迟发性耳聋家系临床表型及相关基因突变研究

发布时间：2018-08-17 09:05

【摘要】：目的：耳聋是导致人类交流障碍最常见的疾病，，其病因中最主要的因素为遗传因素，而听力正常人群中耳聋基因的携带率并不低，这也是耳聋发病率之所以居高不下的重要原因之一。虽然目前国内外学者对遗传性耳聋的研究很多，但仍有一部分耳聋病人无法确定其致病原因。本课题组收集了5个迟发性耳聋家系，拟通过分析和总结其临床表型的特征与基因型的相关性，探讨常见迟发性耳聋家系的分子基因，为这些家庭提供遗传咨询和婚育指导，力争丰富遗传性耳聋基因突变谱。方法：收集病例，采集家系信息，分别编号为：HBZ-01、HBZ-02、HBX、HBD、HBM。收集家系成员血液样本，提取外周静脉血基因组DNA，采取PCR扩增后直接测序，首先筛查国人常见突变基因，包括GJB2、SLC26A4以及线粒体基因（mtDNA12S rRNA）。筛查阳性者，分析其临床表型与基因型的相关性，对耳聋患者及家庭成员提供遗传咨询；筛查阴性者，依据表型相似性原则，在目前已定位的DFNA中筛选可疑致病基因，阳性者，同前；阴性者，进行耳聋基因组高通量测序，分析可疑的致病基因位点，阳性者对该突变点进行PCR扩增，扩增产物进行Sanger测序验证，依旧为阴性者下一步将考虑进行全外显子组测序。结果: 1在1个遗传方式不明的耳聋小家系（HBZ-01家系）中检测出GJB2基因的复合杂合突变c.35insG/c.232GA，家系耳聋成员中相同的突变类型却出现不同的临床表型，患者发病年龄为0~20岁，均为感音神经性耳聋，语后聋患者早期表现为高频听力下降，后逐渐累及中、低频。c.35insG/c.232GA复合杂合突变在HBZ-01家系内与耳聋表型共分离。初步结果显示，在耳聋人群中GJB2基因的这种复合杂合突变较少。 2在1个表现为常染色体显性遗传非综合征型耳聋的河北家系（HBZ-02家系）中，发现该家系的表型中除了听力下降外，部分患者还伴有前庭功能障碍。我们将可能的致病基因锁定在已报道的COCH基因上，筛查后在其第4和11外显子上发现了新的突变位点，其中第4外显子上为致病位点，c.226GA (p.76AT)，该突变与临床表型共分离，在多个物种间亦为保守性序列。第11外显子上为c.1055GA (p.352TS)，后经验证发现该位点为多态性改变。COCH基因是为数不多的耳蜗和前庭共相关基因，对其分子机制的全面认识还需深入的研究。HBZ-02家系的临床表型与以往报道的DFNA9家系相似，这也进一步证实了表型相似性原则，准确判断特征性的遗传性耳聋表型对指导基因型分析具有重要意义。 3在1个常染色体显性非综合征型耳聋家系（HBX家系）中首次发现了中国人群中报道较少见的EYA4基因突变，c.544_545insA，该位点突变位于EYA4基因第8外显子，导致氨基酸的移码突变（p.E182X），c.544_545insA突变在HBX家系家系内与耳聋表型共分离。与以往国外报道的DFNA10家系不同的是，该家系听力曲线图不是典型的“谷型”图而是平坦型或陡降型，发病年龄跨度较大，分布在20~40岁之间，部分成员伴有耳鸣。该突变也是首次发现的新突变。 4其他2个家系(HBM家系、HBD家系)，利用基于下一代测序技术和外显子捕获技术的耳聋基因组高通量测序（含已知的130个耳聋基因），未发现明确的致病基因，提示这两个家系可能由一些少见的或未知的耳聋基因缺陷致聋，下一步可考虑进行全外显子组测序。结论： 1由于遗传性耳聋遗传的异质性，不同基因的突变可以导致同一种临床表型；同一基因不同位点的突变可以导致不同的临床表型，甚至是同一基因同一突变位点在同一家系的不同个体之间表型也可能不同。 2准确识别特征性的耳聋临床表型可以指导基因型的诊断。 3目前仍有许多耳聋患者的致病基因不明，遗传性耳聋基因数据库仍待完善。
[Abstract]:Objective: Deafness is the most common cause of communication disorders in humans. Genetic factors are the main cause of deafness, and the carrying rate of deafness genes is not low in normal hearing population. This is also one of the important reasons for the high incidence of deafness. We collected 5 families with delayed hearing loss to analyze and summarize the correlation between their clinical phenotype and genotype, and to explore the molecular genes of common families with delayed hearing loss, so as to provide genetic counseling and guidance in marriage and childbearing for these families, so as to enrich the genetic hearing loss. Gene mutation spectrum.
Methods: Collect the cases and collect the family information, which are HBZ-01, HBZ-02, HBX, HBD, HBM. Collect the blood samples of family members, extract the genomic DNA of peripheral venous blood, and then sequence it directly after PCR amplification. First, screen the common mutant genes in China, including GJB2, SLC26A4 and mitochondrial gene (mtDNA 12S rRNA). Correlation between clinical phenotype and genotype provides genetic counseling for deaf patients and family members; screening negative patients, according to the principle of phenotypic similarity, screening suspicious pathogenic genes in the currently located DFNA, positive, same; negative people, high-throughput sequencing of the deaf genome, analysis of suspicious pathogenic gene loci, positive pairs The mutation point was amplified by PCR, and the amplified product was verified by Sanger sequencing. Whole exome sequencing would be considered in the next step for those still negative.
Result:
1. Complex heterozygous mutation c.35insG/c.232GA of GJB2 gene was detected in a small family with unknown hereditary pattern of deafness (HBZ-01). The same mutation type in the deaf members of the family showed different clinical phenotypes. The onset age of the patients ranged from 0 to 20 years old, and they were all sensorineural deafness. After gradual involvement, low-frequency. c.35insG/c.232GA complex heterozygous mutation was co-segregated with the deaf phenotype in HBZ-01 family. Preliminary results showed that there were fewer such complex heterozygous mutations in the GJB2 gene in deaf people.
2 In a Hebei pedigree with autosomal dominant nonsyndromic hearing loss (HBZ-02), besides hearing loss, some patients were found to have vestibular dysfunction. The mutation site in exon 4 was the pathogenic site, C. 226GA (p. 76AT), which was co-segregated with clinical phenotype and conserved in many species. The clinical phenotype of HBZ-02 family is similar to that of the previously reported DFNA9 family, which further confirms the principle of phenotypic similarity. It is important to accurately determine the characteristic phenotype of hereditary deafness for guiding genotypic analysis.
3. A rare EYA4 gene mutation, c.544_545insA, was found in an autosomal dominant nonsyndromic deafness family (HBX family) for the first time in China. The mutation was found in exon 8 of the EYA4 gene, resulting in amino acid frameshift mutation (p.E182X), c.544_545insA mutation was separated from the phenotype of deafness in HBX family. Different from the previously reported DFNA 10 family, the audiogram of this family is not a typical "trough" pattern but a flat or steep descending pattern. The onset age span is large, ranging from 20 to 40 years old, and some members have tinnitus.
4 In the other two families (HBM family, HBD family), high-throughput sequencing of the deaf genome (including 130 known deafness genes) based on next-generation sequencing and exon capture techniques was used. No definite pathogenic genes were found, suggesting that these two families may be deaf from some rare or unknown deafness gene defects. The next step may be considered. Whole exon group sequencing.
Conclusion:
Because of the genetic heterogeneity of hereditary deafness, mutations in different genes can lead to the same clinical phenotype; mutations in different loci of the same gene can lead to different clinical phenotypes, and even the same gene at the same mutation locus may lead to different phenotypes among different individuals in the same family.
2 accurately identifying the clinical phenotype of characteristic deafness can guide the diagnosis of genotypes.
3 there are still many unknown genes of deafness patients. The genetic deafness gene database needs to be improved.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R764.43

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