Discovery Of New Susceptibility Loci And Analysis Of Pleiotropic Effects In Leprosy

[Background] Leprosy is a chronic infectious dis-ease caused by Mycobacterium leprae. It affects the skin and peripheral nerves and can cause irreversible impairment of nerve function and consequent chronic disabilities. Because of implementation of multi-drug therapy (MDT) in the 1980s by the World Health Organization, the prevalence of leprosy has declined dramatically. Nonetheless, with more than 200,000 new cases reported annually throughout the world, leprosy remains a public health problem, especially in developing countries. The narrow host range of Mycobacterium leprae and the fact that it is refractory to growth in culture has limited research on and the biologic understanding of leprosy. Host genetic factors are thought to influence susceptibility to infection as well as disease progression. The molecular nature of genetic susceptibility to leprosy has been intensively investigated by candidate gene studies, genome-wide linkage analyses and genome wide association study (GWAS), which have identified 11 susceptibility loci. Most of the implicated susceptibility genes encode proteins involved in immunity, whereas one locus (RAB32 at 6q24.3) suggests a role for autophagocytosis in leprosy pathogenesis. While as a complex trait, these genetic risk loci only partially explain genetic susceptibility to leprosy, and additional genetic risk factors remain to be identified.[Objective] To explore new susceptibility genes of leprosy and improve the understanding of leprosy pathogenesis by discovering novel genetic susceptibility loci for leprosy through genetic association studies in Chinese population.[Methods] Two candidate-gene case controls studies and one GWAS were conducted to identify the new susceptibility genes of leprosy.Study I:In the current study, we performed a candidate gene-based association study on 30 TLR and 47 GARD-related genes that were identified through an UCSC Genome Browser. We first examined the association evidence within the critical regions of these candidate genes in the two published GWAS datasets of matched 1,220 samples and expanded 6,287 samples. In total,4,363 SNPs within these 77 candidate genes were investigated in the two GWAS datasets. After excluding the SNPs located in known susceptibility loci, SNPs with suggestive evidence of association were identified. A moderate threshold of suggestive association was employed here to maximize the power and the chance to discover novel associations within these candidate genes. In total, eight suggestive SNPs were brought forward for further validation. In the first validation analysis (Stage 2), six of the eight selected SNPs were successfully genotyped in 1,504 leprosy cases and 1,502 healthy controls, while the other two SNPs failed in either the assay design or genotyping analysis. In the second validation (Stage 3), the two SNPs showed suggestive association were further genotyped in additional 938 cases and 5,827 controls.Study II:We carried out a comprehensive association study of IBD susceptibility loci in multiple independent leprosy samples from a Chinese population. We reviewed the findings from 12 CD GWASs and seven UC GWASs through the Catalog of Published GWASs and identified within 118 genes a total of 133 SNPs that showed genome-wide significant associations with Crohn’s disease (CD), ulcerative colitis (UC), or both.133 SNPs were selected for multistage association study in the four independent leprosy samples from the Chinese population. We conducted genotyping analyses of the four leprosy samples by using the Sequenom MassArray system. In stage 1, all 133 selected SNPs were genotyped in 1,504 leprosy cases and 1,502 healthy controls from a northern Chinese Han population. Of the 119 SNPs that were successfully designed and genotyped,19 showed suggestive association (p< 0.05) in stage 1 analysis and included nine CD-associated SNPs. In stage 2, the 19 SNPs were further genotyped in an additional three independent leprosy cohorts from the Chinese population:(1) 1,154 casesand 2,605 controls from a northern Chinese Han population, (2) 1,165 cases and 648 controls from a southern Chinese Han population, and (3) 1,148 case and 748 control minorities from southern China.Study III:We conducted a three-stage GWAS of leprosy in the Chinese population. The genome-wide discovery analysis (stage 1) involved 2 independent data sets:a previously published GWAS data set of 706 leprosy cases,1,225 healthy controls and 4,362 individuals with immune-related diseases as population controls from northern China of Chinese Han descent and a new unpublished data set of 842 leprosy cases and 925 controls from northern (Chinese Han descent) and southern (Chinese Han descent and minority ancestry groups) China. To validate the newly associated loci, we selected the top SNPs from 917 independent new loci with suggestive association with leprosy (P<5×10-4) for a follow- up analysis in an additional 2,761 leprosy cases and 3,038 controls from northern China. Of the 88 successfully genotyped SNPs,11 showed association at P< 0.05 in the validation samples and 5 showed consistent effects on risk in the discovery (stage 1) and validation (stage 2) samples. These 16 SNPs were selected for further validation in 5 additional independent sample series from different regions in China with a total of 4,004 cases and 6,467 controls (stage 3)Cochran-Armitage trend test (study Ⅰ and Ⅱ) and logistic regression models (study Ⅲ) were fitted assuming an additive genetic model of inheritance whereby the biallelic SNP genotypes were coded as 0,1 and 2 (study Ⅰ to Ⅲ). To combine multiple independent datasets, fixed-effects (Cochran-Mantel-Haenszel) or random-effects model was employed in studies Ⅰ to Ⅲ.[Results] Totally nine new susceptibility loci were identified:rs2735591 onlp22 (P=1.03×10-9, OR=1.24)、rs6871626 on 5q33.3(P=3.95×10-18,OR=0.75)、的 rs2058660 on 2q12(P=4.57×10-9, OR=1.30)、rs2221593 on lq32.3(P=3.09×10-8, rs73058713 on 5P14.3(P=9.54×10-9, OR=1.19)、 rs10817758 on 9q32(P=1.15×10-8,OR=1.13)、rs58600253 on 10q21.3 (P=3.02×10-12, OR=1.22) rs663743 on 11q3.1(P=8.84×10-14,OR=1.24) and rs77061563 on 16p13.13 (P=6.23×10-15, OR=0.84) implicating BCL10、IL12B、IL1RL1-IL18RAP、BATF3、 CDH18、DEC1、ZNF365、EGR2、CCDC88B、CIITA and SOCS1 as new susceptibility genes of leprosy 。Among of all the identified genes, IL1RL1-IL18RAP、IL12B、 CCDC88B、EGR2、ZNF365、CIITA were shared by leprosy and inflammatory/immune diseases.[Conclusion] In the first study, a candidate gene-based analysis of Toll-like receptor (TLR) and caspase recruitment domain (CARD) genes revealed BCL10 locus as a novel susceptibility locus for leprosy, bringing insight that adaptive immunity, in addition to the NOD2-mediated innate immunity, also play an important role in leprosy. The second study, also under the candidate gene-based study design, investigated SNPs established to be associated with inflammatory bowel disease (IBD) susceptibility in leprosy. This study revealed two novel loci, implicating IL1RL1-IL18RAP and IL12B as susceptibility genes for leprosy and further demonstrated evidence of shared genetic susceptibility between inflammation and infectious diseases. In the third study, where SNPs were tested in a hypothesis free manner under a genome wide association study design, we identified four additional loci; an association 1q32.3 implicating BATF3,9q32 implicating DECI/TNC,10q21.2 implicating ZNF365 and 11ql3.1 implicating CCDC88B. Two of these novel loci once again strongly support the commonality between infection and inflammatory response.Overall, our current study has advanced the genetic understanding of leprosy by substantially increasing the number of confirmed genetic susceptibility loci and has provided further biological insight into the pleiotropic effects of leprosy susceptibility loci on autoimmunity and inflammatory diseases. In particular, our findings suggest that, although molecular sensing of intracellular infection might have similar pathogenic roles in these diseases, immune- related pathways have discordant roles, supporting the concept that strong immune and inflammatory responses are advantageous in defending against infection but increase the risk of autoimmunity and inflammatory diseases. Further sequencing and functional investigations will likely identify true susceptibility genes and causal mutational events within these susceptibility loci, which could help elucidate the molecular mechanisms underlying leprosy development and further understanding of the impact of these loci on autoimmunity and inflammatory diseases.