猪先天性肛门闭锁症致病基因的鉴别

Congenital anal atresia is a common and severe deformation of digestive tract, and genetic factors play key roles in this innate disorder. Human anal atresia causes severe economic and mental pressure to the patients and their families. Porcine anal atresia leads to consideral economic loss in the pig indusrty. To date, few studies on mammalian anal atresia have been reported and the causative genes for this disease are still elusive. The domestic pig is an ideal biomedical model for human genetic diseases because of the high biological similarity between humans and pigs. We have constructed a Large White inbred population, which shows animals affected with anal atresia. Genetic pedigree analysis supports that this disease is controlled by two genes under a recessive model. In this project, we will use this inbred family as experimental animals, and perform a genome scan using Illumia 60K SNP chip to map the underlying loci. Then, bioinformatic analyses will be conducted based on the inheritance pattern of two pairs of recessive genes. Next, deep resequencing of target regions, RNA-seq and functional assays of candidate causal mutations will be implemented, with the aim to identify the pathogenic gene and the causative mutation underlying this disease. The findings will not only provide scientific guidance for selecting nucleus herds of pigs without the deleterous alleles for anal atresia, but also shed novel insight into the genetic mechanism of this disease in humans.

先天性肛门闭锁症是哺乳动物中一种常见的消化道重大畸形，遗传因素在该疾病发生过程中起主导作用。人肛门闭锁症给患者和家属的经济和精神上都造成较大的影响，猪肛门闭锁症对养猪业也造成经济损失。目前哺乳动物中有关肛门闭锁症遗传机理的研究鲜有报告，真正的致病基因仍然不明。家猪因其生理结构与人高度相似，成为研究人类遗传疾病的理想模式动物。课题组前期获得了一个大白猪先天性肛门闭锁的近交群体，并通过系谱分析推测该疾病受两个基因双隐性遗传模式控制。本项目拟利用该家系为研究材料，通过Illumina 60K SNP扫描、基于双基因隐性遗传模式的全基因组扫描、目标区域深度重测序、RNA-seq及突变位点功能验证等研究，鉴别到导致该疾病的致病基因和因果突变。预期研究结果将为养猪生产中选育无肛门闭锁症致病基因的核心群提供科学依据，并将为人类肛门闭锁症研究提供重要的参考。

先天性肝门闭锁症是哺乳动物中一种常见的消化道重大畸形，遗传因素在该疾病发生中起主导作用。本研究首先根据大白猪回交群体的表型分离情况（患病/正常≈1/3）并结合近交群的系谱信息，推测出该群体中的先天性锁肛症呈常染色体双基因隐性遗传模式。接着，利用60K SNP芯片扫描、全基因组关联分析（GWAS）、全基因组连锁定位、SNP基因型与遗传模式一致性分析、后裔同源(IBD)定位分析、断点重组分析，将两个致病基因定位于SSC1 127.5-129.2 Mb或145.8-147.5 Mb和SSC15 29.1-33.9 Mb区域。. 针对不同基因型个体开展全基因组重测序，根据已知个体基因型与SNP基因型一致性筛选分析，在上述置信区间内筛选到15个重要候选致病突变。然后通过对32头患病个体和188头正常个体进行sanger测序，最终鉴别到2个主效候选致病突变，分别是位于SSC1上CAPN3基因下游2 Kb左右的T>A突变和位于SSC15上GLI2基因的内含子1剪接区域上的G>A突变。CAPN3基因与细胞凋亡通路密切相关，GLI2基因在SHH信号通路中起着重要作用；该两个通路出现异常都可能与肛门闭锁症的发生密切相关。qRT-PCR结果表明，与正常个体相比，CAPN3基因在患病个体肛门处皮肤和直肠末端中的表达量均显著上调，而GLI2基因在患病个体的两种组织中的表达量均显著下调。推测GLI2 G>A剪接突变可能形成非正常转录本被降解进而导致GLI2表达水平显著下降，抑制甚至阻断SHH信号传递，而CAPN3 T>A突变可能上调CAPN3基因的表达，导致泄殖腔膜不能正常破裂，两者共同作用从而引起大白猪先天性锁肛症的发生。针对患病个体和全同胞正常个体肛门处皮肤和直肠末端组织开展了RNA测序分析，差异表达基因（DEGs）显著富集于凋亡过程负调控、胚胎发育及器官形成、WNT信号通路、BMP信号通路等可能与肛门直肠发育密切相关的通路。此外，项目组优化了家猪CRISPR/Cas9基因组编辑技术平台，利用优化的技术平台创制了同时敲除致病候选基因GLI2和敲入CAPN3（H11安全港定位整合）的猪胎儿成纤维细胞。. 本研究的结果不仅为种猪遗传病患（先天性锁肛症）选育改良提供了重要的基因标记，更为人类锁肛症的遗传机制及致病分子机理的解析提供了重要借鉴。