中国科学院昆明动物研究所机构知识库

麻风，也称汉森病，是由胞内寄生菌麻风分枝杆菌（Mycobacterium leprae）感染引起的慢性传染病。它影响人类皮肤、眼睛、周围神经等身体系统，导致各种与麻风有关的残疾。麻风的临床表现分为两类，即结核型（TT）和瘤型（LL），以及三种中间过渡类型（BT, BB, BL）。为了治疗目的，世界卫生组织（WHO）将麻风归类为少毛细血管（PB）和多毛细血管（MB）。世界卫生组织的报告显示，全球麻风患病率从80年代中期的500多万例减少到2016年底的22万例以下，然而，每年仍有很多新发病例。截止2016年底，全球共有214,783例新发麻风病例，相当于世界范围内新发病例每10万人2.9例。有大量证据表明宿主遗传因素在麻风发生发展中起着至关重要的作用。前人基于家系的连锁分析、候选基因关联分析、以及全基因组关联研究等已经鉴定了多个与麻风遗传易感相关的遗传变异位点与麻风易感基因。这些风险基因参与先天和适应性免疫系统，神经通路和线粒体相关通路，如NOD2，PARK2/PRK，LRRK2，APOE，PINK1/PRKN和PARL。中国人群在过去十年中使用GWAS进行的大规模分析大大拓宽了我们对麻风病和氨苯砜治疗超敏反应的遗传易感性的认识。然而，GWAS鉴定的变异位点仅占麻风病遗传易感风险的一部分。此外，GWAS鉴定的大部分SNP位于功能未知的非编码区，这些麻风风险相关区域的功能性变异及其致病基因仍有待确认。本研究中，我们通过二代测序技术鉴定出影响麻风遗传易感的功能变异位点，再进行独立验证和表达分析。我们通过对798例麻风患者与990例正常对照进行靶向捕获测序，鉴定出了一个位于LACC1基因的功能变异位点rs3764147 (p. Ile254Val): 该位点的G等位基因与更高的麻风风险相关（P-value = 1.33×10-7, OR = 1.460）。随后，我们通过一代测序，在527例麻风患者与583例正常对照人群中验证了这一结果（P-value = 4.37×10-12, OR = 1.955）。进一步对全世界人群的分析发现，该突变位点对麻风的风险效应，是普遍存在与各世界人群。我们随后进行了表达数量性状位点分析，发现该突变影响LACC1基因的表达水平，而对应的，我们在麻风患者的皮损组织中确实观察到LACC1基因的异常表达。总之，我们提供了系统的遗传数据与表达证据，提示LACC1是麻风的易感基因，而rs3764147还需要进一步在更多独立人群中验证，并对其作用机制进一步的研究。补体系统是机体先天免疫的重要组成部分，在抵抗麻风杆菌感染中发挥多种作用。我们前期基于候选基因策略研究发现，补体组分FCN2, MBL2以及CFH的调节性变异位点通过影响相关补体组分的表达改变，从而影响麻风的遗传易感。然而，其他补体组分与麻风的遗传易感是否有关，是否存在影响氨基酸编码的错义突变促进麻风发病，补体组分在麻风患者中的表达模式如何，还不甚清楚。在本研究的第二部分，我们通过靶向捕获测序与表达谱分析，系统的分析了22个补体系统基因的遗传变异与表达模式。我们验证了前期发现的基因FCN2与麻风的相关性（rs7851696, c.772G>T, p. Ala258Ser, P-value = 2.5×10-6, OR = 0.655）；发现补体核心组分C3（rs2277984, P-value = 2.4×10-5, OR = 1.491）和补体受体CR1（rs2274567, c. 4973A>G, p. His1208Arg; P = 6.4′10-6, OR = 1.381, rs41274776, c. *37A>G, P = 2.1′10-5, OR = 1.35, rs3811381, c. Pro1827Arg; P = 3.4′10-5, OR = 1.348, and rs6691117, c. 6193A>G, p.Ile1615Val; P = 3.6′10-5, OR = 1.342）和CR1L (rs3085, c. 415A>G, p. Ile139Val; P = 6.7′10-10, OR = 1.527, and rs2296158, c. 346A>G, p. Arg116Gal; P = 1.4′10-5, OR = 1.393)与麻风遗传风险有关；同时，我们发现在麻风杆菌感染的细胞及病人皮损组织中，这些补体组分的表达水平也发生改变。这些结果提示我们，补体系统的遗传变异与表达改变，参与了麻风的发生发展。综上，本论文鉴定了LACC1，FCN2，C3, CR1和 CR1L基因上的遗传变异与麻风遗传易感相关。同时，系统分析了补体系统在麻风中的遗传变异与表达模式。结果为鉴定麻风的遗传易感基础，理解这一古老传染疾病，提供了基础数据。 

Leprosy (also known as Hansen’s disease) is a chronic infectious disease caused by the intracellular bacterium Mycobacterium leprae. It affects human skin, eyes, the peripheral nerves, and other body systems causing a variety of disabilities related to leprosy. The clinical manifestation of leprosy falls into two poles, the tuberculoid (TT) and the lepromatous (LL) together with three intermediary types (borderline tuberculoid (BT), borderline borderline (BB) and borderline lepromatous (BL)). For treatment purpose, leprosy was categorized as paucibacillary (PB) and multibacillary (MB) by the World Health Organization (WHO). It is predicted that globally two to three million people are disabled permanently because of leprosy. The global prevalence of leprosy has reduced from more than 5 million cases in the mid-1980s to less than 220,000 cases at the end of 2016 according to WHO reports. However, new leprosy cases continue to occur. A total of 214,783 new leprosy cases were reported at the end of 2016, corresponding to the worldwide new case detection rate of 2.9 per 100 000 population. There is a large body of evidence indicating a crucial role for the host genetic background in disease outcome. Previous genetic studies, either using a family-based linkage analysis, candidate gene or genome-wide association study (GWAS) strategy have identified a variety of risk loci and/or susceptibility genes for leprosy. These risk genes are involved in the innate and adaptive immune systems, neurological pathways and mitochondrion related pathways, such as NOD2, PARK2/PRK, LRRK2, APOE, PINK1/PRKN, and PARL. The large-scale analysis in Chinese populations using a GWAS in the past decade has greatly broadened our knowledge regarding the genetic susceptibility to leprosy and dapsone treatment hypersensitivity. However, the effect size of the GWAS-identified variants was modest and accounted for only part of the genetic heritability of leprosy. Moreover, most of SNPs in the GWAS loci were located in noncoding regions with unknown function and the functional variants and causal genes underlying the GWAS loci remain to be recognized. In this study, we aimed to identify protein-coding variants in leprosy risk genes by using next-generation sequencing (NGS) technology, followed by independent validation and expression analysis. In the NGS stage, we sequenced genes within previously reported GWAS loci in 798 individuals with leprosy and 990 general controls from Wenshan, Yunan, China. We mapped a common missense variant rs3764147 (c.760A>G, p. Ile254Val, P-value = 1.33×10-7, OR = 1.460) in the LACC1 gene as the potentially functional variants within the reported GWAS locus. The result was validated (P-value = 4.37×10-12, OR = 1.955) in 527 individuals with leprosy and 583 general controls from Yuxi, Yunan, China. Further analysis showed that the leprosy-risk allele leads to dysregulation of LACC1 gene expression. Consistently, we observed expression changes of LACC1 in skin lesion of leprosy patients. Taken together, we provided genetic and expressional evidence to indicate LACC1 as susceptibility genes for leprosy. Further studies are needed to validate the associations in more independent populations and to functionally characterize the roles of LACC1 in the development of leprosy.We also determined the genetic effect of the complement system, which is an integral part of innate immunity and plays an important role in host immunity to prevent leprosy infection. Previously, by using a candidate gene approach we have found that genetic variants of complement genes CFH, FCN2, and MBL2 contribute to leprosy via altering the expression level of the complement system. However, many questions, such as whether other complement components are associated with the genetic susceptibility of leprosy, whether there are missense mutants contribute to the development of leprosy, and how the expression pattern of complement components changes in leprosy patients, have not been sufficiently studied. In the second part of this study, we systematically analyzed the genetic variants and mRNA expression patterns of 22 complement system genes through targeted NGS and mRNA expression profiling. We have validated the association of the previously identified FCN2 with susceptibility to leprosy (rs7851696, c.772G>T, p. Ala258Ser, P-value = 2.5×10-6, OR = 0.655). Missense mutations in the complement core component C3 (rs2277984, P-value = 2.4×10-5, OR = 1.491), complement receptor CR1 (rs2274567, c. 4973A>G, p. His1208Arg; P = 6.4′10-6, OR = 1.381, rs41274776, c. *37A>G, P = 2.1′10-5, OR = 1.35, rs3811381, c. Pro1827Arg; P = 3.4′10-5, OR = 1.348, and rs6691117, c. 6193A>G, p.Ile1615Val; P = 3.6′10-5, OR = 1.342) and CR1L (rs3085, c. 415A>G, p. Ile139Val; P = 6.7′10-10, OR = 1.527, and rs2296158, c. 346A>G, p. Arg116Gal; P = 1.4′10-5, OR = 1.393) were associated with the genetic risk of leprosy. Additionally, we found an alteration in mRNA expression of these genes in M. leprae infected cells and skin lesion of leprosy patient.In summary, we found that genetic variants in LACC1, FCN2, C3, CR1, and CR1L are associated with leprosy. These results, together with previous genetic studies of leprosy, indicated that leprosy is genetically determined and provided a basis for understanding this ancient infectious disease.