A Genome-wide Association Study Identifies SERPINB10, CRLF3, STX7, LAMP3, IFNG-AS1, and KRT80 As Risk Loci Contributing to Cutaneous Leishmaniasis in Brazil.

BACKGROUND: Our goal was to identify genetic risk factors for cutaneous leishmaniasis (CL) caused by Leishmania braziliensis. METHODS: Genotyping 2066 CL cases and 2046 controls using Illumina HumanCoreExomeBeadChips provided data for 4 498 586 imputed single-nucleotide variants (SNVs). A genome-wide association study (GWAS) using linear mixed models took account of genetic diversity/ethnicity/admixture. Post-GWAS positional, expression quantitative trait locus (eQTL) and chromatin interaction mapping was performed in Functional Mapping and Annotation (FUMA). Transcriptional data were compared between lesions and normal skin, and cytokines measured using flow cytometry and Bioplex assay. RESULTS: Positional mapping identified 32 genomic loci associated with CL, none achieving genome-wide significance (P < 5 × 10-8). Lead SNVs at 23 loci occurred at protein coding or noncoding RNA genes, 15 with eQTLs for functionally relevant cells/tissues and/or showing differential expression in lesions. Of these, the 6 most plausible genetic risk loci were SERPINB10 (Pimputed_1000G = 2.67 × 10-6), CRLF3 (Pimputed_1000G = 5.12 × 10-6), STX7 (Pimputed_1000G = 6.06 × 10-6), KRT80 (Pimputed_1000G = 6.58 × 10-6), LAMP3 (Pimputed_1000G = 6.54 × 10-6), and IFNG-AS1 (Pimputed_1000G = 1.32 × 10-5). LAMP3 (Padjusted = 9.25 × 10-12; +6-fold), STX7 (Padjusted = 7.62 × 10-3; +1.3-fold), and CRLF3 (Padjusted = 9.19 × 10-9; +1.97-fold) were expressed more highly in CL biopsies compared to normal skin; KRT80 (Padjusted = 3.07 × 10-8; -3-fold) was lower. Multiple cis-eQTLs across SERPINB10 mapped to chromatin interaction regions of transcriptional/enhancer activity in neutrophils, monocytes, B cells, and hematopoietic stem cells. Those at IFNG-AS1 mapped to transcriptional/enhancer regions in T, natural killer, and B cells. The percentage of peripheral blood CD3+ T cells making antigen-specific interferon-γ differed significantly by IFNG-AS1 genotype. CONCLUSIONS: This first GWAS for CL identified multiple genetic risk loci including a novel lead to understanding CL pathogenesis through regulation of interferon-γ by IFNG antisense RNA 1.

Human genetics of leishmania infections.

Identifying genetic risk factors for parasitic infections such as the leishmaniases could provide important leads for improved therapies and vaccines. Until recently most genetic studies of human leishmaniasis were underpowered and/or not replicated. Here, we focus on recent genome-wide association studies of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). For VL, analysis across 2287 cases and 2692 controls from three cohorts identified a single major peak of genome-wide significance (Pcombined = 2.76 × 10-17) at HLA-DRB1-HLA-DQA1. HLA-DRB1*1501 and DRB1*1404/DRB1*1301 were the most significant protective versus risk alleles, respectively, with specific residues at amino acid positions 11 and 13 unique to protective alleles. Epitope-binding studies showed higher frequency of basic AAs in DRB1*1404-/*1301-specific epitopes compared to hydrophobic and polar AAs in DRB1*1501-specific epitopes at anchor residues P4 and P6 which interact with residues at DRB1 positions 11 and 13. For CL, genome-wide significance was not achieved in combined analysis of 2066 cases and 2046 controls across 2 cohorts. Rather, multiple top hits at P < 5 × 10-5 were observed, amongst which IFNG-AS1 was of specific interest as a non-coding anti-sense RNA known to influence responses to pathogens by increasing IFN-γ secretion. Association at LAMP3 encoding dendritic cell lysosomal associated membrane protein 3 was also interesting. LAMP3 increases markedly upon activation of dendritic cells, localizing to the MHC Class II compartment immediately prior to translocation of Class II to the cell surface. Together these GWAS results provide firm confirmation for the importance of antigen presentation and the regulation of IFNγ in determining the outcome of Leishmania infections.

Analysis of expression of FLI1 and MMP1 in American cutaneous leishmaniasis caused by Leishmania braziliensis infection.

FLI1 (Friend leukemia virus integration 1) and IL6 (interleukin 6; IL-6) are associated with Leishmania braziliensis susceptibility. Cutaneous lesions show exaggerated matrix metalloproteinase 1 (MMP1). In other skin diseases, FLI1 promoter methylation reduces FLI1 expression, and low FLI1 down-regulates MMP1. IL-6 increases FLI1 expression. We hypothesized that epigenetic regulation of FLI1 in cutaneous leishmaniasis, together with IL-6, might determine MMP1 expression. While generally low (<10%), percent FLI1 promoter methylation was lower (P=0.001) in lesion biopsies than normal skin. Contrary to expectation, a strong positive correlation occurred between FLI1 methylation and gene expression in lesions (r=0.98, P=0.0005) and in IL-6-treated L. braziliensis-infected macrophages (r=0.99, P=0.0004). In silico analysis of the FLI1 promoter revealed co-occurring active H3K27ac and repressive DNA methylation marks to enhance gene expression. FLI1 expression was enhanced between 3 and 24hour post infection in untreated (P=0.0002) and IL-6-treated (P=0.028) macrophages. MMP1 was enhanced in lesion biopsies (P=0.0002), induced (P=0.007) in infected macrophages, but strongly inhibited by IL-6. No correlations occurred between FLI1 and MMP1 expression in lesions or infected macrophages (with/without IL-6). We conclude that MMP1 is regulated by factors other than FLI1, and that the influence of IL-6 on MMP1 was independent of its effect on FLI1.

The -308 bp TNF gene polymorphism influences tumor necrosis factor expression in leprosy patients in Bahia State, Brazil.

Leprosy or Hansen's disease is a debilitating chronic granulomatous disease caused by Mycobacterium leprae, with high incidence and prevalence in Brazil. The -308 bp G/A single nucleotide polymorphism (SNP rs1800629) in the tumor necrosis factor (TNF) gene promoter is a proposed risk factor for leprosy. In Brazil, Northern India, Egypt and Nepal, the common G allele was associated with leprosy. In Eastern India, Thailand and Malawi the minor A allele was the risk factor. Allele A was previously associated with high TNF. We genotyped rs1800629 in 326 leprosy cases from Bahia State, Brazil, including 72 paucibacillary (PB) and 47 multibacillary (MB) without reactions, and 69 reversal reaction (RR) and 78 erythema nodosum leprosum (ENL) with reactions. Logistic regression was used to compare patient groups with 331 healthy controls. Relative TNF mRNA was determined in peripheral blood leukocytes by QRTPCR, and serum TNF levels measured by ELISA. We found that TNF mRNA expression was higher (P=0.03) in leprosy patients compared to endemic controls, but did not differ significantly between clinical subgroups. Carriage of the minor A allele was associated (P=0.003) with low TNF mRNA across leprosy patients. Nevertheless, we found no evidence for either allele at this SNP as a risk factor for leprosy per se (OR=1.12, 95% CI 0.79-1.60, P=0.52), PB (OR=0.99, 95% CI 0.54-1.81, P=0.97), MB (OR=0.86, 95% CI 0.40-1.83, P=0.70), RR (OR=1.37, 95% CI 0.79-2.38, P=0.27) or ENL (OR=0.76, 95% CI 0.40-1.45, P=0.42) when compared to endemic controls. Further studies are required to determine whether the influence of the minor A allele on TNF mRNA levels determines response to treatment, particularly in the context of ENL reaction treatment with anti-TNF therapies and RR reactions where treatment with prednisolone is known to reduce TNF levels. Our findings contribute to understanding TNF as an important determinant of leprosy immunopathology in Brazil.