid expression vector was constructed.Methods:Two TPRN siRNAs and one negative control siRNA, which were used to be inserted into pGenesil-2plasmids, had been designed and synthesized. The recombinant plasmids were identificated by PCR and restriction endonuclease.Results:TPRN siRNAs were detected in the recombinant plasmids.Conclusion:TPRN siRNA plasmid expression vector, pGenesil-2-tprn-siRNA, has been constructed and identificated successfully.