靶向捕获测序技术检测川崎病冠状动脉损害易感基因研究

Identification of mutations associated with coronary artery lesion susceptibility in Kawasaki disease by targeted enrichment of genomic region sequencing technique

目的应用靶向捕获测序技术对川崎病患者进行基因定制芯片筛查，探讨川崎病及冠状动脉损害易感基因。方法采用病例对照的研究方法，选择2015年12月至2016年11月在上海市儿童医院就诊的川崎病患儿114例作为川崎病组和门诊健康查体的45例健康儿童作为对照组。根据超声心动图检查结果分组。抽取外周静脉血，提取全基因组DNA，制备靶向捕获测序文库并测序，分析后得到川崎病冠状动脉损害易感基因。采用负担检验、Pearson卡方检验或Fisher确切概率法检验易感基因。结果川崎病组和对照组中TNFRSF11B基因（rs2073618）G〉C突变（p.N3K），GG、GC、CC基因型（12、37、65比2、30、13）差异有统计学意义（χ2＝15.52，P＝0.00）。有冠状动脉病变（CAL）组和无CAL组中TNFRSF13B基因（rs34562254）C〉T突变（p.P251L）位点CC、CT、TT基因型频率（19、6、3比29、44、13）差异有统计学意义（χ2＝10.40，P＝0.01）。LEFTY1基因（rs360057）T〉G突变（p.D322A），该位点有CAL组和无CAL组TT、TG、GG基因型频率（25、2、1比52、28、6）差异有统计学意义（χ2＝8.505，P＝0.01）。结论利用靶向捕获测序技术针对中国人群进行川崎病及冠状动脉损害基因易感性的初步筛查，对开展多中心更大样本川崎病高危预测评分标准研究提供理论依据。

Objective To screen and identify the mutations in Kawasaki disease by targeted enrichment of genomic region sequencing technique and investigate susceptibility genes associated with coronary artery lesion.Method This was a case-control study.A total of 114 patients diagnosed as Kawasaki disease treated in Shanghai Children′s Hospital between December 2015 and November 2016 were studied and another 45 healthy children who were physically examined in outpatient department were enrolled as control group. Patients were divided into two groups based on the results of echocardiogram. Peripheral venous blood was obtained from patients and controls. Genomic DNA was extracted. SeqCap EZ Choice libraries were prepared by targeted enrichment of genomic region technology. Then the libraries were sequenced to identify susceptibility genes associated with coronary artery lesion in patients diagnosed as Kawasaki disease.Susceptible genes were identified by Burden test, Pearson chi-square test or Fisher′s exact probability test.ResultThere was statistically significant difference in TNFRSF11B（rs2073618）G〉C（p.N3K）mutation and GG/GC/CC genotype between Kawasaki disease group and control group（χ2=15.52, P=0.00）. There was statistically significant difference in TNFRSF13B（rs34562254）C〉T（p.P251L）mutation（χ2=10.40, P=0.01）and LEFTY1（rs360057）T〉G（p.D322A）mutation（χ2=8.505, P=0.01）between patients with coronary artery lesions and those without.ConclusionTargeted enrichment of genomic region sequencing technology can be used to do primary screening for the susceptible genes associated with coronary artery lesions in Chinese Kawasaki patients and may provide theoretical basis for larger sample investigation of risk prediction score standard in Kawasaki disease.