Genes and Immunity (2007) 8, 539–551 & 2007 Nature Publishing Group All rights reserved 1466-4879/07 $30.00 www.nature.com/gene

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

SMB Jeronimo1, AKB Holst2, SE Jamieson3, R Francis3, DRA Martins1, FL Bezerra1, NA Ettinger2, ET Nascimento1, GR Monteiro1, HG Lacerda1, EN Miller3, HJ Cordell4, P Duggal5, TH Beaty6, JM Blackwell3,7 and ME Wilson2,7 1Department of Biochemistry, Federal University of Rio Grande do Norte, Natal, RN, Brazil; 2Departments of Internal Medicine, Microbiology, Epidemiology and the Molecular Biology Program, University of Iowa and the VA Medical Center, Iowa City, IA, USA; 3Cambridge Institute for Medical Research, University of Cambridge School of Clinical Medicine, Cambridge, UK; 4Institute of Human Genetics, International Centre for Life, Newcastle University, Central Parkway, Newcastle upon Tyne, UK; 5Inherited Disease Research Branch, National Research Institute, NIH, Bethesda, MD, USA and 6Department of Epidemiology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA

Visceral leishmaniasis (VL) caused by Leishmania chagasi is endemic to northeast Brazil. A positive delayed-type hypersensitivity skin test response (DTH þ ) is a marker for acquired resistance to disease, clusters in families and may be genetically controlled. Twenty-three single polymorphisms (SNPs) were genotyped in the cytokine 5q23.3–q31.1 region IRF1-IL5-IL13-IL4-IL9-LECT2-TGFBI in 102 families (323 DTH þ ; 190 DTHÀ; 123 VL individuals) from a VL endemic region in northeast Brazil. Data from 20 SNPs were analyzed for association with DTH þ /À status and VL using family-based, stepwise conditional logistic regression analysis. Independent associations were observed between the DTH þ phenotype and markers in separate linkage disequilibrium blocks in LECT2 (OR 2.25; P ¼ 0.005; 95% CI ¼ 1.28–3.97) and TGFBI (OR 1.94; P ¼ 0.003; 95% CI ¼ 1.24–3.03). VL child/parent trios gave no evidence of association, but the DTHÀ phenotype was associated with SNP rs2070874 at IL4 (OR 3.14; P ¼ 0.006; 95% CI ¼ 1.38–7.14), and SNP rs30740 between LECT2 and TGFBI (OR 3.00; P ¼ 0.042; 95% CI ¼ 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. Genes and Immunity (2007) 8, 539–551; doi:10.1038/sj.gene.6364422; published online 23 August 2007

Keywords: delayed type hypersensitivity; visceral leishmaniasis; genetic control; chromosome 5q31.1

Introduction parasites is quite variable. In most individuals, infection with L. donovani complex parasites is asymptomatic, Leishmaniasis refers to a spectrum of diseases caused by usually accompanied by a positive delayed-type hyper- protozoa belonging to the genus Leishmania. Distinct sensitivity (DTH þ ) skin test response to leishmania clinical forms of leishmaniasis are typically caused by antigen, called a Montenegro response. Progressive different species of the parasite. The leishmaniases are infection is associated with high titers of anti-leishmania caused by the Leishmania spp. protozoa, and parasites are antibodies and a negative DTH (DTHÀ).3–5 Antibody acquired through the bite of infected sand flies. titers are detected by enzyme-linked immunosorbent Leishmaniasis is endemic in 88 countries (Electronic assay (ELISA) using soluble crude leishmania antigen Database Information 1). The clinical form of leishma- lysates or the recombinant antigen k39, which is an niasis that causes the most fatalities is visceral leishma- indicator of acute VL in some populations including niasis (VL), a disease usually caused by members of the Brazil.6–8 Symptomatic VL is accompanied by hepatos- Leishmania donovani species complex, L. infantum or L. plenomegaly, fever, cachexia and progressive suppres- donovani in the Old World and L. infantum/chagasi in Latin sion of cellular immunity, eventually leading to death if America.1,2 However, the outcome of infection with these treatment does not intervene. The death rate from VL varies from 5 to 10% in endemic regions, even where Correspondence: Professor ME Wilson, Department of Internal treatment is available.2,4 Medicine, The University of Iowa, 200 Hawkins Drive, Iowa City, IA Variability in human Leishmania chagasi infection is 52242, USA. explained in part by host factors that increase suscept- E-mail: [email protected] 2,9 7These authors contributed equally to this work. ibility, for example, young age and nutritional status. Received 22 March 2007; revised 18 July 2007; accepted 18 July 2007; Heterogeneity in parasite isolates contribute,10–12 but published online 23 August 2007 there is gathering evidence that host genetic factors also DTH responses in L. chagasi infection SMB Jeronimo et al 540 play a role in determining the outcome of human and routine cell counts, and DNA was extracted from infection with parasites causing VL.13–15 A role for host 10 ml of blood leukocytes as described previously.33 Skin genetics was first suggested by studies in mice showing test results were read 48–72 h after test placement, at that polymorphism at Slc11a1 (formerly Nramp1/Lsh/Ity/ which time subjects were informed of test results and Bcg) determines innate macrophage-mediated resistance referred to the appropriate medical facility for any to infection with L. donovani16 and L. infantum/chagasi,17 medical conditions found. The diagnosis of VL was whereas the murine major histocompatibility complex confirmed by compatible clinical symptoms, response to controls acquired T-cell-mediated recovery.17,18 Candi- glucantime therapy and visualization of the parasite on a date gene studies in humans have confirmed a role for Giemsa stain of a bone marrow aspirate. The antigen SLC11A1 in human VL,19,20 with variable evidence for a for ELISA was prepared from a lysate of whole L. chagasi role of human HLA genes in regulating clinical dis- promastigotes, and/or from recombinant k39 kindly ease.21–25 In addition, human studies show increased provided by Steven Reed, PhD, IDRI, Seattle, WA, circulating levels of the T-helper 2 cytokine -4 USA.6,34 (IL-4) in VL patients,26,27 and candidate gene analysis has also demonstrated association between clinical VL and Phenotype definitions polymorphisms at IL4.28 In a previous report32 of 1106 exposed individuals, we Thus far, the emphasis of human genetic studies of VL described four main phenotypes associated with L. has been on finding associations between genetic chagasi infection in our study area; VL are individuals variants and clinical disease. Epidemiological studies with current or prior symptomatic VL (12%); DTH þ , have shown that a positive DTH response (DTH þ )isa individuals with positive delayed-type hypersensitivity marker for developing acquired resistance to disease.29 In response with no history of VL (35%); Ab þ , individuals Brazil, DTH þ shows marked clustering in families,30 with DTHÀ response and seropositive (2%); and DTHÀ and appears to be under genetic control.31 To investigate are individuals with DTHÀ who are seronegative (51%). genetic factors that might be associated with the DTH þ Upon reviewing the data of 1698 individuals enrolled for phenotype, we have been working in a site in northeast genetic studies between 1996 and 2004, and for whom Brazil where the prevalence of VL in peri-urban areas complete data (clinical, Montenegro and anti-leishmania has been high for the past two decades.32 Using this antibodies) are available, we found a total of 524 subjects population we have been able to discern not only (30.85%) who were DTH þ with no history of VL. Of symptomatic individuals, but also asymptomatic DTH þ these, 51 (9.7%) remained weakly positive for anti- infected persons and individuals who remain DTH leishmania antibodies at last survey. None have devel- negative (DTHÀ) despite prolonged residence in a oped VL. Of the DTH þ subjects followed over 5–10 dwelling where there is demonstrated risk of L. chagasi years after first DTH response, 92% remain DTH þ . infection. Here, we report on a study of candidate genes Hence, a DTH response in the absence of any history of on chromosome 5q23.3–q31.1, near the cluster of genes clinical VL provides a measure of acquired resistance to encoding type II cytokines, and their association with infection. Treated VL subjects also convert to a DTH þ DTH þ and DTHÀ phenotypes in subjects exposed to response at 6 months to 1 year after treatment, L. chagasi. concomitant with a substantial decrease in anti-leishma- nia antibody levels as previously reported.6 Of 155 VL patients studied longitudinally, only one patient with an Methods underlying immunosuppressive condition at the time of second DTH placement has become DTHÀ. Hence, it is Study subjects and study procedures important to have accurate information on history of Brazilian subjects with active VL were identified because symptomatic VL and treatment in the study area to they were admitted to one of three public hospitals in define phenotypes for genetic analysis. Natal, northeast Brazil. Transmission of L. chagasi is both For the present study, VL index cases identified focal and cyclic, and this allowed us to identify through the hospitals were used to ascertain a total of neighborhoods in Natal, Rio Grande do Norte, Brazil 102 leishmaniasis (mixed for VL and DTH þ ), or with ongoing or recent transmission of L. chagasi neighborhood (DTH þ only) pedigrees (156 nuclear infection. Clinical and epidemiological characteristics of families; Table 1). Phenotypes within families (Table 1) an overlapping population from the region have been were defined as previously;32 that is, VL, including published previously.32 Hospitalized patients with VL historic cases or cases current at the time of study; were interviewed for their level of interest in the study, DTH þ , including individuals with a positive skin test and the location of their houses was determined. reaction, defined as X5 mm of induration 48–72 h after Consenting subjects and their family members were test placement; and DTHÀ, including individuals were later visited in their homes where informed consent was negative for leishmania serology with a Montenegro skin obtained from other family members. Families in the test reaction o5 mm in size. DTH þ and DTHÀ subjects endemic neighborhood without a VL case who lived in all had a negative history for VL or comparable illness, houses adjacent to or near VL case families were also and none developed VL during the course of the study. enrolled. These homes were less than 500 m from a case The DTHÀ category included subjects living in house- family home. holds for 43 years with a 440% infection rate but who Family members were interviewed for information on had remained healthy and DTHÀ. As such, all DTHÀ relationships, medical history and current medical individuals included in the genetic study were over 3 status. Consenting subjects were examined, blood was years of age. We examined this phenotype based upon drawn and a leishmania skin test (Montenegro) was the hypothesis that such exposed individuals may clear administered. Blood was tested for leishmania serology the parasite through innate immune mechanisms before

Genes and Immunity DTH responses in L. chagasi infection SMB Jeronimo et al 541 Table 1 Distribution of study subjects by phenotype (VL, DTH+, Ab+ and DTHÀ) as defined in the text

N individuals with phenotypes Total

VL DTH+ Ab+ DTHÀ Unknown

(A) Individuals 123 323 20 190 17 673 Females 46 169 11 110 11 347 Males 77 154 9 80 6 326

VL phenotype DTH+ phenotype DTHÀ phenotype

(B) Number of pedigrees 82 93 68 Number of nuclear families 87 100 76 Nuclear families: 1 sib with phenotype 70 50 45 Nuclear families: 2 sibs with phenotype 17 35 21 Nuclear families: 3 sibs with phenotype 1 8 9 Nuclear families: 4 sibs with phenotype 0 4 1 Nuclear families: 5 sibs with phenotype 0 2 0 Nuclear families: 6 sibs with phenotype 0 1 0 Total number with phenotype 123 323 190 Total number with phenotype in nuclear families 107 176 118

Abbreviations: Ab+, individuals with negative delayed-type hypersensitivity response and seropositive (2%); DTHÀ, individuals with negative delayed-type hypersensitivity response and seronegative (51%); DTH+, individuals with positive delayed-type hypersensitivity response with no history of VL (35%); VL, visceral leishmaniasis. (A) Provides details of the total number of individuals by phenotype included in the study. (B) Shows the distribution of VL, DTH+ and DTHÀ phenotypes by nuclear family. Most nuclear families contained more than one phenotype and as such may be represented in more than one column of the table. Overall, 102 pedigrees containing 156 nuclear families were studied. All subjects were residents of neighborhoods outside or peripheral to Natal, Brazil. an adaptive immune response to parasite antigen performed using 40 ng of genomic DNA and analyzed develops. Although documented (Table 1), there were using an ABI Prism 7000 Sequence Detection System and insufficient numbers of individuals seropositive for anti- associated 7000 System SDS Software v.1.2 (ABI). leishmania antibodies and negative for DTH to permit meaningful analysis of this group as a separate pheno- Statistical analyses type for genetic analysis. The 102 pedigrees provided a All SNP genotypes were checked with the program total of 123 VL cases with 107 child/parent trios, 323 PEDCHECK35 to identify Mendelian inconsistencies. DTH þ individuals with 176 child/parent trios and 190 Since the families used here are a subset of those for DTHÀ individuals with 118 child/parent trios. whom a large number of highly polymorphic micro- satellites had already been genotyped,36 Mendelian Ethical approvals inconsistencies due to mis-paternities or incorrect famil- Approval for work with human subjects was obtained ial relationships had already been removed. Mendelian from the Ethical Research Committee at the Universi- inconsistencies for SNP data, therefore, invariably dade Federal do Rio Grande do Norte, the Comissa˜o represented miscalling of alleles on the Taqman output. Nacional de E´ tica em Pesquisa (CONEP), the University If the call remained ambiguous on re-scoring of the of Iowa, Johns Hopkins University, the University of Taqman output, that individual’s SNP genotype was Virginia and the National Human Genome Research removed from the analysis. Such errors occurred in Institute boards. The Brazilian university has Federal- o0.1% of the total genotypes (23 SNPs  673 in- Wide Assurance, and its Institutional Review Board is dividuals ¼ 15 479 genotypes) undertaken for this study. registered with the NIH. Allele frequencies were determined from unrelated individuals in these families using the computer pro- Genotyping methods gram SPLINK.37 Deviation from Hardy–Weinberg Equi- Twenty-three single nucleotide polymorphisms (SNPs) librium (HWE) was determined using a set of unrelated were selected from chromosome 5q23.3–q31.1 on the individuals from the families (parents and individuals basis of public domain information on allele frequencies, married in), excluding any with VL. The unrelated group proximity to genes and the known functional SNPs in included 48 individuals of unknown phenotype, 241 IL4. HapMap data were not available at the time this individuals with DTH þ and 165 individuals with a study commenced. Taqman SNP genotyping assays were DTHÀ phenotype. Tests for HWE were performed ordered from ABI using both pre-made and custom within STATA 9.1 (Electronic Database Information 3) design assays (that is, ‘Assays-On-Demand’ or ‘Assays- using the GenAssoc package (Electronic Database In- By-Design’, Electronic Database Information 2 (see formation 4). Markers that were out of HWE were Supplementary Information)). Taqman assays were excluded from the analysis. Linkage disequilibrium (LD)

Genes and Immunity DTH responses in L. chagasi infection SMB Jeronimo et al 542 between pairs of markers across chromosome 5q23.3– served noncoding sequences (CNS) that might contain q31.1 was determined for the set of unrelated individuals long-range regulatory elements and transcription factor using Hedrick’s definition of Lewontin’s D0 statistic38 binding sites. CNS are defined as regions of conserved and r2.39 LD plots were generated using the default sequence approaching or similar in magnitude to that settings in Haploview40 available from the HapMap observed at the known in the region under Project site (Electronic Database Information 5). examination. Genomic sequence for the six organisms Power calculations for trios were performed using the was exported in FASTA format from ENSEMBL (NCBI method of Knapp.41 This provides an estimate of power Build 36, Ensembl Genebuild August 2006, Database for independent trios, which would be the case for trios version 42.36d) and associated annotation exported in in the families for a true functional variant, or something the form of a General Feature Format (GFF) file. The in complete LD with it.42 In this case, we determined global alignment tool LAGAN was used to align statistical power on the basis of the number of full child/ genomic sequences using guide trees (((human dog) parent trios for each phenotype. For the DTH þ cow) mouse) and ((human mouse) chicken). SYNPLOT44 phenotype, the 176 child/parent trios had X82.6% was used to visualize the annotated alignment. CNS power to detect an effect corresponding to an odds were analyzed for promoter and enhancer elements ratio ¼ 2 (at P ¼ 0.01) for SNPs with variant allele using PROMO (Electronic Database Information 7), frequencies of X0.2, but only p51.0% power for SNPs AliBaba v2.1 (Electronic Database Information 8) and with variant allele frequencies p0.1. For the VL and MatInspector (Electronic Database Information 9). DTHÀ phenotypes, B110 child/parent trios provided X56.4% power to detect a similar effect size for SNPs with variant allele frequencies of X0.2, and p31.4% Results power for SNPs with variant allele frequencies of p0.1. All SNPs used in the study had minor allele frequencies Subject characteristics X0.2. Since not all markers studied fall into the category The study group consisted of 688 subjects from 102 of functional markers, or markers in complete LD with a families (Table 1). All families included members that functional marker, robust tests were performed to take were infected with L. chagasi, documented by either a account of multiple trios within a pedigree in the history of VL or a positive Montenegro reaction without a history of disease. Some subjects were reported in association tests (see the following text). 32,33 Allelic association was determined using a family- earlier studies. The Montenegro test is a highly based design in which sets of cases and ‘pseudo-sib’ sensitive measure of prior L. chagasi infection because controls are created where the cases comprise the of the low incidence of diseases with the potential to genotypes of the ‘affected’ offspring, and the ‘pseudo- crossreact (for example, Trypanosoma cruzi) and the fact sib’ controls are the other genotypes possible from the that no other Leishmania species with autochthonous parental mating type.43 Odds ratios, 95% confidence transmission have been detected in the vicinity of Natal. intervals and P-values were estimated using conditional Thus, individuals with a positive Montenegro but no logistic regression models employing a robust variance– history of active disease were considered to have had covariance matrix to control for clustering of trios within asymptomatic L. chagasi infection. As outlined above, the a pedigree, and a Wald’s w2 test to indicate overall individuals that defined the DTHÀ phenotype were significance for allelic associations. Correction for multi- those who had remained negative for the Montenegro skin test despite X3 years exposure in households with ple testing was not performed on the analysis for X individuals markers, as groups of these markers were 40% infection rate. in LD and therefore not independent of each other. Instead, a stepwise conditional logistic regression proce- SNP markers dure43 was used to determine which SNPs within and Individuals from the 102 pedigrees were genotyped for between candidate genes contribute independently to the 23 SNPs. Table 1 indicates the distribution of phenotypes observed associations. In the stepwise analysis, Wald’s w2 in pedigrees, and nuclear families included in these tests were used to compare models in which the effect of pedigrees. Locations of SNPs in relation to genes in the adding a second locus is compared with a model where region on chromosome 5q23.3–q31.1 are shown in main effects at the primary locus only are included. Only Figure 1, with the markers further defined in Table 2. pair-wise comparisons were made. A likelihood ratio test One SNP (rs848) was eliminated from the analysis comparing the two models determined whether includ- because it was not polymorphic in our population. Two ing the second locus adds independent main effects, or SNP markers (rs31564 and rs2107506) that deviated from whether after inclusion of the first marker, the second HWE were also excluded. marker no longer accounts for significant association. Robust case/pseudo-control statistical tests, which were Allelic association used throughout to take account of familial clustering, Results of the conditional logistic regression analysis to were developed by Heather Cordell and David Clayton43 look for association between the 20 SNPs on chromo- at the Cambridge Institute for Medical Research (avail- somes 5q23–q33 and either clinical VL, DTH þ or DTHÀ able at Electronic Database Information 6) and were phenotypes are shown in Table 3. No significant implemented within STATA 9.1. associations were observed for clinical VL. This may reflect the limited power to detect association compared Bioinformatics to the other two infection-related phenotypes. Six Global alignment of genomic sequence for the region markers in two clusters (Figure 1) were significantly LECT2 to TGFBI from human, dog, cow, mouse and associated with the DTH þ phenotype; two markers chicken was undertaken to locate evolutionarily con- were associated with the DTHÀ phenotype. There was

Genes and Immunity DTH responses in L. chagasi infection SMB Jeronimo et al 543 = Associated with DTH+ response = Associated with DTH- response

T R AC D H IJKL N PQ SVU 1.9 0.9 MB MB

IRF1 IL5 IL13 SEC24 DCOL IL9 IL4 LECT2O TGFBI 191.9 KB 320.8 KB M 171.6 KB Figure 1 Drawing of the chromosome 5q23.3–q31.1 region showing relative positions of genes and single-nucleotide polymorphisms (SNPs) (not to scale). Boxes indicate the locations of genes including both exons and . Letters referring to SNPs (dotted vertical arrows; open dots are SNPs associated with the delayed-type hypersensitivity negative (DTHÀ) phenotype; gray-filled dots are SNPs associated with the DTH þ phenotype) are defined in Table 2. Distances between the start of the 50-UTR of IRF1 and end of the 30-UTR of IL4, the 50-UTR of SEC24 and 30-UTR of DCOL and the 50-UTR of IL9 and 30-UTR of TGFBI are indicated with dotted horizontal arrows. Solid horizontal arrows indicate the direction of the coding region for selected genes. Brackets refer to markers in linkage disequilibrium (LD) clusters (H to O; R to V) as defined by D0 (see Figure 2). Markers not shown on the figure were not analyzed statistically, because either there was only one allele or they were out of Hardy–Weinberg Equilibrium (HWE) (see Methods for details).

Table 2 Details of genomic bp location, functional position relative to genes, allelic variants and minor allele frequencies for the 18 SNPs at 5q23.3–q31.1 used in the association analyses

Gene SNP Location (bp)a Functional position Letter on mapb SNPc alleles Strand Minor allele frequency

5q23.3 IRF1 131 846 679–131 854 333d rs2070727 131 848 174 8 A T/G À 0.348 rs11242120 131 898 588 Intergenic C T/C + 0.378 IL-5 131 905 035–131 907 113 IL-13 131 021 764–132 024 700 IL-4 131 037 272–132 046 267 rs2070874 132 037 609 50-UTR D C/T À 0.34 5q31.1 IL-9 135 255 834–135 259 415 rs31564 135 258 152 Intron 3 E G/T + Not used LECT2 135 310 499–135 318 622 rs31530 135 310 529 30-UTR H C/T À 0.394 rs31517 135 314 928 Nonsynonymous 3 I G/A À 0.398 rs248166 135 320 607 Upstream J T/C À 0.211 rs248163 135 321150 Upstream K G/A À 0.17 rs248158 135 321 969 Upstream L C/T À 0.378 rs2526146 135 327 750 Intergenic M A/G À 0.422 rs2526152 135 340 932 Intergenic N A/G À 0.446 rs30740 135 354 489 Intergenic O A/G À 0.249 rs12520800 135 357 814 Intergenic P T/G + 0.325 rs13157444 135 364 460 Intergenic Q A/G + 0.443 rs4976360/ 135 382 223 Intergenic R T/A + 0.431 rs2282790 135 391 774 Upstream S G/A + 0.36 TGFBI 135 392 597–135 427 406 rs1054124 135 416 562 Synonymous exon 8 T G/A + 0.465 rs4669 135 420 325 Synonymous exon 12 U T/C + 0.41 rs7725447 135 434 433 Intergenic V G/A 0.406

Abbreviations: IL, interleukin; SNP, single-nucleotide polymorphism. aLocation on human (ENSEMBL website; NCBI Build 36, Ensembl Genebuild August 2006, Database version 42.36d). bLetter code refers to Figure 1. cMajor over minor allele for this Brazilian population. dBold signifies locations of genes. no overlap in markers associated with DTH þ versus determine allelic association across 5q23.3–q31.3 in DTHÀ phenotypes. Brazil. These closely mimic LD blocks across this region generated using public domain HapMap–CEU data for Linkage disequilibrium Caucasians (data not shown). For the DTH þ associa- Two major LD blocks, as defined by D0 (Figure 2a) and r2 tions, the three SNPs (rs248158, rs2526146 and rs2526152; (Figure 2b), were observed for the 18 markers used to labeled L, M and N in Figure 1) that lie immediately

Genes and Immunity DTH responses in L. chagasi infection SMB Jeronimo et al 544 Table 3 Results of robust conditional logistic regression analysis to look for association between SNPs on chromosome 5q23.3–q31.1 and three leishmaniasis-related phenotypes (VL, DTH+ and DTHÀ)

Label SNP Phenotype Robust allele-wise global test P-value Allele Odds ratio 95% CI

D rs2070874 DTH+ 0.382 C 1.24 0.77–2.01 L rs248158 DTH+ 0.005 G 2.25 1.28–3.97 M rs2526146 DTH+ 0.015 A 1.65 1.10–2.47 N rs2526152 DTH+ 0.015 A 1.53 1.08–2.16 O rs30740 DTH+ 0.139 T 1.47 0.89–2.45 R rs4976360 DTH+ 0.003 T 1.94 1.24–3.03 T rs1054124 DTH+ 0.013 A 1.91 1.15–3.18 U rs4669 DTH+ 0.029 C 1.69 1.06–2.71 D rs2070874 DTHÀ 0.006 C 3.14 1.38–7.14 L rs248158 DTHÀ 0.281 G 1.56 0.69–3.52 M rs2526146 DTHÀ 0.408 A 1.57 0.54–4.58 N rs2526152 DTHÀ 0.202 A 1.31 0.86–1.99 O rs30740 DTHÀ 0.042 T 3.00 1.04–8.65 R rs4976360 DTHÀ 0.722 T 0.87 0.39–1.91 T rs1054124 DTHÀ 0.536 A 1.25 0.62–2.53 U rs4669 DTHÀ 0.722 C 0.89 0.49–1.65 D rs2070874 VL 0.123 C 1.60 0.88–2.91 L rs248158 VL 0.869 G 1.05 0.60–1.82 M rs2526146 VL 0.881 A 1.04 0.63–1.70 N rs2526152 VL 1.000 A 1.00 0.64–1.55 O rs30740 VL 0.140 T 2.33 0.76–7.19 R rs4976360 VL 1.000 T 1.00 0.46–2.17 T rs1054124 VL 0.794 A 1.10 0.55–2.17 U rs4669 VL 0.874 C 0.96 0.55–1.65

Abbreviations: CI, confidence interval; DTHÀ, individuals with negative delayed-type hypersensitivity response and seronegative (51%); DTH+, individuals with positive delayed-type hypersensitivity response with no history of VL (35%); VL, visceral leishmaniasis; SNP, single- nucleotide polymorphism. Data are shown for 8 of the 18 SNPs analyzed (see Figure 1) that showed association with at least one phenotype. Bold indicates a significant allele-wise association.

upstream of LECT2 (Figure 1) were located in the first LD this is that R, or a variant in strong LD with R, is the block, and were in strong LD with each other (D0 ¼ 1; causal variant. This is supported by the observations that r240.82) but not with the other three DTH þ associated (1) R is the locus with the more significant single-SNP SNPs (rs4976360, rs1054124 and rs4669; labeled R, T and association (Table 3), and (2) once R is included in the U in Figure 1) that lie immediately upstream or within model, U was no longer required. In contrast, when U is TGFBI. Conversely, the three SNPs at TGFBI (rs4976360, put into the model first, R was still required to achieve rs1054124 and rs4669; labeled R, T and U in Figure 1) significance, that is, U alone does not explain all of the were located in the second LD block and were in strong association with DTH þ in this LD block, and therefore LD with each other as defined by D0 (D0 ¼ 0.76–0.91), but U is not likely to be the causal locus. SNP rs1054124 (T) with reduced r2 between marker rs4976360 (R) upstream also does not add significantly to R (Table 4), suggesting of TGFBI and the two SNPs (rs1054124 and rs4669; that in block 2, locus R is sufficient to explain all the labeled T and U) within TGFBI (r2 ¼ 0.20–0.21). For the statistical association with DTH þ . DTHÀ associations, SNP rs2070874 (D) lies within IL4 Strikingly, all of the markers in LD block 1 add (3.3 Mb proximal to the start of the first LD block), and significant independent effects to all of the markers in was not in LD with any other genotyped SNP across the block 2, and vice versa (Table 4). Together with the region. Note that SNP rs30740 (labeled O) was located in evidence for two distinct LD blocks in this population LD block 1 as defined by D0 (Figure 2a) but not by r2 (Figure 2), this stepwise analysis strongly supports the (Figure 2b). presence of two separate genes controlling the DTH þ phenotype across this region of chromosome 5. Stepwise conditional logistic regression analysis The data also provide evidence of association for the Stepwise conditional logistic regression analysis was single SNP in IL4 (rs2070874) influencing the DTHÀ undertaken to determine, in particular, whether SNPs phenotype, plus borderline evidence for SNP rs30740 located within and between the two LD blocks in this (labeled O) between LECT2 and TGFBI (see Table 3). genomic region exerted independent main effects on the However, evidence for any independent effects of either risk of having the DTH þ phenotype. Stepwise analysis of these two SNPs given the other was only marginally within block 1 indicated that none of the three markers significant when stepwise logistic models was examined (L, M and N) showed significant independent main on the DTHÀ phenotype (Table 4). effects (Table 4), suggesting that they are all in strong LD (Figure 2) with a causal variant in this LD block. Bioinformatic analysis Stepwise analysis of SNPs within block 2 indicates that SNPs L, M and N (rs248158, rs2526146 and rs2526152, rs4976360 (R) adds significantly to rs4669 (U), but U does respectively) in LD block 1 lie in the intergenic region not add to R (Table 4). The most likely explanation for upstream of the LECT2 gene (Figure 3). Similarly, SNP

Genes and Immunity DTH responses in L. chagasi infection SMB Jeronimo et al 545 D′

r2

Figure 2 Linkage disequilibrium (LD) plot generated using the default settings in Haploview available from the HapMap Project site (http://www.hapmap.org/). LD between pairs of markers across chromosome 5q23.3–q31.1 was determined within Haploview using (a) D0 or (b) r2 statistics. LD values between markers are indicated at the intercept of the two markers on the matrix. Empty boxes indicate that the LD value is 1. Intensity of color on the red/pink or black/gray scale indicates the degree of confidence in the LD value. Two main haplotype blocks outlined within the black triangles lie within this region of 5q23.3–q31.1: rs31530 (H)-rs30740 (O) encompassing LECT2; and rs4976360 (R)-rs7725447 (V) encompassing TGFBI. All single-nucleotide polymorphisms (SNPs) used had minor allele frequencies 40.1 (see Table 2).

Genes and Immunity DTH responses in L. chagasi infection SMB Jeronimo et al 546 Table 4 Results of robust stepwise conditional logistic regression analysis to look for independent associations between SNPs on chromosome 5q23.3–q31.1 and two leishmaniasis-related phenotypes (DTH+ and DTHÀ)

Null model Alternative model Na Wald w2 d.f. P-valueb

DTH+ phenotype—within LD blocks LD block 1 SNPs Adding block 1 SNP to block 1 (L) rs248158 (L+M) 182 0.00 1 0.985 (L) rs248158 (L+N) 212 0.03 1 0.863 (M) rs2526146 (M+L) 182 2.36 1 0.125 (M) rs2526146 (M+N) 220 3.13 1 0.077 (N) rs2526152 (N+L) 212 2.80 1 0.094 (N) rs2526152 (N+M) 220 0.06 1 0.811 LD block 2 Adding block 2 SNP to block 2 (R) rs4976360 (R+T) 154 2.07 1 0.150 (R) rs4976360 (R+U) 192 1.99 1 0.160 (T) rs1054124 (T+R) 154 2.63 1 0.105 (T) rs1054124 (T+U) 180 0.35 1 0.554 (U) rs4669 (U+R) 192 4.60 1 0.032 (U) rs4669 (U+T) 180 1.60 1 0.206

DTH+ phenotype—between LD blocks LD Block 1 SNPs Adding block 2 SNP to block 1 (L) rs248158 (L+R) 176 9.93 1 0.002 (L) rs248158 (L+T) 152 5.12 1 0.024 (L) rs248158 (L+U) 180 10.02 1 0.002 (M) rs2526146 (M+R) 180 9.24 1 0.002 (M) rs2526146 (M+T) 158 7.11 1 0.008 (M) rs2526146 (M+U) 202 4.54 1 0.033 (N) rs2526152 (N+R) 188 4.39 1 0.036 (N) rs2526152 (N+T) 178 7.78 1 0.005 (N) rs2526152 (N+U) 214 4.71 1 0.030 LD block 2 SNPs Adding block 1 SNP to block 2 (R) rs4976360 (R+L) 176 6.23 1 0.013 (R) rs4976360 (R+M) 180 9.20 1 0.002 (R) rs4976360 (R+N) 188 1.21 1 0.272 (T) rs1054124 (T+L) 152 8.33 1 0.004 (T) rs1054124 (T+M) 158 12.06 1 0.001 (T) rs1054124 (T+N) 178 4.28 1 0.039 (U) rs4669 (U+L) 180 11.51 1 0.001 (U) rs4669 (U+M) 202 7.95 1 0.005 (U) rs4669 (U+N) 214 6.60 1 0.010

DTHÀ phenotype (D) rs2070874 (D+O) 116 2.80 1 0.095 (O) rs30740 (O+D) 116 3.61 1 0.057

Abbreviations: DTHÀ, individuals with negative delayed-type hypersensitivity response and seronegative (51%); DTH+, individuals with positive delayed-type hypersensitivity response with no history of VL (35%); LD, linkage disequilibrium; SNP, single nucleotide polymorphism. aN ¼ number of observations in the conditional logistic regression stepwise comparison based on the number of cases and ‘pseudo-sib’ controls from trios fully genotyped for both markers being compared. bBold indicates significant independent effects for the SNP when added to the alternative model.

rs4976360 (R) that appears to absorb all of the association sequence tag (EST) data, and none were conserved across within LD block 2 lies upstream of TGFBI. LECT2 and all mammalian species (Figure 3) or with chicken (data TGFBI are encoded on opposite strands. Hence, all of not shown). Hence, it is unlikely that further functional these SNPs lie within potential upstream regulatory genes occur within this region that could account for the regions of these two genes. Bioinformatic analysis was associations observed in our analysis of the DTH þ or undertaken to determine whether any of these SNPs lie DTHÀ phenotypes. within CNS that could represent potential regulatory SNPs rs248158 (L) and rs2526146 (M) do not them- elements. Figure 3 displays the multiple alignment selves fall within regions of strong CNS (Figure 2), but a generated by LAGAN as viewed using SYNPLOT, the number of functionally relevant transcription factor SNP positions and exons encoding LECT2 and TGFBI. binding sites are gained or lost with alternative variants Also indicated are regions of CNS in between LECT2 and at these two sites (Table 5), indicating that they could be TGFBI for the four mammalian species human, dog, cow functional. Of further interest are several peaks of strong and mouse. Although GENSCAN predictions in EN- CNS between SNPs rs2526146 (M) and rs2526152 (N). N SEMBL have annotated additional potential coding itself lies in a CNS, and functionally relevant transcrip- sequences across this region (green blocks, Figure 3), tion factor binding sites are predicted to be present for none of these were supported by RNA or expressed the DTH þ associated allele A, but not for alternative G

Genes and Immunity DTH responses in L. chagasi infection SMB Jeronimo et al 547 LECT2

CNS with chicken

TGFBI

Figure 3 The figure shows the SYNPLOT output for the multiple alignment of (top to bottom) human, dog, cow and mouse sequences performed across the region LECT2 to TGFBI on chromosome 5q31.1 using LAGAN. On the four-species alignments, red boxes define exons at the two genes, blue boxes indicate repetitive sequence, green boxes show GENSCAN auto-annotated putative exons and vertical black bars show the positions of single-nucleotide polymorphisms (SNPs) (J to U, see Table 2) used in this study. The plot shows degree of conservation of sequence across all four species, on a vertical scale 0–100%. Noncoding sequences (CNS) are defined as regions of conserved sequence that are approaching or similar in magnitude to that observed at the known exons for the two genes. Examples of potential regions of CNS of interest are indicated by vertical red arrows, including the single CNS showing high conservation between these mammalian species and chicken (data not shown). allele. One peak immediately distal to rs2526152 (N), in binding sites (Table 5) for functionally relevant transcrip- particular, was the only region highly conserved when tion factors. Loss or gain of transcription factors with mammalian and chicken alignments were made (peak marker R SNP alleles is delineated in Table 5. labeled in Figure 3; chicken data not shown). These CNS could contain elements regulating LECT2 expression that may contain important functional polymorphic variants. The region upstream of TGFBI including SNP Discussion rs4976360 (R) was also analyzed for CNS and transcrip- Previous studies have highlighted the region of chromo- tion factor binding sites. SNP rs4976360 (R) itself lies some 5q23.3–q31.1 as containing a cluster of immune within a region of strong CNS with the two alleles at this response genes important in regulating types I and II SNP also showing association with gain or loss of immune responses, and controlling susceptibility to

Genes and Immunity DTH responses in L. chagasi infection SMB Jeronimo et al 548 Table 5 Analysis of putative transcription factor binding sites lost or gained by carriage of alternative alleles at SNPs in the upstream regions of LECT2 and TGFBI that show allelic association with the DTH+ phenotype

SNP Allele TFBS Description Tool

rs248158_L G CEBPA CCAAT enhancer binding , a; myeloid TF crucial for granulopoiesis AliBaba C/EBPa

rs2526146_M G CEBPB NF-IL-6, nuclear factor triggered by IL-6 PROMO C/EBPb rs2526146_M G c-Jun Component of AP1 TF PROMO rs2526146_M G LXRE Highly conserved DR1 element selected by LXR-b/RXR heterodimers MatInspector rs2526146_M G LEF1 Lymphoid enhancer-binding factor-1 expressed in pre-B and T cells, MatInspector involved in the Wnt signal transduction pathway

rs2526152_N A EVI1 Ecotropic viral integration site 1 encoded factor, N-terminal MatInspector zinc-finger domain; myeloid TF rs2526152_N A YY1 Yin and Yang 1 ubiquitous TF MatInspector rs2526152_N A SMARCA3 SWI/SNF related, matrix associated, actin dependent regulator MatInspector of chromatin, subfamily a, member 3

rs4976360_R T HSF Heat shock TF AliBaba rs4976360_R T E2F Critical determinant of G1/S-phase transition during the mammalian cell cycle MatInspector

rs4976360_R A CEBPA CCAAT enhancer binding protein, a; myeloid TF crucial for granulopoiesis AliBaba C/EBPa rs4976360_R A STAT3 TF for acute phase response gene MatInspector

Abbreviations: IL, interleukin; SNP, single-nucleotide polymorphism; TF, transcription factor. Under allele, bold indicates the DTH+ associated allele, normal type indicates the allele not associated with the DTH response. Bioinformatic analysis demonstrated that the transcription factor binding site (TFBS) was only present with the allele indicated, and was lost with the alternative allele at each SNP shown.

parasitic diseases.28,45,46 Consistent with these observa- high IL-4 production may predict onset of clinical tions, we demonstrate here that multiple genes across disease. More recent studies in mice49 demonstrate that this region are associated with different phenotypic treatment of susceptible mice early with IL-4 may induce manifestations of L. chagasi infection in northeast Brazil. a resistant phenotype. Functional studies in a larger In a previous study of clinical VL caused by a different sample for both exposed DTHÀ and VL phenotypes of species (L. donovani) in Sudan, associations were ob- known genotypes in Brazil will be required to under- served with polymorphisms at IL4 but not IL9.28 We stand more about the role of IL-4 in determining disease could not replicate this finding for the clinical VL outcome. phenotype in northeast Brazil, possibly due to the fact The DTH þ phenotype has been used in previous that different species of Leishmania caused the infections, studies in Brazil as a measure of infection with L. chagasi. and/or due to the small sample sizes and limited power. Epidemiological data on Montenegro skin test data in a Nonetheless, we did find evidence of allelic association sample of 502 individuals from 94 families from between IL4 (OR 3.14; P ¼ 0.006; 95% CI 1.38–7.14) and Jacobina, State of Bahia, Brazil, were analyzed using DTHÀ phenotype in individuals who were negative for complex segregation analysis. The results provide the Leishmania-specific Montenegro skin test response evidence for a major gene controlling infection, as despite an extended period (43 years) of exposure to L. determined by a DTH þ phenotype, with a common chagasi. We hypothesize that this group of individuals allele frequency under a recessive (or additive) single may have innate immunity leading to an early elimina- gene model (approximately 0.45).31 These results are tion of Leishmania parasites before an acquired T-cell- important, as a DTH þ phenotype is not only a measure mediated immune response can be induced, at least as of acquired resistance to natural infection,4 but is also the measured by the DTH response. Early studies in mice only correlate to date of protection in human vaccine suggested that there is rapid IL-4 production in response trials.50–54 Indeed, the only group of vaccinees in any to L. major infection, possibly due to priming by vaccine trials undertaken to date that showed measur- microbial antigens from intestinal flora, and may drive able vaccine-induced protection was that where a DTH the acquired T-cell response down a type II pathway.47 In skin test response also converted following the vaccina- humans, innate immunity cytokine profiles, including an tion. Furthermore, protection observed in unvaccinated increase in IL-4 positive and natural killer control groups also occurred only among individuals cells, were associated with early active disease.48 One whose skin test converted due to natural exposure hypothesis to account for our observation of an IL4 during the vaccine trials. Hence, a genetic component association with the DTHÀ phenotype in Brazil could be regulating ability to acquire resistance to Leishmania that innately low IL-4 producers are resistant to devel- parasites has important implications both for natural oping clinical VL, thus representing the corollary of infection and ability to respond to vaccination. Histolo- associations observed earlier in Sudan,28 where early gically, the DTH reactions among vaccinated individuals

Genes and Immunity DTH responses in L. chagasi infection SMB Jeronimo et al 549 are indistinguishable from those with naturally acquired LXR agonists reduce in a model of contact infections.55 The mechanisms that determine acquisition dermatitis.64 Further work will be required to validate of a DTH þ phenotype may reflect mechanisms provid- these leads based on analysis in silico to determine ing resistance to leishmaniasis. whether these are functional variants in the 50 region of Although a clear relationship exists between acquisi- LECT2 that contribute to the association with the DTH þ tion of a DTH þ phenotype and measurement of type I phenotype that we have observed here. (IFN-g) cytokines following re-stimulation of whole Associations with the DTH þ phenotype, observed for blood or peripheral blood mononuclear cells with either SNPs upstream of TGFBI and intragenic SNPs, were leishmanial56 or mycobacterial57,58 antigen in vitro, independent of those observed with markers in LECT2. association with type II (IL-4, -5, -9, -10, -13) cytokines TGFBI encodes the protein keratoepithelin, which is has not been documented. In concordance with these upregulated by transforming growth factor-b (TGF-b), is observations, we found no evidence for an association expressed in many tissues including the eye, and is between the DTH þ phenotype and the type II cytokine mutated in several different types of corneal dystro- gene IL4 at 5q23.3. We did not saturate this region with phies.65 This protein contains an N-terminal signal haplotype-tagging SNPs, but our data failed to provide peptide and a C-terminal Arg-Gly-Asp (RGD) motif any evidence of association using SNPs at IRF1 and IL5, similar to other adhesion . The RGD motif is which lie proximal to IL4. IL13 lies immediately adjacent found in many extracellular matrix proteins that mod- to, and is in strong LD with, IL4. Hence, it seems unlikely ulate cell adhesion and migration, and TGFBI has been that genes in this region (Figure 1) contribute to the shown to inhibit cell attachment in vitro and suppress DTH þ phenotype. Instead, we found strong evidence growth of CHO cells in nude mice.66 It is possible that for independent associations between the DTH þ phe- TGFBI expressed in skin epithelial cells could modulate notype and SNPs in two strong LD blocks located the DTH phenotype. Other candidates are SMAD5 and around the genes LECT2 and TGFBI. We have not its antisense transcript DAMS, which are located formally ruled out IL9, which lies proximal to and hence B100 kb distal to TGFBI on chromosome 5q31.1. SMAD5 downstream of LECT2, as a candidate gene in the region. plays a critical role in the signaling pathway by which However, there is no evidence from any of the popula- TGF-b inhibits cell proliferation, and there is some tions analyzed in the HapMap (Electronic Database evidence from the HapMap (Electronic Database Infor- Information 5) that SNPs at LECT2 tag haplotypes that mation 5; data not shown) for long range LD between extend to IL9. TGFBI and DAMS/SMAD5 in Caucasians. Nevertheless, LECT2 encodes leukocyte-cell-derived chemotaxin 2, our own data indicated independent associations with which is chemotactic for neutrophils.59 It shows strong SNPs both upstream of, and within, the TGFBI gene. expression in fetal and adult liver,59 but is also generally Similar to LECT2, functionally relevant transcription expressed in vascular, endothelial and smooth muscle factor binding sites are lost or gained with alternative cells, adipocytes, cerebral nerve cells, apical squamous alleles at the 50 SNP associated with the DTH þ pheno- epithelia, parathyroid cells, sweat and - type, including CEPBA discussed above and STAT3 ular epithelia, Hassall’s bodies and some mononuclear which is an important regulator of acute phase responses. cells in immunohematopoietic tissue.60 Further research In summary, this study provides evidence that poly- is required to determine whether LECT2 is expressed at morphism at IL4 may contribute to innate immunity to L. the site of injection of the Montenegro skin test antigen, chagasi infection in northeast Brazil. In addition, we have and whether this contributes to chemotactic activity that found evidence that genes not previously associated with brings immune cells to the site.55 An early influx of immune response, LECT2 and TGFBI, are associated with neutrophils has been demonstrated at the site of ability to mount an acquired immune response to L. cutaneous injection of L. donovani promastigotes into chagasi infection as measured by the Montenegro or hamsters.61 Primer extension analysis shows that several DTH þ phenotype. These data, therefore, provide novel transcription initiation sites occur within 70–230 nucleo- insight into the mechanisms of resistance to parasite tides upstream of the translation initiation codon.62 infection. Further analysis of the role of these genes in Several transcriptional control sequences relevant to the determining ability to mount a DTH þ response has liver-specific expression have also been identified at the important implications not only for our understanding of 50-untranslated region of the human LECT2 gene.62 In our acquired resistance to natural infection, but in under- study, no associations were observed for SNPs located standing the immune mechanisms required to mount a within the LECT2 gene with the DTH þ phenotype, but protective immune response to vaccination. with SNPs found in the region immediately upstream, that is, distal to LECT2 on 5q31.1. Bioinformatic analysis demonstrated that a number of transcription factor binding sites are gained or lost in the presence of Acknowledgements alternative alleles at these SNPs, including CEBPA that is crucial for normal granulopoiesis, and LEF1 that is active This research was supported by National Institutes of in skin and is known to bind directly to, and regulate, Health grants AI048822 (MEW), AI45540 (MEW), NIH CEBPA to control neutropenia.63 Similarly, liver X TMRC grant AI30639 (SMBJ), Conselho Nacional de receptor (LXR) transcription factors for which binding Desenvolvimento Cientı´fico e Tecnolo´gico (SMBJ), Merit sites were lost or gained for SNPs associated with the Review and Gulf War grants from the Department of DTH þ phenotype are known64 regulators of macro- Veterans’ Affairs (MEW), The Wellcome Trust (JMB and phage inflammatory mediators such as inducible nitric HJC) and in part by the Intramural Research Program of oxide synthase, cyclooxygenase-2 and IL-6 gene expres- the National Human Genome Research Institute, sion, which are all relevant to the DTH þ phenotype. National Institutes of Health.

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