本文選題：痛風(fēng)　切入點：全外顯子組測序　出處：《昆明理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：痛風(fēng)是患有高尿酸血癥后關(guān)節(jié)內(nèi)尿酸結(jié)晶沉積而引起的一種常見的關(guān)節(jié)炎。在強大的基因參與調(diào)控的疾病中,痛風(fēng)是一種發(fā)病率最高的炎癥性關(guān)節(jié)疾病。格外值得注意的是,痛風(fēng)在中國人群中已經(jīng)成為第二大代謝性疾病。對痛風(fēng)(Gout)病例組和健康對照組進(jìn)行全外顯子高通量測序(Whole Exome Sequencing,WES),在全外顯子范圍內(nèi)探尋痛風(fēng)疾病新的易感基因位點。本研究共納入33名中國成年人,病例組包含了 11名被昆明醫(yī)科大學(xué)第一附屬醫(yī)院風(fēng)濕免疫科專家臨床診斷為痛風(fēng)的成人患者;健康對照組由來自體檢中心的22名健康成年人組成。分別從患者和健康人的全血中提取基因組DNA。利用Agilent SureSelect Human All Exon Kit對研究對象進(jìn)行了全基因組外顯子捕獲,然后用HiSeq 3000高通量測序儀進(jìn)行全外顯子組測序。對比分析各種測序數(shù)據(jù)分析軟件的優(yōu)缺點后,詳細(xì)地論述SeqMule軟件的特點及操作,利用SeqMule對研究對象的外顯子測序數(shù)據(jù)進(jìn)行自動化比對與SNP分析。對所有的變異體進(jìn)行功能注釋,篩選出可能對基因功能有顯著影響的非同義突變(Nonsysnonymous,NS)。使用R軟件對得到的基因數(shù)據(jù)進(jìn)行生物信息學(xué)分析和統(tǒng)計學(xué)分析:過濾掉同義突變等不影響基因功能的突變,只保留影響基因功能的非同義突變;過濾掉公共遺傳突變數(shù)據(jù)庫(dbSNP)正常人攜帶的常見突變、已發(fā)表的正常對照個體的突變、以及該項目實驗測序的正常對照個體的突變;然后在所有患病個體的突變集合中取交集;利用Fisher精確檢驗篩選具有統(tǒng)計學(xué)意義(P0.05)的單核苷酸變異,減少候選致病突變的數(shù)量。經(jīng)過全外顯子測序、生物信息學(xué)分析、統(tǒng)計學(xué)分析、以及蛋白質(zhì)功能預(yù)測后,我們找到23個罕見突變基因包含了 24個罕見的單核苷酸變異。通過在HGMD、Pubmed、snp138等公共數(shù)據(jù)庫的篩查,發(fā)現(xiàn)兩個可能與痛風(fēng)疾病發(fā)展顯著相關(guān)的SNPs(單核苷酸多態(tài)性)的遺傳變異,分別是KLRC2和NCOR1基因在外顯子區(qū)域的 rs3419537(c.56CG,P = 0.00417)和 rs73281920(c.59TG,P =0.00000143)位點。這兩個位點都被蛋白質(zhì)功能預(yù)測軟件SIFT和PolyPhen-2預(yù)測為“有害”。KLRC2(c.56CG)位于定位區(qū)域12p13.2上,6名患者中檢測到突變,健康對照組均未突變。NCOR1(c.59TG)位于定位區(qū)域17p12-p11.2上,9名患者中檢測到突變,健康對照組均未突變。利用全外顯子組測序技術(shù),使用自編R程序,經(jīng)過生物信息學(xué)分析和統(tǒng)計學(xué)分析后,從11名痛風(fēng)患者中篩選出2個可能致病突變基因位點,為痛風(fēng)的診斷提供了更加快速、準(zhǔn)確的方法。與多個分析軟件的對比,發(fā)現(xiàn)了一站式全基因組和外顯子組測序數(shù)據(jù)自動分析軟件(SeqMule)能夠優(yōu)化許多分析軟件存在的一些缺點,能夠?qū)ν怙@子組和全基因組測序數(shù)據(jù)完成全面、靈活、高效地自動化分析,可以更好地分析高通量測序數(shù)據(jù),最終提升數(shù)據(jù)分析的一致性和準(zhǔn)確性。
[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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TP311.56;R589.7

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