基于全外显子组测序技术筛选痛风相关基因的研究

发布时间：2018-03-03 15:20

本文选题：痛风　切入点：全外显子组测序　出处：《昆明理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：痛风是患有高尿酸血症后关节内尿酸结晶沉积而引起的一种常见的关节炎。在强大的基因参与调控的疾病中,痛风是一种发病率最高的炎症性关节疾病。格外值得注意的是,痛风在中国人群中已经成为第二大代谢性疾病。对痛风(Gout)病例组和健康对照组进行全外显子高通量测序(Whole Exome Sequencing,WES),在全外显子范围内探寻痛风疾病新的易感基因位点。本研究共纳入33名中国成年人,病例组包含了 11名被昆明医科大学第一附属医院风湿免疫科专家临床诊断为痛风的成人患者;健康对照组由来自体检中心的22名健康成年人组成。分别从患者和健康人的全血中提取基因组DNA。利用Agilent SureSelect Human All Exon Kit对研究对象进行了全基因组外显子捕获,然后用HiSeq 3000高通量测序仪进行全外显子组测序。对比分析各种测序数据分析软件的优缺点后,详细地论述SeqMule软件的特点及操作,利用SeqMule对研究对象的外显子测序数据进行自动化比对与SNP分析。对所有的变异体进行功能注释,筛选出可能对基因功能有显著影响的非同义突变(Nonsysnonymous,NS)。使用R软件对得到的基因数据进行生物信息学分析和统计学分析:过滤掉同义突变等不影响基因功能的突变,只保留影响基因功能的非同义突变;过滤掉公共遗传突变数据库(dbSNP)正常人携带的常见突变、已发表的正常对照个体的突变、以及该项目实验测序的正常对照个体的突变;然后在所有患病个体的突变集合中取交集;利用Fisher精确检验筛选具有统计学意义(P0.05)的单核苷酸变异,减少候选致病突变的数量。经过全外显子测序、生物信息学分析、统计学分析、以及蛋白质功能预测后,我们找到23个罕见突变基因包含了 24个罕见的单核苷酸变异。通过在HGMD、Pubmed、snp138等公共数据库的筛查,发现两个可能与痛风疾病发展显著相关的SNPs(单核苷酸多态性)的遗传变异,分别是KLRC2和NCOR1基因在外显子区域的 rs3419537(c.56CG,P = 0.00417)和 rs73281920(c.59TG,P =0.00000143)位点。这两个位点都被蛋白质功能预测软件SIFT和PolyPhen-2预测为“有害”。KLRC2(c.56CG)位于定位区域12p13.2上,6名患者中检测到突变,健康对照组均未突变。NCOR1(c.59TG)位于定位区域17p12-p11.2上,9名患者中检测到突变,健康对照组均未突变。利用全外显子组测序技术,使用自编R程序,经过生物信息学分析和统计学分析后,从11名痛风患者中筛选出2个可能致病突变基因位点,为痛风的诊断提供了更加快速、准确的方法。与多个分析软件的对比,发现了一站式全基因组和外显子组测序数据自动分析软件(SeqMule)能够优化许多分析软件存在的一些缺点,能够对外显子组和全基因组测序数据完成全面、灵活、高效地自动化分析,可以更好地分析高通量测序数据,最终提升数据分析的一致性和准确性。
[Abstract]:Gout is a common form of arthritis caused by the deposition of uric acid in the joint after hyperuricemia. Gout is one of the most common inflammatory joint diseases with the highest incidence of hyperuricemia. Gout has become the second largest metabolic disease in Chinese population. The whole Exome sequencingand the whole exon sequence sequence were carried out in the gout case group and the healthy control group to search for the new susceptible gene locus of gout disease in the whole exon. A total of 33 Chinese adults were included in this study. The case group consisted of 11 adult patients who were clinically diagnosed as gout by the rheumatic immunologists in the first affiliated Hospital of Kunming Medical University. The healthy control group consisted of 22 healthy adults from the physical examination center. Genomic DNAs were extracted from the whole blood of the patient and the healthy person respectively. The whole genome exon was captured by Agilent SureSelect Human All Exon Kit. Then the whole exon group sequencing was carried out with HiSeq 3000 high-throughput sequencer. After comparing and analyzing the advantages and disadvantages of all kinds of sequencing data analysis software, the characteristics and operation of SeqMule software were discussed in detail. Automated alignment and SNP analysis of exon sequencing data were performed using SeqMule. All variants were annotated. Nonsynonymous mutations were screened out which may have a significant effect on gene function. Bioinformatics analysis and statistical analysis of the obtained gene data using R software: filtering out synonymous mutations and other mutations that do not affect gene function. Only non-synonymous mutations affecting gene function were retained, common mutations carried by normal individuals in the common genetic mutation database (DBSNP), mutations in published normal control individuals and mutations in normal control individuals sequenced by this project were filtered out. Then the mutation sets of all the diseased individuals were selected, and the single nucleotide mutation with statistical significance (P0.05) was screened by Fisher accurate test, and the number of candidate pathogenic mutations was reduced. After the whole exon sequencing, bioinformatics analysis was carried out. After statistical analysis and functional prediction of proteins, we found 23 rare mutation genes containing 24 rare single nucleotide mutations. Two SNPs (single nucleotide polymorphisms) were found to be significantly associated with the development of gout disease. The KLRC2 and NCOR1 loci at rs3419537C. 56CGN P = 0.00417) and rs73281920 c. 59TGGGG P + 0.00000143) loci were detected for mutations in 6 patients located at 12p13.2 by the protein function prediction software SIFT and PolyPhen-2 as "harmful" .KLRC2c.56CG. None of the healthy controls had any mutation. NCor1C. 59TG) was detected in 9 patients in the locational region 17p12-p11.2, but not in the healthy control group. Using the whole exon sequencing technique, using the self-made R program, the mutation was analyzed by bioinformatics and statistics. Two possible mutation gene loci were screened from 11 patients with gout, which provides a more rapid and accurate method for the diagnosis of gout. It is found that the one-stop automatic analysis software Seq Mule can optimize some disadvantages of many software, and can complete comprehensive, flexible and efficient automated analysis of exon and exon group sequencing data. It can better analyze high-throughput sequencing data and ultimately improve the consistency and accuracy of data analysis.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP311.56;R589.7

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