双生子样本全外显子组测序筛查非综合征型唇腭裂易感基因研究

Non-syndromic cleft lip or palate (NSCL/P) are one of the most common birth defects in humans, with a high birth prevalence and the substantial burden for the families and the society. It is a kind of the congenital diseases that Chinese government has intended to make great efforts to reduce the incidence in next twenty years. The etiology of NSCL/P is complex and considered to be caused by the interaction between genetic and environmental factors. Our previous work has accomplished a program of National 863 Projects to conduct a GWAS of NSCL/P in Chinese population, which made a progress in identifying candidate genes in genetic etiology of the disease. Based on this research, we have collected 10 pairs of NSCL/P twins to take a further study on genetic factors. The classical twin design has made a great contribution in dissecting the genetic and environmental contributions to human diseases and complex traits. These advantages will provide higher reliability of the results. Using the strategy of genomic research and next generation sequencing technology, the genomic DNA of the twins were detected the genetic variants by whole exome sequencing in this study. The sequencing results were blasted in the 1000 Genomes, UCSC databases and were analyzed by bioinformatics software to identify candidate genes and variations including mutations, SNPs and indels. Furthermore, 500 nuclear family samples were genotyped for the variants of the candidate genes to validate the association between the genes or variations and NSCL/P. Our aim is to find and confirm new susceptible genes and variants, which will help us to explore the mechanism of NSCL/P.

非综合征型唇腭裂（NSCL/P）是人类最常见的出生缺陷之一，已纳入我国政府今后二十年要努力降低发生率的主要先天性疾病。该疾病病因复杂，被认为是遗传因素和环境因素共同作用导致的。本课题组前期完成了国家863项目子项目中国人群NSCL/P 的GWAS，取得了一定的突破。在此基础上，本研究选择NSCL/P双生子样本作为研究对象（因其具有相同的发育和生长环境，而且又具有相似的遗传背景，是更为理想的研究样本），结合基因组学研究思路及二代测序技术，对已收集到的10对NSCL/P双生子样本进行全外显子组测序，将结果应用1000G、UCSC等人类基因数据库和相关软件进行比对及生物信息学分析，筛选出疾病易感的基因及变异（mutation，SNPs，Indels）。并进一步在500例三人核心家系样本中进行验证，找到与NSCL/P发病密切相关的基因/新基因和变异，探讨其在疾病发生机制中的作用。

本项目对已收集的8对双生子非综合征型唇腭裂家系样本完成了全外显子组测序，共筛选出1007个SNPs，29个Indels突变，通过生物信息学筛选，将易感基因定位在ARHGAP29、IFT88、ADAMTS17、GLI2、MATN1、TERT、ENTPD5、USP7和PBX1等基因上。针对这些易感基因，我们进行了验证。.ARHGAP29基因上选择rs7543337、rs12408537、rs11164988、rs1004197进行病例对照研究，结果提示rs7543337和rs12408537等位基因及基因型频率分布有统计学差异；IFT88 基因选择了rs2442455、rs9509307、rs17055251、rs75494891进行验证。其中，rs17055251 位点在唇腭裂组、rs75494891位点在单纯腭裂组与对照组基因型及等位基因频率比较具有统计学差异（P＝0.007）；ADAMTS17基因选取了常见的341个SNP，其中rs79564799、rs7171763的基因型频率和等位基因频率在病例组和对照组中均具有统计学差异（P＜0.05）；TERT基因选择rs10069690，rs2736100，MATN1基因选择rs12410878，rs1022490，ENTPD5基因上选择rs10138789，rs11626122，rs35067250进行病例对照研究，只有MATN1基因上的rs1022490位点的基因型频率分布是有统计学差异的；USP7基因上选择了31个常见SNP，其中，rs1677480、rs1677490、rs12929193的基因型频率和等位基因频率在病例组和对照组中均具有统计学差异（P＜0.05）；PBX1基因共筛查出常见变异5个，和两个突变（第6外显子区域为: chr1:164781411_T>G和3’UTR区域： chr1:164818560_G>A）。只有rs2275558和rs3835581两个位点基因型和等位基因频率分布具有统计学差异。位于第6外显子区域的chr1:164781411_T>G 和3’UTR区域的chr1:164818560_G>A属于新的突变。.本研究筛选出的易感基因在人群中进行了很好地验证，其后续还需要通过动物实验等探讨其功能和机制。

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