Detection of gene hot spots mutation in genetic deafness associated gene by polymerase chain reaction-reverse dot blot technique

Abstract

Objective To verify the accuracy and effect of polymerase chain reaction-reverse dot blotting (PCR-RDB) technique in genetic deafness associated gene test. Methods A total of 250 patients with nonsyndromic hearing impairment(NSHI) who were diagnosed in Guangdong Women and Children Hospital from January 2014 to October 2016, were chosen as research objects. With PCR-RDB technique, we detected several genes associated with NSHI in genome DNA from peripheral blood cells of all the patients. To carry out PCR-RDB, the gene chip kit for hereditary hearing loss was used, which can detect 12 hot spots mutation of four genes in Chinese, including GJB2 (35del G, 176_191del 16, 235del C, 299_300del AT), GJB3 (538C>T), SLC26A4 (1174A>T, 1226G>A, 1229C>T, IVS7-2A>G, 2168A>G) and mitochondrion MTRNR1 (1494C>T, 1555A>G). Meanwhile, all the samples were also tested by Sanger sequencing, in order to compare and validate the results to that of PCR-RDB technique. This study was in line with the World Medical Association Declaration of Helsinki revised in 2013 and informed contents were obtained from all patients. Results ①There was 51.6%(129/250) genetic deafness associated gene mutations in DNA sample of 250 patients of NSHI, and some of which were 28.4%(71/250) from GJB2 gene, 19.2%(48/250) from SLC26A4 gene, 4.4%(11/250) from mitochondrion MTRNR1 gene and 2.0%(5/250) from GJB3 gene, respectively. Among 129 cases of deafness associated gene mutations, 86.8%(112/129) was met with pathogenicity characteristics of hereditary disease. ② All detection results of 12 hot spots mutations of deafness associated gene by PCR-RDB were consistent with those of by Sanger sequencing method. ③The sensitivity, specificity, total consistency, positive predictive value and negative predictive value of detection results of 12 hot spots mutations of deafness associated gene by PCR-RDB technique were all 100.0%. Conclusions Although detection results of gene hot spots mutation in genetic deafness associated gene by PCR-RDB technique were consistent with those of by Sanger sequencing method, whether it is worthy of clinical application, multicenter and large sample randomized controlled trial should be conducted as sample size of this study is still small. Key words: Nonsyndromic deafness; Polymerase chain reaction; Oligonucleotide array sequence analysis; Genotype; Nucleic acid hybridization; Mutation