Genome-wide meta-analysis and fine-mapping prioritize potential causal variants and genes related to leprosy.

To date, genome-wide association studies (GWASs) have discovered 35 susceptible loci of leprosy; however, the cumulative effects of these loci can only partially explain the overall risk of leprosy, and the causal variants and genes within these loci remain unknown. Here, we conducted out new GWASs in two independent cohorts of 5007 cases and 4579 controls and then a meta-analysis in these newly generated and multiple previously published (2277 cases and 3159 controls) datasets were performed. Three novel and 15 previously reported risk loci were identified from these datasets, increasing the known leprosy risk loci of explained genetic heritability from 23.0 to 38.5%. A comprehensive fine-mapping analysis was conducted, and 19 causal variants and 14 causal genes were identified. Specifically, manual checking of epigenomic information from the Epimap database revealed that the causal variants were mainly located within the immune-relevant or immune-specific regulatory elements. Furthermore, by using gene-set, tissue, and cell-type enrichment analyses, we highlighted the key roles of immune-related tissues and cells and implicated the PD-1 signaling pathways in the pathogenetic mechanism of leprosy. Collectively, our study identified candidate causal variants and elucidated the potential regulatory and coding mechanisms for genes associated with leprosy.

Single-cell sequencing analysis reveals development and differentiation trajectory of Schwann cells manipulated by M. leprae.

BACKGROUND: M. leprae preferentially infects Schwann cells (SCs) in the peripheral nerves leading to nerve damage and irreversible disability. Knowledge of how M. leprae infects and interacts with host SCs is essential for understanding mechanisms of nerve damage and revealing potential new therapeutic strategies. METHODOLOGY/PRINCIPAL FINDINGS: We performed a time-course single-cell sequencing analysis of SCs infected with M. leprae at different time points, further analyzed the heterogeneity of SCs, subpopulations associated with M. leprae infection, developmental trajectory of SCs and validated by Western blot or flow cytometry. Different subpopulations of SCs exhibiting distinct genetic features and functional enrichments were present. We observed two subpopulations associated with M. leprae infection, a stem cell-like cell subpopulation increased significantly at 24 h but declined by 72 h after M. leprae infection, and an adipocyte-like cell subpopulation, emerged at 72 h post-infection. The results were validated and confirmed that a stem cell-like cell subpopulation was in the early stage of differentiation and could differentiate into an adipocyte-like cell subpopulation. CONCLUSIONS/SIGNIFICANCE: Our results present a systematic time-course analysis of SC heterogeneity after infection by M. leprae at single-cell resolution, provide valuable information to understand the critical biological processes underlying reprogramming and lipid metabolism during M. leprae infection of SCs, and increase understanding of the disease-causing mechanisms at play in leprosy patients as well as revealing potential new therapeutic strategies.

IL-23/IL-23R Promote Macrophage Pyroptosis and T Helper 1/T Helper 17 Cell Differentiation in Mycobacterial Infection.

Pathogen-induced epigenetic modifications can reshape anti-infection immune processes and control the magnitude of host responses. DNA methylation profiling has identified crucial aberrant methylation changes associated with diseases, thus providing biological insights into the roles of epigenetic factors in mycobacterial infection. In this study, we performed a genome-wide methylation analysis of skin biopsies from patients with leprosy and healthy controls. T helper 17 differentiation pathway was found to be significantly associated with leprosy through functional enrichment analysis. As a key gene in this pathway, IL-23R was found to be critical to mycobacterial immunity in leprosy, according to integrated analysis with DNA methylation, RNA sequencing, and GWASs. Functional analysis revealed that IL-23/IL-23R-enhanced bacterial clearance by activating caspase-1/GSDMD-mediated pyroptosis in a manner dependent on NLRP3 through signal transducer and activator of transcription 3 signaling in macrophages. Moreover, IL23/IL-23R promoted T helper 1 and T helper 17 cell differentiation and proinflammatory cytokine secretion, thereby increasing host bactericidal activity. IL-23R knockout attenuated the effects and increased susceptibility to mycobacterial infection mentioned earlier. These findings illustrate the biological functions of IL-23/IL-23R in modulating intracellular bacterial clearance in macrophages and further support their regulatory effects in T helper cell differentiation. Our study highlights that IL-23/IL-23R might serve as potential targets for the prevention and treatment of leprosy and other mycobacterial infections.

Massively Parallel Sequencingof the Filaggrin Gene Reveals an Association Between FLG Loss-of-function Mutations and Leprosy.

Filaggrin, encoded by the FLG gene, plays a crucial role in the barrier function of epidermis, but the association between FLG loss-of-function mutations and infectious skin diseases has not been systematically studied. FLG coding sequences from 945 patients with leprosy and 916 healthy controls were captured and enriched using an array-based high-throughput system, and subjected to next-generation sequencing. The loss-of-function mutations found were further validated by Sanger sequencing. A total of 21 loss-of-function mutations were found in 945 patients with leprosy, with a carrier rate of 17.53%, while the prevalence of these mutations in 916 healthy controls was 14.77%, which was significantly lower than in patients. Two individual FLG loss-of-function mutations (K4022X and Q1790X) were found to be significantly associated with leprosy. These results suggest a possible role for filaggrin in defending against leprosy pathogens.

The role of an active surveillance strategy of targeting household and neighborhood contacts related to leprosy cases released from treatment in a low-endemic area of China.

OBJECTIVE: Early diagnosis remains the primary goal for leprosy management programs. This study aims to determine whether active surveillance of patients with leprosy and their contact individuals increased identification of latent leprosy cases in the low-endemic areas. METHODS: This cross-sectional survey was carried out between October 2014 and August 2016 in 21 counties throughout Shandong Province. The survey was conducted among patients with leprosy released from treatment (RFT) and their contacts from both household and neighbors. RESULTS: A total of 2,210 RFT patients and 9,742 contacts comprising 7877 household contacts (HHCs), including 5,844 genetic related family members (GRFMs) and 2033 non-genetic related family members and 1,865 contacts living in neighboring houses (neighbor contacts, NCs), were recruited. Among identified individuals, one relapsed and 13 were newly diagnosed, giving a detection rate of 0.12%, corresponding to 120 times the passive case detection rate. Detection rates were similar for HHCs and NCs (0.114% vs. 0.214%, P = 0.287). Analysis of the family history of leprosy patients revealed clustering of newly diagnosed cases and association with residential coordinates of previously-diagnosed multibacillary leprosy cases. CONCLUSION: Active case-finding programs are feasible and contributes to early case detection by tracking HHCs and NCs in low-endemic areas.

Prediction of leprosy in the Chinese population based on a weighted genetic risk score.

Genome wide association studies (GWASs) have revealed multiple genetic variants associated with leprosy in the Chinese population. The aim of our study was to utilize the genetic variants to construct a risk prediction model through a weighted genetic risk score (GRS) in a Chinese set and to further assess the performance of the model in identifying higher-risk contact individuals in an independent set. The highest prediction accuracy, with an area under the curve (AUC) of 0.743 (95% confidence interval (CI): 0.729-0.757), was achieved with a GRS encompassing 25 GWAS variants in a discovery set that included 2,144 people affected by leprosy and 2,671 controls. Individuals in the high-risk group, based on genetic factors (GRS > 28.06), have a 24.65 higher odds ratio (OR) for developing leprosy relative to those in the low-risk group (GRS≤18.17). The model was then applied to a validation set consisting of 1,385 people affected by leprosy and 7,541 individuals in contact with leprosy, which yielded a discriminatory ability with an AUC of 0.707 (95% CI: 0.691-0.723). When a GRS cut-off value of 22.38 was selected with the optimal sensitivity and specificity, it was found that 39.31% of high risk contact individuals should be screened in order to detect leprosy in 64.9% of those people affected by leprosy. In summary, we developed and validated a risk model for the prediction of leprosy that showed good discrimination capabilities, which may help physicians in the identification of patients coming into contact with leprosy and are at a higher-risk of developing this condition.

A pathway-based association analysis identified FMNL1-MAP3K14 as susceptibility genes for leprosy.

The nuclear transcription factor-κB (NF-κB) plays a pivotal role in controlling both innate and adaptive immunity and regulates the expressions of many immunological mediators. Abundant evidences have showed the importance of NF-κB pathway in the host immune responses against Mycobacterium leprae in the development of leprosy. However, no particular association study between leprosy and NF-κB pathway-related gene polymorphisms was reported. Here, we performed a large-scale and two-stage candidate association study to investigate the association between 94 NF-κB pathway-related genes and leprosy. Our results showed that rs58744688 was significantly associated with leprosy (P = 7.57 × 10-7 , OR = 1.12) by combining the previous genomewide association data sets and four independent validation sample series, consisting of a total of 4631 leprosy cases and 6413 healthy controls. This founding implicated that MAP3K14 and FMNL1 were susceptibility genes for leprosy, which suggested the involvement of macrophage targeting and NF-κB pathway in the development of leprosy.

Genome-Wide Analysis of Protein-Coding Variants in Leprosy.

Although genome-wide association studies have greatly advanced our understanding of the contribution of common noncoding variants to leprosy susceptibility, protein-coding variants have not been systematically investigated. We carried out a three-stage genome-wide association study of protein-coding variants in Han Chinese, of whom were 7,048 leprosy patients and 14,398 were healthy control subjects. Seven coding variants of exome-wide significance were discovered, including two rare variants: rs145562243 in NCKIPSD (P = 1.71 × 10-9, odds ratio [OR] = 4.35) and rs149308743 in CARD9 (P = 2.09 × 10-8, OR = 4.75); three low-frequency variants: rs76418789 in IL23R (P = 1.03 × 10-10, OR = 1.36), rs146466242 in FLG (P = 3.39 × 10-12, OR = 1.45), and rs55882956 in TYK2 (P = 1.04 × 10-6, OR = 1.30); and two common variants: rs780668 in SLC29A3 (P = 2.17 × 10-9, OR = 1.14) and rs181206 in IL27 (P = 1.08 × 10-7, OR = 0.83). Discovered protein-coding variants, particularly low-frequency and rare ones, showed involvement of skin barrier and endocytosis/phagocytosis/autophagy, in addition to known innate and adaptive immunity, in the pathogenesis of leprosy, highlighting the merits of protein-coding variant studies for complex diseases.

A Review on Dapsone Hypersensitivity Syndrome Among Chinese Patients with an Emphasis on Preventing Adverse Drug Reactions with Genetic Testing.

AbstractDapsone is a bactericidal and bacteriostatic against Mycobacterium leprae, a causative agent of leprosy. Dapsone is also applied in a range of medical fields because of its anti-inflammatory and immunomodulatory effects. Dapsone hypersensitivity syndrome (DHS) is a rare yet serious adverse drug reaction (ADR) caused by dapsone involving multiple organs. We performed a systematic review of published articles describing dapsone-induced hypersensitivity syndrome, including all Chinese articles and the latest literature available in online databases published between October 2009 and October 2015. We determined the prevalence, clinical characteristics, and mortality rate of DHS. Importantly, we also summarized the recent advances in genetic testing allowing prediction of ADRs. In an initial systematic electronic search, we retrieved 191 articles. Subsequently, these articles were further filtered and ultimately 84 articles (60 Chinese case reports, 21 non-Chinese articles, and three epidemiological studies) were selected, which included 877 patients. The prevalence of DHS among Chinese patients was 1.5% with a fatality rate of 9.6%. Early withdrawal of dapsone and appropriate treatment reduced the fatality rate. Most importantly, genetic screening for the HLA-B*13:01 allele among high-risk populations showed a significant utility as a useful genetic marker to DHS. In conclusion, this review discusses the epidemiological and clinical characteristics of DHS among Chinese patients, which may help physicians to understand this syndrome.

A large-scale genome-wide association and meta-analysis identified four novel susceptibility loci for leprosy.

Leprosy, a chronic infectious disease, results from the uncultivable pathogen Mycobacterium leprae (M. leprae), and usually progresses to peripheral neuropathy and permanent progressive deformity if not treated. Previously published genetic studies have identified 18 gene/loci significantly associated with leprosy at the genome-wide significant level. However as a complex disease, only a small proportion of leprosy risk could be explained by those gene/loci. To further identify more susceptibility gene/loci, we hereby performed a three-stage GWAS comprising 8,156 leprosy patients and 15,610 controls of Chinese ancestry. Four novel loci were identified including rs6807915 on 3p25.2 (P=1.94 × 10-8, OR=0.89), rs4720118 on 7p14.3 (P=3.85 × 10-10, OR=1.16), rs55894533 on 8p23.1 (P=5.07 × 10-11, OR=1.15) and rs10100465 on 8q24.11 (P=2.85 × 10-11, OR=0.85). Altogether, these findings have provided new insight and significantly expanded our understanding of the genetic basis of leprosy.

Tuberculosis risk-associated single nucleotide polymorphisms do not show association with leprosy in Chinese population.

OBJECTIVE: Leprosy and tuberculosis (TB) are chronic granulomatous infectious diseases. As well as pathogen and environmental factors, host genetic factors make a substantial contribution to susceptibility to both diseases. More importantly, leprosy and TB also have pathogenic mechanisms and clinical features in common. In this study, the genetic association between leprosy and TB was investigated in a Chinese Han population. METHODS: A genetic association study that included 46 TB susceptibility single nucleotide polymorphisms (SNPs) was performed, involving 1150 leprosy cases and 1150 controls from the Chinese Han population. The Sequenom MassARRAY system was used. RESULTS: No significant association was found between the 46 SNPs and leprosy. Therefore, according to the present study, there is no shared susceptibility locus between leprosy and TB in the Chinese Han population. CONCLUSIONS: Although leprosy and TB have a number of similar characteristics, no shared susceptibility loci were found in the Chinese Han population. Thus, this study demonstrated that the genetic basis of the pathogenesis of the two diseases may vary greatly.

Discovery of six new susceptibility loci and analysis of pleiotropic effects in leprosy.

Genome-wide association studies (GWAS) have led to the discovery of several susceptibility loci for leprosy with robust evidence, providing biological insight into the role of host genetic factors in mycobacterial infection. However, the identified loci only partially explain disease heritability, and additional genetic risk factors remain to be discovered. We performed a 3-stage GWAS of leprosy in the Chinese population using 8,313 cases and 16,017 controls. Besides confirming all previously published loci, we discovered six new susceptibility loci, and further gene prioritization analysis of these loci implicated BATF3, CCDC88B and CIITA-SOCS1 as new susceptibility genes for leprosy. A systematic evaluation of pleiotropic effects demonstrated a high tendency for leprosy susceptibility loci to show association with autoimmunity and inflammatory diseases. Further analysis suggests that molecular sensing of infection might have a similar pathogenic role across these diseases, whereas immune responses have discordant roles in infectious and inflammatory diseases.

An association study of TOLL and CARD with leprosy susceptibility in Chinese population.

Previous genome-wide association studies (GWASs) identified multiple susceptibility loci that have highlighted the important role of TLR (Toll-like receptor) and CARD (caspase recruitment domain) genes in leprosy. A large three-stage candidate gene-based association study of 30 TLR and 47 CARD genes was performed in the leprosy samples of Chinese Han. Of 4363 SNPs investigated, eight SNPs showed suggestive association (P < 0.01) in our previously published GWAS datasets (Stage 1). Of the eight SNPs, rs2735591 and rs4889841 showed significant association (P < 0.001) in an independent series of 1504 cases and 1502 controls (Stage 2), but only rs2735591 (next to BCL10) showed significant association in the second independent series of 938 cases and 5827 controls (Stage 3). Rs2735591 showed consistent association across the three stages (P > 0.05 for heterogeneity test), significant association in the combined validation samples (Pcorrected = 5.54 × 10(-4) after correction for 4363 SNPs tested) and genome-wide significance in the whole GWAS and validation samples (P = 1.03 × 10(-9), OR = 1.24). In addition, we demonstrated the lower expression of BCL10 in leprosy lesions than normal skins and a significant gene connection between BCL10 and the eight previously identified leprosy loci that are associated with NFκB, a major regulator of downstream inflammatory responses, which provides further biological evidence for the association. We have discovered a novel susceptibility locus on 1p22, which implicates BCL10 as a new susceptibility gene for leprosy. Our finding highlights the important role of both innate and adaptive immune responses in leprosy.

Identification of IL18RAP/IL18R1 and IL12B as leprosy risk genes demonstrates shared pathogenesis between inflammation and infectious diseases.

Of eight leprosy susceptibility loci identified by genome-wide association studies, five have been implicated in Crohn disease, suggesting a common genetic fingerprint between leprosy and inflammatory bowel disease (IBD). Here, we conducted a multiple-stage genetic association study of 133 IBD susceptibility loci in multiple leprosy samples (totaling 4,971 leprosy cases and 5,503 controls) from a Chinese population and discovered two associations at rs2058660 on 2q12.1 (p = 4.57 × 10(-19); odds ratio [OR] = 1.30) and rs6871626 on 5q33.3 (p = 3.95 × 10(-18); OR = 0.75), implicating IL18RAP/IL18R1 and IL12B as susceptibility genes for leprosy. Our study reveals the important role of IL12/IL18-mediated transcriptional regulation of IFN-γ production in leprosy, and together with previous findings, it demonstrates the shared genetic susceptibility between infectious and inflammatory diseases.