Genetic polymorphisms of IL17A associated with Chagas disease: results from a meta-analysis in Latin American populations

Genetic factors and the immunologic response have been suggested to determine the susceptibility against the infection and the outcome of Chagas disease. In the present study, we analysed three IL17A genetic variants (rs4711998, rs8193036 and rs2275913) regarding the predisposition to Trypanosoma cruzi infection and the development of chronic Chagas cardiomyopathy (CCC) in different Latin American populations. A total of 2,967 individuals from Colombia, Argentina, Bolivia and Brazil, were included in this study. The individuals were classified as seronegative and seropositive for T. cruzi antigens, and this last group were divided into asymptomatic and CCC. For T. cruzi infection susceptibility, the IL17A rs2275913*A showed a significant association in a fixed-effect meta-analysis after a Bonferroni correction (P = 0.016, OR = 1.21, 95%CI = 1.06–1.41). No evidence of association was detected when comparing CCC vs. asymptomatic patients. However, when CCC were compared with seronegative individuals, it showed a nominal association in the meta-analysis (P = 0.040, OR = 1.20, 95%CI = 1.01–1.45). For the IL17A rs4711998 and rs8193036, no association was observed. In conclusion, our results suggest that IL17A rs2275913 plays an important role in the susceptibility to T. cruzi infection and could also be implicated in the development of chronic cardiomyopathy in the studied Latin American population.

It's well known that, genetic factors and immunologic response may determine the susceptibility against the infection and the outcome of Chagas disease [16][17][18][19] . Thus, polymorphisms in genes encoding cytokines may influence the level of cytokines production and, consequently, cause different immunological responses [20][21][22] . Specifically, IL17A polymorphisms, located in the promoter region of the gene, have been associated with plasma IL-17A levels in cell cultures 20 and in healthy infants 23 . Several studies have found associations between the IL17A gene polymorphisms with infectious diseases, such as, cutaneous leishmaniasis 24 , brucellosis 25 , and tuberculosis 26 .
A previous study performed, by our group, in a Colombian cohort, observed nominal significant associations between variants of IL17A gene with the susceptibility to T. cruzi infection and with the development of chronic cardiomyopathy 27 . An additional study conducted in the South and Southeast regions of Brazil also found an association with IL17A and IL17F variants and the susceptibility to the development of chronic Chagas cardiomyopathy 28 .
Given the limited information about the role of IL17A gene variants in Chagas disease, this study aimed to analyse the association of three IL17A genetic variants with the predisposition to T. cruzi infection, the development of chronic cardiomyopathy and chronic Chagas cardiomyopathy, in different Latin American populations.

Results
A total of 2,967 patients were included in the study. The demographic characteristics of the studied cohorts are reported in Table 1.
The three single nucleotide polymorphisms (SNPs) of IL17A gene: rs4711998, rs8193036 and rs2275913, selected for this study, were in Hardy-Weinberg equilibrium in all the analysed cohorts (P > 0.01). The genotyping success rate was over 95% and the allele frequencies in all cases were similar to those described for the Americans sub-populations of the 1000 Genomes Project phase III (http://www.1000genomes.org) 29 (Table S1).
T. cruzi infection susceptibility. The allelic and genotypic frequencies of seronegative and seropositive individuals from the Colombian cohort were compared in Tables S3-1 The minor allele frequency (MAF), in rs8193036*C was higher in seronegative than in seropositive individuals, being nominally significant after the adjustment by sex and age [P = 0.043, odds ratio (OR) = 0.83, 95% confidence interval (CI) = 0.70-0.99]. No associations between the three IL17A genetic variants and susceptibility to T. cruzi infection were found after performing logistic regression adjusted by sex and age in the Argentinian cohort (Tables S3-2). In contrast, interestingly, the SNP IL17A rs2275913*A, that was studied in a Brazilian cohort, was found statistically significant for the risk to T. cruzi infection 28 . Furthermore, a meta-analysis combining each individual cohort (Colombian, Argentinian and Brazilian) was performed ( Table 2). The IL17A rs2275913*A allele effect was consistent in the three cohorts and the association improved after the meta-analysis, showing statistically significant results (P = 0.016, OR = 1.21, 95% CI = 1.06-1.41, under a fixed-effects meta-analysis) after Bonferroni correction. No association was observed for the IL17A rs4711998 and rs8193036. chronic chagas cardiomyopathy susceptibility. To  patients from the Colombian, Argentinian and Bolivian cohorts were not statistically significant after the logistic regression adjusted by sex and age (Tables S4-1-3), consistent with previous findings in the Brazilian cohort 28 . Moreover, no significant associations were detected for the available SNPs when the meta-analysis was performed combining these cohorts (Table 3). Finally, in order to evaluate the possible association between IL17A genetic variants and chronic cardiomyopathy we compared CCC patients and seronegative individuals, as previously performed 27,28 . In the Colombian and Argentinian cohort (Tables S5-1 and 2) and no associations were found after applying logistic regression adjusted by sex and age. In contrast, a previous study, in the Brazilian cohort the IL17A rs2275913*A allele was nominally significant 28 . As can be observed in Table 4 the IL17A rs2275913 allele effect was consistent in the Colombian, Argentinian and Brazilian cohorts, and the association with chronic cardiomyopathy susceptibility improved after the meta-analysis showing nominally statistical differences (P = 0.040, OR = 1.20, 95% CI = 1.01-1.45, under a fixed-effects meta-analysis). These results suggest that rs2275913*A allele was associated with the risk of cardiomyopathy in the analysed population.
In silico functional characterization of IL17A gene variants. Since the IL17A rs2275913 showed a statistical association with the risk to T. cruzi infection and the development of chronic cardiomyopathy, we perform an in silico functional analysis of this SNP and the ones in high LD (R 2 ≥ 0.8) on peripheral mononuclear blood in American population from the 1000 Genomes Project ( Table 5). The annotation indicates that these SNPs map in enhancer regions and marks of histone modifications (H3K4me1, H3K4me3, H3K27ac and chromatin marks), potentially modulating gene expression.

Discussion
Association studies offer a potentially powerful approach to identify genetic variations that are involved in the immunopathogenesis of Chagas disease [16][17][18][19] . However, individual genetic association studies frequently have limitations and the results may be specific to the population of the study. The meta-analysis approach has been proposed to resolve these limitations, to increase the power of statistical analyses 30,31 and to reach to more conclusive results in order to improve our understanding of the genetic basis underlying Chagas disease. In this study, three IL17A genetic variants were analysed in four Latin American populations, and our results evidenced the implication of rs2275913 associated with the risk to T. cruzi infection and the development of chronic cardiomyopathy, in Colombian, Argentinian and Brazilian population.
In the early stages of the infection, the IL-17A is a crucial cytokine secreted by a wide range cell types such as Th17, B cells, innate lymphoid cells, CD4+, CD8+, gamma-delta T and invariant NKT [10][11][12][13]32 . The rs2275913, which was associated with the risk to T. cruzi infection in the Latin American population studied, is a functional polymorphism that modifies the binding of the transcriptional nuclear factor of activated T cells (NFAT) in the IL-17A promoter. Moreover, as observed in the in silico analysis, the associated variant is located in promoter histone marks, potentially modulating gene expression. In addition, it has been demonstrated that the substitution of the G by an A allele in the IL17A rs2275913 gene promoter was significantly associated with autoimmune diseases and cancer [33][34][35][36] . However, controversial results have been reported regarding the levels of IL-17A in serum, where the A allele was associated with a higher 20,36,37 , lower 38,39 or no significant 40 levels of transcription and synthesis of the protein. In this work, we could hypothesized that the individuals who carry the A allele in the IL17A rs2275913 would be more susceptibility to the T. cruzi infection, probably due to a variation in the gene   www.nature.com/scientificreports www.nature.com/scientificreports/ expression and therefore lower IL-17A production, which would impede a rapid proinflammatory activation of chemokines and cytokines for the resolution of T. cruzi infection 12,13 . However, further studies are required to understand the complexity of IL17A rs2275913 polymorphism functional effect.
Several studies showed that IL-17A has an important immunomodulatory role in the chronic phase of the disease 8,14,15,41 . IL-17 expression by Th17 cells and B cells were found in patients with cardiac involvement more frequently, compared to asymptomatic patients, correlating with worse cardiac function 14 ; IL-17 exacerbated produce a proinflammatory environment in Chagas severe heart disease 42,43 . However controversially, others groups suggested a protective role of IL-17 and Th17 in the chronic cardiac form 44,45 . Regarding IL17A genetic variants, in our study, no significant association was detected when CCC and asymptomatic patients from Colombia, Argentina, Bolivia and Brazil were compared. This lack of association could be a consequence of an insufficient statistical power (Table S1) or genetic heterogeneity among the studied cohorts. The lack of replication may occur if the studied polymorphism is not the causal variant but is rather in LD with it. LD patterns depend on the genetic background of the founder population and population history, always challenging in Latin American population due to their genetic diversity and recent admixture [46][47][48] .
Interestingly, at the present time IL-17A have become a relevant drug target in various forms of autoimmune and inflammatory diseases, mainly as negative modulators of the secreted protein 13,49 . Two antibodies are currently in Phase IV of drug development for the treatment of immune system diseases, namely, Secukinumab and Ixekizumab (Anatomical Therapeutic Chemical [ATC] code L04AC10 and L04AC13, respectively). Given the role of IL-17A as a key cytokine in the pathogenesis of Chagas disease, the opportunities for drug repurposing becomes very important for this neglected disease, as there are only two treatments available: Benznidazole and Nifurtimox, with high rates of adverse effects and treatment withdrawal 50,51 .
In conclusion, in this work, we found an association of IL17A rs2275913 with Chagas disease in a large cohort composed of different Latin American countries, validating previous findings 27,28 . Finally, further studies on this gene accounting for functional analyses and heterogeneity among populations, would be necessary for the complete understanding of IL17A polymorphisms in Chagas disease.

Study design and patient population. A candidate-gene case-control study was performed in
Colombian, Argentinian and Bolivian cohorts in order to replicate previous findings 27,28 . In addition, to improve the statistical power of the study a meta-analysis was performed combining the results from all the available cohorts.
A total of 2,967 individuals from Latin American countries (Argentina, Bolivia Colombia and Brazil) were studied. In all cohorts, patients were classified as seropositive for T. cruzi antigens (n = 2,099) and seronegative (n = 868) on the basis of results of at least 2 of 3 independent tests. Among seropositive individuals, based on electrocardiographic, echocardiographic, chest X-ray and clinical findings, patients were classified as chronic Chagas cardiomyopathy (CCC, n = 1,070) and asymptomatic (ASY, n = 1,029).
Colombian cohort. A total of 406 Colombian individuals from the same population as the study by Leon Rodriguez et al. 27 were recruited by the health care team from the Industrial University of Santander and Cardiovascular Foundation from Colombia. In order to increase the sample size, these individuals were included with the previously published Colombian cohort, making a total of 1,577 individuals. From this, 937 were classified as seropositive for T. cruzi antigens and 640 were classified as seronegative (according to the serological tests: recombinant antigen ELISA and commercial indirect hemagglutination test). Subsequently, and after a clinical evaluation, an electrocardiogram (ECG) and an echocardiogram (Echo) were recorded to detect any conduction and/or structural alteration in seropositive patients. Based on complementary tests and clinical findings, seropositive patients were classified as CCC = 576 and ASY = 361. The mean age of participants was 45.55 ± 17.19 years for seronegative individuals, CCC = 61.44 ± 12.82 and ASY = 51.90 ± 14.18. The sex distribution was 58% female and 42% male 52 .
Argentinian cohort. A total of 350 Argentinian individuals from an endemic region for Chagas disease (Cordoba province) were included in this study. The study subjects were recruited from the National Hospital of Clinics and Sucre Clinic, Cordoba city. The population in this region of Argentina is a homogeneous mixture, with no specific concentration of any ethnicity. All participants underwent a serological diagnosis for T. cruzi infection  Statistical analysis. For the candidate gene study, the statistical analyses were performed with the software Plink V1.9 (https://www.cog-genomics.org/plink2) 54 . Deviance from Hardy-Weinberg equilibrium was determined at the 1% significance level in all groups of individuals. Individuals that did not achieved an SNP completion rate of 95% were filtered out. To test for possible allelic association, logistic regression model and Fisher's exact test were assessed in seronegative vs. seropositive individuals and asymptomatic vs. chronic Chagas cardiomyopathy individuals, using age and sex as covariates; and odds ratios (OR) and 95% confidence intervals (CI) were calculated. P-values lower than 0.05 were considered as statistically significant.
To assess the consistency of effects across the cohorts, a meta-analysis was performed with METASOFT (http://genetics.cs.ucla.edu/meta/) based on inverse-variance-weighted effect size. Heterogeneity across studies was assessed using the Cochran's Q statistic (Q test P < 0.05) and I 2 heterogeneity index 55 . A fixed-effects model was applied for those SNPs without evidence of heterogeneity (Cochran's Q test P > 0.05), and a random-effects model was applied for SNPs displaying heterogeneity of effects between studies (Cochran's Q test P ≤ 0.05). The significance threshold for the meta-analyses was estimated based on the Bonferroni correction (0.05/3 = 0.017) 56 .
The statistical power of the studies was estimated with the Power Calculator for Genetic Studies 2006 (CaTS) software (Tables S1 and S2) (http://www.sph.umich.edu/csg/abecasis/CaTS/) 57 . In silico functional characterization of IL17A gene variants. Evaluation of functionality of the statistically significant associated SNP with Chagas disease, was performed with the online software HaploReg v4.1 58 . (https://pubs.broadinstitute.org/mammals/haploreg/ haploreg.php) based on empirical data from the ENCODE project (http://www.genome.gov/encode/). Specifically, we focused our attention on experiments performed on blood in the American population. Moreover, updated information related to expression Quantitative Trait Loci (eQTL) were inspected for IL17A rs2275913 and for SNPs in high LD (R 2 ≥ 0.8) ( Table 5).

Data availability
All relevant data are within the paper and its Supporting Information files.