| Background:Deafness mostly due to genetic factors is one of the most common diseases with defective sensory system, genes related deafness which has a major feature of high genetic heterogeneity has more than 200. According to the different phenotype, hereditary deafness is divided into two categories, one is syndromic type deafness( SHL) accompanied by other systems’ symptoms in addition to hearing loss; another kind which accounted for more than 70% is non syndrome type deafness(NSHL) without other clinical symptoms in addition to hearing loss. Now many studies have showed that deafness is mainly related to4 genes(GJB2, GJB3, SLC26A4, mt DNA), the clinical deafness gene diagnosis also focus on hotspot mutations of these 4 genes. However, these hotspot mutations were not found in a considerable part of the NSHL patients, namely these patients can not get early diagnosis, so how to improve the rate of genetic diagnosis of deafness become a hot problem.Objective:The technology of matrix-assisted laser desorption ionization time of flight mass spectrometry accompanied by high-throughput gene trap technology and Sanger sequencing was applied to get statistics of hotspot mutations in common disease-causing genes in patients from Shanxi with NSHL and to look for new disease-causing hotspot mutations, which can provide evidence for early diagnosis and treatment of deafness.Methods:300 blood samples with NSHL from different region schools for deaf in Shanxi province were collected by Taiyuan City Central Hospital. Firstly, matrix-assisted laser desorption ionization time of flight mass spectrometry was used to screen 20 hotspot mutations in the 4 common disease-causing genes for 300 collected samples, and then get statistics of 20 common hotspot mutations in sporadic population with NSHL.Secondly,we take advantage of high-through gene trap technology to detect 20 samples selected in patients without mutations in 20 hotspot mutations and then get other hotspot statistics.Aiming at new hotspot mutations,online feature predictive software were applied to predict the damaging to protein function. Finally, hotspot mutations which found by next-generation sequencing were detected by Sanger sequencing in remaining samples to identify the hotspot mutations, at the same time, there were 200 cases of normal specimens for comparison to new hotspot mutations.Results:1. 152 cases with common mutations were found in 300 cases by matrix-assisted laser desorption ionization time of flight mass spectrometry; No mutations in GJB3 gene were found in 300 samples.2. In 148 remaining cases, 20 cases were detected by high-through gene trap technology; Three hotspot mutations were found: c.1334 T>G(p.L445W) in SLC26A4 gene 、c.2596 G> A(p.D866N) in WFS1 gene、c.5662 C>T(p.H1888Y) in MYO7 A gene;The two mutations of c.1334 T>G and c.2596 G> A have been reported as causative mutations, and the other mutation of c.5662 C>T is the first discovery as suspicious pathogenic mutation.3. Three hotspot mutations found by next-generation sequencing were detected by Sanger sequencing in remaining cases; Mutation of c.2596 G> A was found in 3 cases, and mutations of c.1334 T>G and c.5662 C>T were found in no cases, in addition to, normal control group were not found mutation of c.5662 C>T,pathogenicity of the new mutation needs further study.Conclusion:1. The major causative genes for hereditary deafness in Shanxi province are GJB2、SLC26A4、mt DNA,but the spectrum of mutations is obviously different in respective sites;Mutations of c.235 delC and c.299-300 delAT are the hotspot mutations in GJB2 gene,mutations of IVS7-2A>G and c.2168A>G are the hotspot mutations in SLC26A4 gene,mutation of c.1555A>G is the hotspot mutation in mitochondrial 12 S r RNA gene.2. The mutation of c.2596 G> A( p.D866N) in WFS1 gene is related with NSHL in Shanxi province; Mutation of c.1334 T>G carries different rate in different races.3. The mutation of c.5662C>T(p.H1888Y)in MYO7 A gene is first discovered as suspicious pathogenic mutation. |
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