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目的 :在多年围绕1个常染色体显性遗传性非综合征型听神经病家系开展系统分子遗传学研究的基础上,进一步探讨该家系耳聋的致病机制,以期发现新的听神经病致病基因和突变位点。方法:对3例耳聋患者和1例配偶进行全外显子组测序,初步筛选出与家系耳聋相关的候选致病基因。采用PCR-Sanger测序法,检测上述候选基因变异是否与家系表型共分离。最后,以50例与研究家系无关的听力正常人为对照,检测候选致病突变在正常群体中的突变频率和SNPs遗传多态性。结果:全外显子测序分析得到41个候选致病基因突变;用PCR-Sanger测序法对核心家系的9名成员和2名家系外听力正常人进行验证,仅发现1个基因突变(ALOX15B 7942797 C>T)与家系耳聋表型共分离。选取50例家系外正常对照的DNA样本对ALOX15B基因进行PCR扩增和序列分析,结果显示有2例听力正常人也检测到该基因的同一变异,提示该变异为SNPs遗传多态性。结论:对核心家系成员的全外显子组测序分析和Sanger测序法验证未发现有意义的突变位点,排除了该家系耳聋由基因编码区突变及Indels致病的可能性。
OBJECTIVE: To further investigate the pathogenesis of deafness in this pedigree based on systematic molecular genetics research on a pedigree of autonomic dominant nonsyndromic auditory neuropathy for many years, with a view to discovering new pathogenic genes of auditory neuropathy and Mutation site. Methods: Three extremities deafness patients and one spouse were enrolled in the whole exome sequencing, and the candidate pathogenic genes related to family deafness were screened out. PCR-Sanger sequencing was used to determine whether the above candidate gene mutations were co-segregated with family phenotypes. Finally, the mutation frequencies and SNPs genetic polymorphisms of candidate disease-causing mutations in normal population were tested in 50 cases of normal hearing-unrelated people. RESULTS: Forty-one candidate mutations were detected by whole-exome sequencing. Nine members of the nuclear pedigree and two non-family members of the hearing-impaired family were verified by PCR-Sanger sequencing and only one mutation was found (ALOX15B 7942797 C> T) co-segregation with family deafness phenotype. DNA samples of 50 normal controls were selected to amplify and sequence the ALOX15B gene. The results showed that 2 of the normal controls also detected the same mutation of this gene, suggesting that this mutation is a SNP genetic polymorphism. Conclusion: There is no significant mutation locus in the whole exome sequencing and Sanger sequencing of core family members, which excludes the possibility of deafness mutation in the pedigree and Indels pathogenicity.