FLI1 polymorphism affects susceptibility to cutaneous leishmaniasis in Brazil.

Mapping murine genes controlling cutaneous leishmaniasis (CL) identified Fli1 as a candidate influencing resistance to L. major and enhanced wound healing. We examine FLI1 as a gene controlling CL and mucosal leishmaniasis (ML) caused by L. braziliensis in humans. Intron 1 single nucleotide polymorphisms tagging promoter and enhancer elements were analysed in 168 nuclear families (250 CL; 87 ML cases) and replicated in 157 families (402 CL; 39 ML cases). Robust case-pseudocontrol logistic regression analysis showed association between allele C (odds ratio (OR) 1.65; 95% confidence interval 1.18-2.29; P=0.003) of FLI1_rs7930515 and CL in the primary sample that was confirmed (OR 1.60; 95% confidence interval 1.10-2.33; P=0.014) in the replication set (combined P=1.8 × 10(-4)). FLI1_rs7930515 is in linkage disequilibrium with the functional GAn microsatellite in the proximal promoter. Haplotype associations extended across the enhancer, which was not polymorphic. ML associated with inverse haplotypes compared with CL. Wound healing is therefore important in CL, providing potential for therapies modulating FLI1.

Evidence for associations between the purinergic receptor P2X(7) (P2RX7) and toxoplasmosis.

Congenital Toxoplasma gondii infection can result in intracranial calcification, hydrocephalus and retinochoroiditis. Acquired infection is commonly associated with ocular disease. Pathology is characterized by strong proinflammatory responses. Ligation of ATP by purinergic receptor P2X(7), encoded by P2RX7, stimulates proinflammatory cytokines and can lead directly to killing of intracellular pathogens. To determine whether P2X(7) has a role in susceptibility to congenital toxoplasmosis, we examined polymorphisms at P2RX7 in 149 child/parent trios from North America. We found association (FBAT Z-scores +/-2.429; P=0.015) between the derived C(+)G(-) allele (f=0.68; OR=2.06; 95% CI: 1.14-3.75) at single-nucleotide polymorphism (SNP) rs1718119 (1068T>C; Thr-348-Ala), and a second synonymous variant rs1621388 in linkage disequilibrium with it, and clinical signs of disease per se. Analysis of clinical subgroups showed no association with hydrocephalus, with effect sizes for associations with retinal disease and brain calcifications enhanced (OR=3.0-4.25; 0.004

Genetics and visceral leishmaniasis: of mice and man.

Ninety per cent of the 500,000 annual new cases of visceral leishmaniasis (VL) occur in India/Bangladesh/Nepal, Sudan and Brazil. Importantly, 80-90% of human infections are sub-clinical or asymptomatic, usually associated with strong cell-mediated immunity. Understanding the environmental and genetic risk factors that determine why two people with the same exposure to infection differ in susceptibility could provide important leads for improved therapies. Recent research using candidate gene association analysis and genome-wide linkage studies (GWLS) in collections of families from Sudan, Brazil and India have identified a number of genes/regions related both to environmental risk factors (e.g. iron), as well as genes that determine type 1 vs. type 2 cellular immune responses. However, until now all of the allelic association studies carried out have been underpowered to find genes of small effect sizes (odds ratios or OR < 2), and GWLS using multicase pedigrees have only been powered to find single major genes, or at best oligogenic control. The accumulation of large DNA banks from India and Brazil now makes it possible to undertake genome-wide association studies (GWAS), which are ongoing as part of phase 2 of the Wellcome Trust Case Control Consortium. Data from this analysis should seed research into novel genes and mechanisms that influence susceptibility to VL.

Genes at human chromosome 5q31.1 regulate delayed-type hypersensitivity responses associated with Leishmania chagasi infection.

Visceral leishmaniasis (VL) caused by Leishmania chagasi is endemic to northeast Brazil. A positive delayed-type hypersensitivity skin test response (DTH+) is a marker for acquired resistance to disease, clusters in families and may be genetically controlled. Twenty-three single nucleotide polymorphisms (SNPs) were genotyped in the cytokine 5q23.3-q31.1 region IRF1-IL5-IL13-IL4-IL9-LECT2-TGFBI in 102 families (323 DTH+; 190 DTH-; 123 VL individuals) from a VL endemic region in northeast Brazil. Data from 20 SNPs were analyzed for association with DTH+/- status and VL using family-based, stepwise conditional logistic regression analysis. Independent associations were observed between the DTH+ phenotype and markers in separate linkage disequilibrium blocks in LECT2 (OR 2.25; P=0.005; 95% CI=1.28-3.97) and TGFBI (OR 1.94; P=0.003; 95% CI=1.24-3.03). VL child/parent trios gave no evidence of association, but the DTH- phenotype was associated with SNP rs2070874 at IL4 (OR 3.14; P=0.006; 95% CI=1.38-7.14), and SNP rs30740 between LECT2 and TGFBI (OR 3.00; P=0.042; 95% CI=1.04-8.65). These results indicate several genes in the immune response gene cluster at 5q23.3-q31.1 influence outcomes of L. chagasi infection in this region of Brazil.

Analysis of the BTNL2 truncating splice site mutation in tuberculosis, leprosy and Crohn's disease.

The region on chromosome 6 encoding the major histocompatibility complex (MHC) is associated with a number of autoimmune and infectious diseases. Primary susceptibility to many of these has been localized to a region containing the human leukocyte antigen (HLA)-DR and -DQ genes. A recent study of sarcoidosis has provided evidence of an independent effect, associated with a truncating single nucleotide polymorphism (SNP) of a nearby gene, BTNL2. This gene may encode an immune receptor involved in costimulation. Sarcoidosis, tuberculoid leprosy, tuberculosis (TB) and Crohn's disease all have similar immunological features, including a Th1 response with granuloma formation. In addition mycobacteria have been identified or suggested to be causative pathogens in such conditions. We genotyped the truncating BTNL2 SNP in 92 TB and 72 leprosy families from Brazil and carried out family-based association studies. We could not find evidence of overtransmission of the truncating allele in TB. There was an association with susceptibility to leprosy (P=0.04), however, this is most likely due to linkage disequilibrium with HLA-DR. We also genotyped 476 UK Caucasian cases of Crohn's disease with 760 geographically matched controls and found no evidence of a disease association. We conclude that the truncating BTNL2 SNP is not important in this group of Th1 dominated granulomatous diseases.

Genome-wide scan for visceral leishmaniasis susceptibility genes in Brazil.

A genome-wide scan was conducted for visceral leishmaniasis (VL) in Brazil. Initially, 405 markers were typed in 22 multicase pedigrees (28 nuclear families; 174 individuals; 66 affected). Non-parametric multipoint analysis detected nine chromosomal regions with provisional evidence (logarithm of the odds (LOD) scores 0.95-1.66; 0.003

Genome-wide scan for loci influencing quantitative immune response traits in the Belém family study: comparison of methods and summary of results.

Here we report the results from a genome-wide linkage scan to identify genes and chromosomal regions that influence quantitative immune response traits, using multi-case leprosy and tuberculosis families from north-eastern Brazil. Total plasma IgE, antigen-specific IgG to Mycobacterium leprae soluble antigen (MLSA), M. tuberculosis soluble antigen (MTSA) and M. tuberculosis purified protein derivative (PPD), and antigen-specific lymphocyte proliferation (stimulation index or SI) and interferon-gamma (IFN-gamma) release to MLSA and PPD, were measured in 16 tuberculosis (184 individuals) and 21 leprosy (177 individuals) families. The individuals were genotyped at 382 autosomal microsatellite markers across the genome. The adjusted immune-response phenotypes were analysed using a variety of variance components and regression-based methods. These analyses highlighted a number of practical issues and problems with regard to implementation of the methods and, interestingly, differences were observed between several standard statistical and genetic analysis packages used. From this we determined that, for this set of traits in these pedigrees, significant p values for linkage using variance components analysis, supported by significance using the Visscher-Hopper modification of the Haseman-Elston method, provided the most compelling evidence for linkage. Using these criteria, linkage (5.8 x 10(-5) < p < 0.008) was seen for: total plasma IgE on chromosome 2; IgG to MLSA on chromosomes 8, 17 and 21; IgG to PPD on chromosome 12; SI to PPD on chromosome 1; IFN-gamma to MLSA on chromosomes 6, 7, 10, 12 and 14; and IFN-gamma to PPD on chromosomes 1, 16 and 19.

Evidence for a cluster of genes on chromosome 17q11-q21 controlling susceptibility to tuberculosis and leprosy in Brazilians.

The region of conserved synteny on mouse chromosome 11/human 17q11-q21 is known to carry a susceptibility gene(s) for intramacrophage pathogens. The region is rich in candidates including NOS2A, CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL5/RANTES, CCR7, STAT3 and STAT5A/5B. To examine the region in man, we studied 92 multicase tuberculosis (627 individuals) and 72 multicase leprosy (372 individuals) families from Brazil. Multipoint nonparametric analysis (ALLEGRO) using 16 microsatellites shows two peaks of linkage for leprosy at D17S250 (Z(lr) score 2.34; P=0.01) and D17S1795 (Z(lr) 2.67; P=0.004) and a single peak for tuberculosis at D17S250 (Z(lr) 2.04; P=0.02). Combined analysis shows significant linkage (peak Z(lr) 3.38) at D17S250, equivalent to an allele sharing LOD score 2.48 (P=0.0004). To determine whether one or multiple genes contribute, 49 informative single nucleotide polymorphisms were typed in candidate genes. Family-based allelic association testing that was robust to family clustering demonstrated significant associations with tuberculosis susceptibility at four loci separated by intervals (NOS2A-8.4 Mb-CCL18-32.3 kb-CCL4-6.04 Mb-STAT5B) up to several Mb. Stepwise conditional logistic regression analysis using a case/pseudo-control data set showed that the four genes contributed separate main effects, consistent with a cluster of susceptibility genes across 17q11.2.

Genome-wide scans for leprosy and tuberculosis susceptibility genes in Brazilians.

Genome-wide scans were conducted for tuberculosis and leprosy per se in Brazil. At stage 1, 405 markers (10 cM map) were typed in 16 (178 individuals) tuberculosis and 21 (173 individuals) leprosy families. Nonparametric multipoint analysis detected 8 and 9 chromosomal regions respectively with provisional evidence (P<0.05) for linkage. At stage 2, 58 markers from positive regions were typed in a second set of 22 (176 individuals) tuberculosis families, with 22 additional markers typed in all families; 42 positive markers in 50 (192 individuals) new leprosy families, and 30 additional markers in all families. Three regions (10q26.13, 11q12.3, 20p12.1) retained suggestive evidence (peak LOD scores 1.31, 1.85, 1.78; P=0.007, 0.0018, 0.0021) for linkage to tuberculosis, 3 regions (6p21.32, 17q22, 20p13) to leprosy (HLA-DQA, 3.23, P=5.8 x 10(-5); D17S1868, 2.38, P=0.0005; D20S889, 1.51, P=0.004). The peak at D20S889 for leprosy is 3.5 Mb distal to that reported at D20S115 for leprosy in India. (151 words).

Evidence for a cluster of genes on chromosome 17q11-q21 controlling susceptibility to tuberculosis and leprosy in Brazilians

The region of conserved synteny on mouse chromosome 11/human 17q11-q21 is known to carry a susceptibility gene(s) for intramacrophage pathogens. The region is rich in candidates including NOS2A, CCL2/MCP-1, CCL3/MIP-1 alpha, CCL4/MIP-1 beta, CCL5/RANTES, CCR7, STAT5A/5B. To examine the region in man, we studied 92 multicase tuberculosis (627 individuals) and 72 multicase leprosy (372 indiciduals) families from Brazil. Multipoint nonparametric analysis (ALLEGRO) using 16 microsatellites shows two peaks of linkage for leprosy at D17S250 (Zir score 2.34; P=0.01) and D17S1795 (Zir 2.67; P=O.004) and a single peack for tuberculosis at D17S250 (Zir 2.04; P=0.02). Combined analysis shows significant linkage (peak Zir 3.38) at D17S250, equivalent to an allele sharing LOD score 2.48 (P=0.0004). To determine whether one or multiple genes contribute, 49 informative single nucleotide polymorphisms were typed in candidate genes. Family-based allelic association testing that was robust to family clustering demonstrated significant associations with tuberculosis susceptibility at four loci separated by intervals (NOS2A-8.4 Mb-CCL 18-32.3 kb-CCL4-6.04 Mb-STAT5B) up to several Mb. Stepwise conditional logistic regression analysis using a case/pseudo-control data set showed that the four genes contributed separate main effects, consistent with a cluster of susceptibilitty genes acros 17q11.2

Genome-wide scans for leprosy and tuberculosis susceptibility genes in Brazilians

Genome-wide scans were conducted for tuberculosis and leprosy per se in Brazil. At stage 1,405 markers (10 cM map) were typed in 16 (178 individuals) tuberculosis and 21 (173 individuals) leprosy families. Nonparametric multipoint analysis detected 8 and 9 chromosomal regions respectively with provisional evidence (P<0.05) for linkage. A stage 2, 58 markers from positive regions were typed in a second set of 22 (176 individuals) tuberculosis families, with 22 additional markers types in all families; 42 positive markers in 50 (192 individuals) new leprosy families, and 30 additional markers in all families. Three regions (10q26.13, 11q12.3, 20p12.1) retained suggestive evidence (peak LOD scores 1.31, 1.78, 1.78; P=0.007, 0.0018, 0.0021) for linkage to tuberculosis, 3 regions (6p21.32, 17q22, 20p13) to leprosy (HLA-DQA, 3.23, P=5.8 x 10-5; D17S1868.2.38, P=0.0005; D20S889, 1.51, P=0.004). The peak at D20S889 for leprosy is 3.5 Mb distal to that reported at D20S115 for leprosy in India