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目的分析不同听力水平的中老年人GJB2基因的突变类型和基因型频率,探讨GJB2基因的各种突变和单核苷酸多态(SNPs)是否与年龄相关性耳聋的遗传易感性相关联。方法通过普查共收集到648例中老年人的听力学资料和血样,根据听力学检查结果将其分为四组。提取基因组DNA,经聚合酶链反应(Polymerase chainreaction,PCR)扩增GJB2基因编码区,利用直接测序方法获得所有样本的基因型,区分致病突变和多态性改变,利用统计学分析方法研究各种突变和多态在各组的分布情况。结果根据听力学结果将所有样本分为四组:正常对照组(157人)、轻度听力下降组(199人)、中度听力下降组(226人)、重度听力下降组(66人)。通过直接测序的方法,发现4种移码突变,包括235delC杂合突变16例、299-300delAT杂合突变3例、176-191del16杂合突变1例、512insAACG杂合突变2例;1种错义突变109G>A;6种多态,包括79G>A、341A>G、608 T>C、457G>A、368C>A、571T>C;3种已报道但与疾病关系不确定的突变11G>A、187G>T、558G>A;5种没有报道过的突变,包括14C>T、253T>C、377T>C、478 G>A、594G>A。4种移码致病突变,1种错义突变和3种常见多态在各组间的分布未发现显著性统计学差异(P>0.05)。结论虽然未发现GJB2基因与年龄相关性聋具有显著的关联性,但伴随着各组间听阈的逐渐升高,致病突变所占的比率也逐渐升高,提示GJB2基因可能是年龄相关性聋遗传易感性的微效致病基因之一;可能因关联性比较微弱,因此在此研究样本量的基础上未能检出。
Objective To analyze the type and genotype frequency of GJB2 gene in middle and old aged people with different hearing levels and to explore whether various mutations and single nucleotide polymorphisms (SNPs) of GJB2 gene are associated with the genetic susceptibility to age-related deafness. Methods The audiology data and blood samples of 648 middle-aged and old people were collected through census, which were divided into four groups according to the results of audiological examination. The genomic DNA was extracted and the coding region of GJB2 gene was amplified by polymerase chain reaction (PCR). The genotypes of all the samples were obtained by direct sequencing. The pathogenic mutations and polymorphisms were identified. Statistical analysis Mutation and polymorphism in each group distribution. Results According to the results of audiology, all samples were divided into four groups: normal control group (157), mild hearing loss group (199), moderate hearing loss group (226) and severe hearing loss group (66). Four kinds of frameshift mutations were found by direct sequencing, including 235delC heterozygous mutation, 299-300delAT heterozygous mutation, 176-191del16 heterozygous mutation and 512insAACG heterozygous mutation. One missense Mutations 109G> A; six polymorphisms, including 79G> A, 341A> G, 608T> C, 457G> A, 368C> A, 571T> A, 187G> T, 558G> A; 5 kinds of unreported mutations, including 14C> T, 253T> C, 377T> C, 478G> A and 594G> A. Four kinds of frameshift disease-causing mutations, a missense mutation and three common polymorphisms in the distribution of the group did not find a significant statistical difference (P> 0.05). Conclusions Although GJB2 gene was not found to be significantly associated with age-related deafness, with the gradual increase of hearing threshold in each group, the proportion of pathogenic mutation also gradually increased, suggesting GJB2 gene may be age-related deafness Genetic susceptibility to one of the microscopic pathogenic genes; may be due to the weak association, therefore, based on the sample size of this study failed to detect.