Sequence Analysis Of The Upstream Regulation Sequences Of GJB2Based On GJB2235delC Heterozygous Deaf Patients

PartI: Mutational analysis of GJB2and mitochondrial12S rRNA genesin patients with non-syndromic hearing lossObjective: To investigate the mutation types and frequencies of both GJB2andmitochondrial12SrRNA genes in patients with non-syndromic hearing loss; and toprovide theoretical basis for molecular testing and genetic counseling of deafness.Methods: The blood samples were obtained from190clinic patients withnon-syndromic hearing loss. Genomic DNA was isolated from the peripheralleukocytes using the Puregene DNA Isolation Kits. The subject’s DNA fragmentsspanning the coding area of GJB2gene and mitochondrial12S rRNA gene werePCR amplified. Each fragment was purified and analyzed by direct sequencing toidentify deafness-associated mutations.Results: There were totally23kinds of GJB2sequence variants and43kinds ofmitochondrial12SrRNA sequence variants identified in190patients. Of which,5kinds of GJB2variants and3kinds of mitochondrial12SrRNA variants were knowndisease-causing mutations, with highest frequencies found in235delC of GJB2gene(16.32%) and A1555G in the mitochondrial12SrRNA gene (6.32%) respectively.Deafness-related heterozygous GJB2mutations occurred in25patients (13.16%), ofwhich14were GJB2235delC heterozygote (7.37%). In addition,4cases carried bothGJB2235delC monoallelic mutation and mitochondrial12S rRNA mutation.Conclusions: Both GJB2gene and mitochondrial12SrRNA gene are hot spots for mutations causing non-syndromic hearing loss. Genetic testing for these genesmay be helpful in identifying the molecular etiology of deafness patients. As GJB2235delC monoallelic variant was detected in a high frequency, it seems needed tofurther clarify the molecular mechanisms underling the hearing loss with thismutation. PartII: Sequence analysis of the upstream regulation sequences of GJB2gene in patient carrying the GJB2235delC monoallelic mutationObjective: To explore the correlation between mutations of the upstreamregulation sequences of the GJB2gene and deafness in patients carrying the GJB2235delC monoallelic mutation.Methods:14deaf patients who harbored heterozygous GJB2235delC mutation,20patients without virulence gene, and25normal-hearing controls were included.PCR amplification and mutation analysis of the upstream regulation sequences of theGJB2gene in these individuals were conducted.Results: Two kinds of sequence variants-195T>C and-843A>C were found inthe14deaf patients. However, none of them was deafness-causing mutation ascompared with control sequences.Conclusion: The mutation in the upstream regulation sequences of GJB2geneseems not a common co-factor of deafness in patients carrying GJB2heterozygousmutation.