Leprosy is a chronic infection of the skin and peripheral nerves caused by Mycobacterium leprae. Despite recent improvements in disease control, leprosy remains an important cause of infectious disability globally. Large-scale genetic association studies in Chinese, Vietnamese and Indian populations have identified over 30 susceptibility loci for leprosy. There is a significant burden of leprosy in Africa, however it is uncertain whether the findings of published genetic association studies are generalizable to African populations. To address this, we conducted a genome-wide association study (GWAS) of leprosy in Malawian (327 cases, 436 controls) and Malian (247 cases, 368 controls) individuals. In that analysis, we replicated five risk loci previously reported in China, Vietnam and India; MHC Class I and II, LACC1 (2 independent loci) and SLC29A3. We further identified a novel leprosy susceptibility locus at 10q24 (rs2015583: combined p=8.81x10-9; OR=0.51 [95% CI 0.40-0.64]). The leprosy risk locus is a determinant of ACTR1A RNA expression in CD4+ T cells (posterior probability of colocalization - PP=0.96). Furthermore, it demonstrates pleiotropy with established risk loci for inflammatory bowel disease and atopic disease. Reduced ACTR1A expression decreases susceptibility to leprosy and atopy but increases risk of inflammatory bowel disease. A shared genetic architecture for leprosy and inflammatory bowel disease has been previously described. We expand on this, strengthening the evidence that selection pressure driven by leprosy has shaped the evolution of autoimmune and atopic disease in modern populations. More broadly, our data highlights the importance of defining the genetic architecture of disease across genetically diverse populations, and that disease insights derived from GWAS in one population may not translate to all affected populations.

Leprosy is a chronic infection of the skin and peripheral nerves caused by Mycobacterium leprae . Despite recent improvements in disease control, leprosy remains an important cause of infectious disability globally. Large-scale genetic association studies in Chinese, Vietnamese and Indian populations have identified over 30 susceptibility loci for leprosy. There is a significant burden of leprosy in Africa, however it is uncertain whether the findings of published genetic association studies are generalizable to African populations. To address this, we conducted a genome-wide association study (GWAS) of leprosy in Malawian (327 cases, 436 controls) and Malian (247 cases, 368 controls) individuals. In that analysis, we replicated four risk loci previously reported in China, Vietnam and India; MHC Class I and II, LACC1 and SLC29A3 . We further identified a novel leprosy susceptibility locus at 10q24 (rs2015583; combined p = 8.81 × 10 −9 ; OR = 0.51 [95% CI 0.40 − 0.64]). Using publicly-available data we characterise regulatory activity at this locus, identifying ACTR1A as a candidate mediator of leprosy risk. This locus shows evidence of recent positive selection and demonstrates pleiotropy with established risk loci for inflammatory bowel disease and childhood-onset asthma. A shared genetic architecture for leprosy and inflammatory bowel disease has been previously described. We expand on this, strengthening the hypothesis that selection pressure driven by leprosy has shaped the evolution of autoimmune and atopic disease in modern populations. More broadly, our data highlights the importance of defining the genetic architecture of disease across genetically diverse populations, and that disease insights derived from GWAS in one population may not translate to all affected populations.

BackgroundA large, double-blind, randomised, placebo-controlled trial of repeat BCG found 49% efficacy against leprosy but no protection against tuberculosis after 6–9 years’ follow-up in 1995. We report here additional follow-up, which resulted in greatly increased tuberculosis case numbers, and allowed subgroup analysis.MethodsNearly 47 000 individuals of all ages living in northern Malawi with a BCG vaccine scar were randomly assigned (1:1) between 1986 and 1989 to receive a second BCG or placebo. The investigators and project staff remained masked to all interventions. Enhanced passive surveillance ensured ascertainment of tuberculosis and leprosy to the end of 2018. Tuberculosis case definitions included rigorous microbiological or histological confirmation. Prespecified subgroup analyses were by tuberculosis type, age at vaccination, time since vaccination, previous tuberculin reactivity, HIV status and Mycobacterium tuberculosis lineage. The original trial is registered with ISRCTN registry, ISRCTN11311670.FindingsIn follow-up until Dec 31, 2018, 824 participants had developed tuberculosis, including 786 with pulmonary disease, of whom 383 (63%) of 607 with known HIV status were HIV positive. There was no effect of a second BCG overall (odds ratio [OR] 0·92; 95% CI 0·80–1·05), or for pulmonary (0·93; 0·81–1·07), or lymph node tuberculosis (0·60; 0·31–1·17). The OR was lower for those with known HIV-negative tuberculosis (0·77; 0·59–1·00), for those vaccinated as children (aged <5 years, 0·74; 0·41–1·35; aged 5–14 years, 0·77; 0·60–0·99), and for cases arising at least 20 years after vaccination (0·79; 0·63–1·01). There were no differences by tuberculin status at vaccination, or lineage. There was no evidence of protection against leprosy beyond 10 years after vaccination (although there have been only nine diagnostically certain cases since 1995).InterpretationThere was no evidence that repeat BCG vaccination provides appreciable protection against overall tuberculosis in this rural African population with a high prevalence of HIV. Subgroup effects should not be overinterpreted given the multiple analyses done. However, the evidence for modest protection against HIV-negative tuberculosis, and for a delayed benefit in those vaccinated as children, is consistent with other observations in the literature.FundingLEPRA, Wellcome Trust, Bill & Melinda Gates Foundation.

We combined two tuberculosis (TB) genome-wide association studies (GWAS) from Ghana and The Gambia with subsequent replication totalling 11,425 participants. A significant association with disease was observed at SNP rs4331426 located in a gene-poor region on chromosome 18q11.2 (P=6.8×10−9, OR=1.19, 95%CI=1.13-1.27). Our finding shows that GWAS can identify novel loci for infectious causes of mortality even in Africa where levels of linkage disequilibrium are particularly low. Tuberculosis (TB) causes significant morbidity and mortality world-wide1,2 with a high disease burden in sub-Saharan Africa. Although previous studies have indicated that susceptibility to pulmonary TB has a substantial genetic component3,4, progress in the determination of contributing genetic variants has been slow. While genome-wide association studies (GWAS) have successfully identified many common variants associated with a variety of diseases5, studies of infectious diseases have so far largely failed to identify novel causative variants, partly due to the small sample sizes studied6,7. We here present a combined analysis of two West African genome-wide association studies, a new data set from Ghana and The Wellcome Trust Case Consortium (WTCCC) tuberculosis study of Gambians (WTCCC, unpublished) with further replication series analyzing in all 11,425 African individuals. The two GWA studies were performed with TB cases and controls recruited from Ghana and The Gambia (study flow-chart in Supplementary Methods) The Ghanaian GWA study included 921 cases and 1,740 controls genotyped using the Affymetrix SNP Array 6.0 with 743,635 autosomal SNPs included in the analysis (Supplementary Methods). The Gambian WTCCC GWA study included 1,316 cases and 1,382 controls who were genotyped using the Affymetrix GeneChip 500K array with 354,607 autosomal SNPs (Supplementary Methods) (WTCCC, unpublished). In total, 333,754 SNPs were included in the combined analysis of 2,237 cases and 3,122 controls from Ghana and The Gambia, achieving 90% power to detect significant association at genotype relative risks of 1.4 or greater. Multidimensional scaling (MDS) analysis was carried out to investigate the population structure (Supplementary Methods)8. The first six MDS components were incorporated as covariates into the logistic regression association analysis in the combined study. The subsequent quantile-quantile plot showed inflation at a level comparable to other GWA studies after correction with the MDS components (λ=1.05, Supplementary Methods). In order to look at these findings more globally we subjected the Ghanaian and Gambian results to an MDS analysis, including available data from the Nigerian Yoruba population, and also non-African populations (Han Chinese, Japanese, CEPH) from the HapMap Project. As expected, differences to the Chinese/Japanese and CEPH populations were large, but the three populations originating from West Africa could also be distinguished clearly (Supplementary Methods). These population-specific differences complicate accurate imputation of the Ghanaian or Gambian genotypes using the Nigerian Yoruba HapMap data set. Fst values for genetic distances for each pair of Ghanaian populations, Gambian populations and between Ghanaian and Gambian populations as a whole are given in Supplementary Methods. Thye et al. Page 2 Nat Genet. Author manuscript; available in PMC 2016 August 05. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts In the combined analysis, we identified 17 SNPs with P≤10−5 with the same direction of effect in both the Ghanaian and WTCCC Gambian study groups (Supplementary Table 1). We attempted to replicate these 17 SNPs in an additional 1,076 cases and 1,611 controls from Ghana (Replication I). The addition of the results obtained in Replication I to the two GWA studies revealed two SNPs (rs2335704 and rs4331426) with P<5×10−7 (Supplementary Table 1). To corroborate the findings, we further genotyped these two SNPs in additional cohorts from Ghana (150 cases, 2,214 controls) and Malawi (236 cases, 779 controls), (Replication II), as well as in 332 family trios/duos from Ghana, whereby cases of nuclear families were part of the complete association analysis. For the two hit regions, neighbouring SNPs were studied in an attempt to fine-map the association signals (Supplementary Table 2). When combining the GWA study data with those from Replications I and II, SNP rs4331426, which maps to chromosome 18q11.2, obtained the highest association signal with an overall P=6.8×10−9 (OR=1.19, 95%CI=1.13-1.27), or PGC =1.6×10−8 after genomic control correction (Fig. 1, Table 1). Consistent estimates of ORs as assessed by the Breslow Day test were observed (P=0.95) across the studies and ethnic groups, and no difference in ORs was seen between the logistic regression and Mantel-Haenszel tests. Heterogeneity between the studies was negligible with I2=0.0%. The analysis of Ghanaian nuclear families supported the association, P=0.016 (OR=1.33, 95%CI=1.05-1.68), although this statistic was not included in the final P value because the cases were part of the association analyses (Table 1). The associated chromosome 18 variant (SNP rs4331426) is common in African populations but much rarer in all other populations and has a remarkably consistent OR across African cohorts, including the East African samples from Malawi. Additional work is required to ascertain the causative variant, its functional significance and any possible counterbalancing selective pressure. The nearest genes to this SNP are GATA6, CTAGE1, RBBP8, CABLES1, as well as a number of, as yet, un-annotated open reading frames. However, the generally low linkage disequilibrium on 18q11.1-q11.2 suggests that rs4331426 is within a genedesert region that is punctuated by evolutionarily conserved domains with regulatory potential. In addition to rs4331426, a second variant, rs2335704 on chromosome 2 was found associated after Replication I (P=3×10−7, OR=1.23, 95%CI=1.14 -1.34). However, the significance decreased to P=2.1×10−6 (OR=1.29, 95%CI=1.11-1.28) after adding results of Replication II (Supplementary Fig. 1, Supplementary Table 3). The Mantel-Haenszel test, stratified for ethnic groups, revealed heterogeneity in ORs between the Ghanaian ethnic group of the Ga-Adangbe compared to all other ethnicities. This heterogeneity suggests that the result should be interpreted with caution. GWA studies in African populations are, in general, limited by extensive genetic diversity and shorter LD ranges, and to date no novel loci of genome wide statistical significance (P<5×10−8) have been reported using the GWA approach in African studies (WTCCC, unpublished). A recent malaria GWA study in The Gambia found substantial population Thye et al. Page 3 Nat Genet. Author manuscript; available in PMC 2016 August 05. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts complexity6, indicating marked diversity among African ethnicities and emphasizing the need for careful population structure correction and ethnicity stratification. In general, finding convincing non-MHC susceptibility loci across populations for infectious diseases has been difficult, even in individuals of European ancestry. A recent GWA study on HIV viral set-point revealed strong signals within the HLA-B and HLA-C loci, but not any novel non-MHC loci7. Pathogen variation may underlie some of the difficulties in finding loci for a given infectious disease and M. tuberculosis displays substantial geographic variation in genotype frequencies9. However, as we have shown previously and also here9-13, combined analyses with increased aggregate sample sizes may help in the identification of novel genetic variants, perhaps particularly those less sensitive to pathogen variation. Analyzing African individuals poses specific challenges, and genetic differences between populations, even within West Africa, are large enough to complicate standard imputation procedures. Assessing imputation accuracy using the YRI as reference we found a mean genotype error rate across all chromosomes of 8.2% for the combined Ghanaian and Gambian sample, which raises some concern about the validity of imputation in this setting. We performed a genome wide imputation analysis with stringent quality criteria (Rsq>0.7) followed by association analysis of the imputed SNPs (Supplementary Table 4). In these calculations, no variant reached a significance of P<10−6. However, many variants did reach the significance level of the rs4331326 SNP, and these will be carried forward in future studies. We analyzed variants of the Affymetrix SNP arrays located in candidate genes previously found to be associated with resistance or susceptibility to TB. We observed weak to moderate signals at several genes including the HLA-DQ region (rs9469220, P=0.0017) (Supplementary Table 5). Considering the a priori associations for these regions there appears to be an over-representation of nominally significant results, suggesting that a number of the analyzed SNPs might be true TB susceptibility loci. However, more work will be necessary to replicate positive findings of previous association studies and to identify the true causative variants of these candidate gene analyses. This work demonstrates that a novel non-MHC locus can be identified for a major fatal infectious disease caused by a highly polymorphic pathogen and suggests that many further loci may be identifiable with GWA studies of sufficient sample size, even in African populations, who suffer the greatest burden of communicable diseases. Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Nat. Genet. 42, 739–741 (2010); published online 8 August 2010; corrected after print 22 September 2011 In the version of this article initially published, an acknowledgment was missing. The study was supported by funding from the NIHR Oxford Biomedical Research Centre programme. The error has been corrected in the HTML and PDF versions of the article.

We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-gamma) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-gamma was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.

Few studies have investigated the risk factors for tuberculosis (TB) infection in highly endemic countries. We conducted a household study in The Gambia, in which a tuberculin skin test (TST) was performed in members of the households of 315 smear-positive pulmonary TB cases and 305 community control subjects. The risk of being TST positive (10 mm or more) was higher in contacts of cases than in contacts of control subjects. It increased with age, male sex, and duration of stay in the household but was not associated with the presence of a bacille de Calmette-Guérin scar. Within the households of the TB cases, the risk of TST positivity was higher in males and was increased with age, social proximity to the case, and the radiologic extent of the disease in the case's chest X-ray. Adjusting on these, the risk of TST positivity was higher in first-degree relatives compared with more distant relatives and nongenetically related household members, but the effect was not statistically significant. In highly endemic areas, the risk of TB infection in contacts of TB infectious cases is associated with age, sex, intensity of exposure to the case, and severity of disease in the case, but it is possible that genetic factors contribute to the susceptibility to Mycobacterium tuberculosis infection.

Evaluation ofdisease outcome iscentral totheassessment oftuberculosis (TB)control programmes. Inthe study reported inthis article weexamined thefactors influencing themeasurement ofoutcome, survival rates during andafter treatment, smearconversion rates, andrelapse rates forpatients diagnosed withTBina rural areaofMalawi between 1986andmid-1994. Patients with lesscertain diagnoses ofTBweremorelikely todiethanthose with confirmed TB,both amongthosewhowereseropositive andthose whowereseronegative tohumanimmunodeficiency virus (HIV). Themortality rate amongsmear-positive patients with aseparate culture-positive specimen washalf that ofpatients with nosuchdiagnostic confirmation. Patients notregistered bytheMinistry ofHealth had muchhigher mortality anddefault rates thandidregistered patients. Amongsmear-positive patients, HIV serostatus wasthemostimportant influence onmortality bothduring andafter treatment (crude hazard ratios (95%confidence intervals) = 5.6(3.0-10) and7.7(3.4-17), resp.), butHIVserostatus didnotinfluence smearconversion rates. Theinitial degree ofsmearpositivity influenced smearconversion rates, butnot mortality rates. Nosignificant predictors ofrelapse wereidentified. Unless considerable careistaken toinclude allTBpatients, andtoexclude nontuberculous patients, recorded TBoutcome statistics aredifficult tointerpret andmaybemisleading. Inpopulations with highrates ofHIVinfection, TBtarget curerates of85%areunrealistic. Whennewinterventions areassessed itcannot beassumedthat factors whichinfluence thesmearconversion ratewill alsoinfluence themortality rate.

Since the mid-1980s tuberculosis (TB) case numbers and HIV seroprevalence have both risen sharply in sub-Saharan Africa. Estimates for the relative risk of TB in those infected with HIV have ranged from less than five to more than 20. The proportion of TB cases attributable to HIV (the population attributable fraction) has been calculated for several populations but is difficult to interpret if no account is taken of the age and sex distribution of the cases. In a rural area of Malawi we have studied the proportion of TB attributable to HIV over time. Nearly 40 per cent of smear-positive TB cases in this rural area of Malawi can now be attributed directly to HIV. The actual effect of HIV on TB is even greater than this because increased case numbers increase transmission of tuberculosis infection to both HIV-infected and non-infected sections of the population. We compare our findings with others from sub-Saharan Africa and discuss reasons for the differences, and methodological issues in interpretation In the presence of the HIV epidemic the number of cases of tuberculosis (TB) reported in subSaharan Africa has increased sharply (De Cock et al. 1992). Increases in TB of more than ten per cent per year have been reported from several countries (Cantwell and Binkin 1996). In Malawi notified cases of TB rose from 5334 in 1985 to 19,496 in 1994 (Harries et al. 1996). Increases in TB have also been reported in many other parts of the world, but the reasons for the increases are not the same in all populations. In Africa the increases are assumed to be due to HIV, but increases are also found in Eastern Europe, in areas relatively untouched by the HIV epidemic (Raviglione et al. 1994). Reasons for reported increases in TB other than through HIV include changes in ascertainment and reporting, and real increases in TB due to poverty, inadequate TB control programs and increasing drug resistance. These effects are not * The Karonga Prevention Study has been funded primarily by LEPRA (The British Leprosy Relief Association) and ILEP (The International Federation of Anti-Leprosy Organizations) with contributions from the WHO/UNDP/World Bank Special Program for Research and Training in Tropical Diseases and the WHO Tuberculosis Program. J.R. Glynn is supported by the British Overseas Development Administration. We thank the Government of the Republic of Malawi for their interest in and support of the Project. We thank Dr P.A. Jenkins and Prof. S. Lucas for their help with the bacteriology and histology, and Drs L. Rodrigues and C. Ronsmans for helpful comments.