基于目标基因靶向捕获和高通量测序解析麻风易感相关重要通路和关键基因

Leprosy is a chronic infectious and neurological disease that is caused by Mycobacterium leprae (M. leprae) infection. The bacterium can affect human peripheral nerve and skin, leading to inflammatory and nerve damage. Therefore, leprosy is considered as a good model to study the interaction between bacterium infection, innate immune, acquired immune and nerves system. During the past years, accumulating evidence showed that host genetic background can influence the infection of M. leprae and its clinical manifestation. However, almost all of these studies were based on the hypothesis of common disease-common-variants (including genome-wide association study (GWAS)), and ignored the role of rare variants, which may have major contribution to genetic susceptibility to common diseases. Here, we will use targeted gene capture and next-generation sequencing technologies to decipher the pattern of rare variants/mutations of key pathways and/or genes that confers to the genetic susceptibility to leprosy. Over 3,000 genes will be covered in this study, including genes associated with mitochondrial function, lipid metabolism, inflammatory responses, nerves system, reported susceptibility genes of leprosy and some complex diseases sharing similar clinical phenotypes with leprosy, as well as risk genes identified from our unpublished whole exome sequencing of leprosy families. In discovery stage, 400 leprosy patients and 400 healthy subjects from Southwest China will be enrolled in targeted gene capture coupled to high-throughput sequencing. Then we will perform a validation stage study to refine the rare variants/mutations distilled from the first stage in 1000 leprosy patients and 1000 healthy subjects. We will further characterize the potential functional change of the candidate rare variants/mutations at the cellular level. Our results will provide essential information for rare variants/mutations in genetic susceptibility to leprosy. And it can provide some helpful information for leprosy prevention and treatment.

麻风分枝杆菌感染机体后，患者会产生一系列免疫炎症和外周神经系统损伤，所以麻风被认为是研究感染、机体免疫和外周神经系统互作的良好模型。目前对于麻风的遗传研究包括大规模的GWAS分析，仅限于对常见变异的分析而忽视了稀有变异在该病中的重要作用。本研究将筛选3000多个与麻风感染、发病等相关系统或通路基因，包括线粒体相关基因、脂类代谢相关基因、免疫系统基因、神经系统基因、已报道的麻风和具有与麻风相似表型疾病的相关易感基因、我们前期基于麻风家系全外显子组测序分析获得的潜在易感基因等，基于这些目的基因进行800例样本的高通量靶向捕获测序，再在1000病例与1000例对照中进行精细的筛查，发现与麻风临床分型、免疫逃逸相关的易感稀有变异。同时对获得的易感基因或变异开展体内、体外功能实验，阐明其潜在的作用机制。研究结果有望进一步理解麻风易感的遗传基础，并对麻风的防治提供更多的科学依据。

麻风是由麻风分枝杆菌（M. Leprae）感染引起的慢性传染病，主要累及皮肤、粘膜、四肢与外周神经等。作为最古老的流行病之一，麻风在全球大部分地区已基本灭绝（患病率<0.01‰）。然而，在我国云南省部分地区，麻风仍然保持着较低而稳定的流行。虽然大部分个体对麻风天然免疫，但仍存在一定比例的易感群体，这些易感群体是否发病？发展为哪一种麻风亚型？是受个体遗传背景、免疫状态和代谢状态等影响。针对麻风相关的遗传因素，目前领域内已开展了较充分的全基因组关联分析研究，揭示了可靠的麻风遗传风险相关的变异位点。然而，全基因组关联分析主要研究对象为人群中常见的变异，且这些变异主要位于非编码区，功能未知。人群中频率稀有的、位于编码区的潜在功能变异，则将依靠近年来发展的高通量测序得到进一步的解析。本项目基于麻风的生物学特征，围绕“重要生物学通路相关基因的编码区遗传变异与麻风遗传风险”这一主题，进行了系统的遗传分析。我们筛选了3000多个与麻风感染相关系统或通路的基因：包括线粒体、脂代谢、免疫系统、神经系统等相关基因，与麻风相似表型疾病的相关基因，以及我们前期基于麻风家系全外显子组测序分析获得的潜在易感基因等，在来自云南文山的大样本的麻风病例与正常对照人群中，开展了全外显子组测序、靶向捕获测序与候选基因的基因分型。我们发现了系列与线粒体、脂代谢相关的基因中的常见变异影响麻风的易感性。尤其值得提出的是，我们鉴定到一个效应量较高的HIF1A基因稀有变异，能显著提高人群的麻风易感风险；同时，我们确定了一个效应量大、频率高的功能性突变，可能是麻风的主效基因之一。本项目的实施产生了SCI论文7篇，其中3篇发表于高水平期刊（Brit J Dermatol 2018; Am J Hum Genet 2018; PNAS 2020）。我们的研究系统的分析了机体的遗传变异在麻风发生发展中的作用，丰富了我们对于麻风的遗传基础与相关通路的认识。研究结果大大促进了云南麻风群体的遗传基础解析与感染性疾病的分子机制阐释，为麻风风险群体的识别及相关防治提供了重要依据。