全外显子组测序寻找Usher综合征致病基因位点
发布时间：2018-08-20 20:06
【摘要】：目的寻找患有先天性耳聋及视网膜变性同胞姐弟的致病基因位点。方法应用全外显子捕获测序技术,对人类基因组上1%的基因编码区进行捕获,体外进行扩增,并在hiseq2500高通量测序仪上进行并行测序,运用生物信息学方法分析测序数据与测序质量,并计算外显子区的覆盖度与测序深度,使用主流的数据分析流程GATK检测变异位点,并使用Annovar对位点进行注释,采用最新的致病位点算法pVAAST分析排序致病位点,分析致病位点在数据库中的频率、危害性及保守性,一代测序验证位点在家系中是否共分离。结果1.一家四口全外显子测序数据都在10G以上,80%的碱基测序质量在Q30以上;2.对外显子区域测序深度分析,平均测序深度在110X以上,99%区域大于10X;3.pVAAST分析显示MYO7A基因的两个位点为最可能致病基因;4.MYO7A两位点在公共数据库中均未见报导,其中一个位点c.5994GA为无义突变,使蛋白翻译提前终止,另一位点c.849+2TC在剪切位点附近,该位点经scSNV算法评估影响外显子剪切;5.通过PhastCons算法评估两个位点均非常保守,一代Sanger测序验证,父母各携带一个位点杂合子,姐弟为复合杂合突变,符合隐性遗传传递模式。结论应用全外显子测序技术寻找到一个Usher综合征核心家系的两个MYO7A致病位点,为临床进行核心家系的遗传致病位点鉴定提供了有义的探索。
[Abstract]:Objective to search for the genetic loci of siblings with congenital deafness and retinal degeneration. Methods A total exon capture and sequencing technique was used to capture 1% of the coding region of the human genome and amplify it in vitro. Parallel sequencing was performed on a hiseq2500 high throughput sequencing apparatus. The sequence data and quality were analyzed by bioinformatics method, and the coverage and sequencing depth of exon region were calculated. GATK was used to detect the mutation sites, and Annovar was used to annotate the sites. The latest pathogenetic loci algorithm (pVAAST) was used to analyze the sequence of pathogenicity loci. The frequency, harmfulness and conservatism of the pathogenicity loci in the database were analyzed. A generation of sequencing was used to verify whether the loci were coisolated at home. Result 1. The total exon sequencing data of four members of a family were all above 10G and 80% of the base sequence quality was above Q30. The analysis of exon region sequencing depth showed that the average sequencing depth was above 110X and 99% was more than 10XG 3.pVAAST analysis showed that the two loci of MYO7A gene were the most likely pathogenetic gene. The two loci of MYO7A were not reported in the public database. One locus, c.5994GA, was a nonsense mutation, resulting in early termination of protein translation, and another locus, c.849 2TC, was located near the shear site. The site was evaluated by scSNV algorithm for the effect of exon shearing. The PhastCons algorithm was used to evaluate the two loci. The results of Sanger sequencing showed that each parent carried one heterozygote and the sibling was a compound heterozygous mutation, which was in accordance with the recessive transmission pattern. Conclusion two MYO7A pathogenicity loci of a nuclear family with Usher syndrome were found by using total exon sequencing technique, which provides a meaningful exploration for clinical identification of genetic pathogenicity sites in nuclear families.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R764.43;R774.1
本文编号：2194843
[Abstract]:Objective to search for the genetic loci of siblings with congenital deafness and retinal degeneration. Methods A total exon capture and sequencing technique was used to capture 1% of the coding region of the human genome and amplify it in vitro. Parallel sequencing was performed on a hiseq2500 high throughput sequencing apparatus. The sequence data and quality were analyzed by bioinformatics method, and the coverage and sequencing depth of exon region were calculated. GATK was used to detect the mutation sites, and Annovar was used to annotate the sites. The latest pathogenetic loci algorithm (pVAAST) was used to analyze the sequence of pathogenicity loci. The frequency, harmfulness and conservatism of the pathogenicity loci in the database were analyzed. A generation of sequencing was used to verify whether the loci were coisolated at home. Result 1. The total exon sequencing data of four members of a family were all above 10G and 80% of the base sequence quality was above Q30. The analysis of exon region sequencing depth showed that the average sequencing depth was above 110X and 99% was more than 10XG 3.pVAAST analysis showed that the two loci of MYO7A gene were the most likely pathogenetic gene. The two loci of MYO7A were not reported in the public database. One locus, c.5994GA, was a nonsense mutation, resulting in early termination of protein translation, and another locus, c.849 2TC, was located near the shear site. The site was evaluated by scSNV algorithm for the effect of exon shearing. The PhastCons algorithm was used to evaluate the two loci. The results of Sanger sequencing showed that each parent carried one heterozygote and the sibling was a compound heterozygous mutation, which was in accordance with the recessive transmission pattern. Conclusion two MYO7A pathogenicity loci of a nuclear family with Usher syndrome were found by using total exon sequencing technique, which provides a meaningful exploration for clinical identification of genetic pathogenicity sites in nuclear families.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R764.43;R774.1
【参考文献】
相关期刊论文 前1条
1 Wei Zhai;Xin Jin;Yan Gong;Ling-Hui Qu;Chen Zhao;Zhao-Hui Li;;Phenotype of Usher syndrome type Ⅱ assosiated with compound missense mutations of c.721 C>T and c.1969 C>T in MYO7A in a Chinese Usher syndrome family[J];International Journal of Ophthalmology;2015年04期
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