基于高通量测序的2型糖尿病易感基因靶向测序panel设计及临床应用
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摘要
目的利用高通量测序(NGS)和多重PCR技术设计基于扩增子的靶向测序panel,对T2DM易感基因进行测序,并评估其与T2DM患者临床特征的关系。方法通过Illumina测序平台,设计多重PCR扩增的靶向测序panel,对16个T2DM易感基因21个SNP位点进行测序。收集1043份确诊T2DM患者的外周血DNA样本进行检测。结果 PCR扩增子在不同样本之间的测序深度具有良好的可重复性,且不同扩增子测序数据分布具有高度一致性,除rs864745和rs712523外,测序深度达到1000×以上。18个SNP位点差异均有统计学意义(P<0. 05)。结论通过NGS技术,利用基于扩增子的靶向测序panel进行T2DM易感基因检测的可行性和实用性,验证了KCNQ1、CDKAL1、CDKN2B是T2DM发病的风险基因。
Objective To design amplicons-based target sequencing panels using high-throughput next generation sequencing(NGS) and multiplex PCR techniques to sequence type 2 diabetes susceptibility genes and to assess their relationship with clinical characteristics in patients with type 2 diabetes. Methods Multiplex PCR-targeted sequencing panels were designed by Illumina sequencing platform, and 16 SNPs of16 type 2 diabetes susceptibility genes were sequenced. Results The sequencing depth of PCR amplicon between different samples was well reproducible and the distribution of sequencing data of different amplicon was highly consistent. Except rs864745 and rs712523,the sequencing depth reached more than 1000X. 18 SNPs were significantly different(P<0.05). Conclusion The detection of T2DM candidate genes using the amplicon-based targeted sequencing panel by NGS technology was feasible and practical. KCNQl,CDKALI,CDKN2B gene were further confirmed to be risk genes of T2DM.
Objective To design amplicons-based target sequencing panels using high-throughput next generation sequencing(NGS) and multiplex PCR techniques to sequence type 2 diabetes susceptibility genes and to assess their relationship with clinical characteristics in patients with type 2 diabetes. Methods Multiplex PCR-targeted sequencing panels were designed by Illumina sequencing platform, and 16 SNPs of16 type 2 diabetes susceptibility genes were sequenced. Results The sequencing depth of PCR amplicon between different samples was well reproducible and the distribution of sequencing data of different amplicon was highly consistent. Except rs864745 and rs712523,the sequencing depth reached more than 1000X. 18 SNPs were significantly different(P<0.05). Conclusion The detection of T2DM candidate genes using the amplicon-based targeted sequencing panel by NGS technology was feasible and practical. KCNQl,CDKALI,CDKN2B gene were further confirmed to be risk genes of T2DM.
引文
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[13]裴智勇,刘满娇,陈禹保.不同人群2型糖尿病的易感基因分析研究进展.国际检验医学杂志,2017, 38:3434-3439.
[14] Hattersley A, Bruining J, Shield J, et al. The diagnosis and management of monogenic diabetes in children and adolescents. Pediatr Diabetes, 2009,10:S33-S42.
[15] Redondo MJ, Rodriguez LM, Escalante M, et al. Types of pediatric diabetes mellitus defined by anti-islet autoimmunity and random c-peptide at diagnosis. Pediatr Diabetes, 2013, 14:333-340.
[16] Qi L, Cornelis MC, Zhang C, et al. Genetic predisposition, westem dietary pattern, and the risk of type 2 diabetes in men. Am J Clin Nutr, 2009, 89:1453-1458.
[17] Huaixing Li,Wei Gan,Ling Lu,et al. A genome-wide association study identifies GRK5 and RASGRP1 as type 2 diabetes loci in Chinese Hans. Diabetes, 2013,62:291-298.
[2] Xu Y,Wang LM,He J,et al. Prevalence and control of diabetes in Chinese adults. JAMA.2013,310:948-959.
[3] van Hoek M,Dehghan A, Witteman JC, et al. Predicting type 2diabetes based on polymorphisms from Genome-Wide Association Studies. Diabetes, 2008,57:2911-2914.
[4] Diabetes Genetics Initiative of Broad Institute of Harvard andMIT, Lund University, and Novartis Institutes of BioMedical Research.Genome wide association analysis identifies loci for type2 diabetes and triglyceride levels. Science, 2007, 316:1331-1336.
[5] Zeggini E, Weedon MN,Lindgren CM, et al. Replication of genome-wide association signals in UK samples reveal s risk loci for type 2 diabetes. Science,2007,316:1336-1341.
[6] ScoR LJ, Mohlke KL, Bonnycastle LL, et al. A genome-wide association study of type 2 diabetes in F inns detects multiple susceptibility variants. Science,2007,316:1341-1345.
[7] Sandhu MS, Weedon MN, Fawcett KA, et al. Common variants in WFS 1 confer risk of type 2 diabetes. Nat Genet, 2007,39:951-953.
[8]Sladek R, Rocheleau G, Rung J, et al. A genome-wide association study identifies novel risk loci for type 2 diabetes. Nature,2007, 445:881-885.
[9]Steinthorsdottir V, Thorleifsson G, Reynisdottir I, et al. A variant in CDKAL 1 influences insulin response and risk of type 2 diabetes. Nat Genet,2007,39:770-775.
[10] Wellcome trust case control consortium. Genome-wide associationstudy of 14000 cases of seven common diseases and 3000shared controls. Nature, 2007, 447:661-678.
[11] Gudmundsson J, Sulem P, Steinthorsdottir V, et al. Two variants on chromosome 17 confer prostate cancer risk, and the one inTCF2 protects against type 2 diabetes. Nat Genet, 2007, 39:977-983.
[12] Frayling, TM,Timpson NJ,Weedon MN,et al. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science, 2007, 316:889-894.
[13]裴智勇,刘满娇,陈禹保.不同人群2型糖尿病的易感基因分析研究进展.国际检验医学杂志,2017, 38:3434-3439.
[14] Hattersley A, Bruining J, Shield J, et al. The diagnosis and management of monogenic diabetes in children and adolescents. Pediatr Diabetes, 2009,10:S33-S42.
[15] Redondo MJ, Rodriguez LM, Escalante M, et al. Types of pediatric diabetes mellitus defined by anti-islet autoimmunity and random c-peptide at diagnosis. Pediatr Diabetes, 2013, 14:333-340.
[16] Qi L, Cornelis MC, Zhang C, et al. Genetic predisposition, westem dietary pattern, and the risk of type 2 diabetes in men. Am J Clin Nutr, 2009, 89:1453-1458.
[17] Huaixing Li,Wei Gan,Ling Lu,et al. A genome-wide association study identifies GRK5 and RASGRP1 as type 2 diabetes loci in Chinese Hans. Diabetes, 2013,62:291-298.