Association of IL18 genetic polymorphisms with Chagas disease in Latin American populations.

Host genetic factors have been suggested to play an important role in the susceptibility to Chagas disease. Given the influence of interleukin 18 (IL-18) in the development of the disease, in the present study, we analyzed three IL18 genetic variants (rs2043055, rs1946518, rs360719) regarding the predisposition to Trypanosoma cruzi infection and the development of chronic Chagas cardiomyopathy (CCC), in different Latin America populations. Genetic data of 3,608 patients from Colombia, Bolivia, Argentina, and Brazil were meta-analyzed to validate previous findings with increased statistical power. Seropositive and seronegative individuals were compared for T. cruzi infection susceptibility. In the Colombian cohort, the allelic frequencies of the three variants showed a significant association, with adjustment for sex and age, and also after applying multiple testing adjustments. Among the Colombian and Argentinean cohorts, rs360719 showed a significant genetic effect in a fixed-effects meta-analysis after a Bonferroni correction (OR: 0.76, CI: 0.66-0.89, P = 0.001). For CCC, the rs2043055 showed an association with protection from cardiomyopathy in the Colombian cohort (OR: 0.79, CI: 0.64-0.99, P = 0.037), with adjustment for sex and age, and after applying multiple testing adjustments. The meta-analysis of the CCC vs. asymptomatic patients from the four cohorts showed no evidence of association. In conclusion, our results validated the association found previously in the Colombian cohort suggesting that IL18 rs360719 plays an important role in the susceptibility to T. cruzi infection and no evidence of association was found between the IL18 genetic variants and CCC in the Latin American population studied.

Introduction

Chagas disease is an intracellular and hematic disease caused by the parasite Trypanosoma cruzi. Around 6 to 7 million people are estimated to be infected worldwide, most of them being in the poorest rural and urban areas of Latin America, where is endemic [1, 2]. Nowadays, large-scale migrations to other countries have turned Chagas disease into a global health problem [1].

Chagas disease clinical course includes an acute and a chronic phase. The acute phase is characterized by an increase of parasitic load in blood. In this stage, the parasitic load is controlled by the activation of the innate immune response by Th1 pro-inflammatory cytokines such as tumor necrosis factor α (TNF) and interferon γ (IFN-G) [3]. IFN-G activates phagocytic cells to destroy intracellular parasites by inducing TNF and nitric oxide (NO) production [4]. After 8–12 weeks from the infection, individuals evolve into the chronic phase of the disease, in which most of them remain asymptomatic for the rest of their lives. However, around 30–40% of chronically infected patients can develop cardiomyopathy or/and megaviscera. The cardiac involvement is the most frequent manifestation of the disease that occurs in 14–45% of chronically infected patients and affects mainly the conduction system and myocardium [5].

Host genetic factors have been suggested to play an important role in the susceptibility to Chagas disease. [6-9]. Polymorphism in genes encoding cytokines may influence the level of cytokines production and, consequently, cause different immunological responses [10, 11]. Interleukin-18 (IL-18), a pro-inflammatory cytokine produced mainly by macrophages, has been proposed to influence the development of Chagas disease. This cytokine is involved in both innate and adaptive immune response and induces IFN-G production by T cells and NK cells, promoting the Th1 response [12]. Previous genetic studies performed in a Brazilian and Colombian cohort found associations between variants of IL18 gene and the predisposition to T. cruzi infection and chronic Chagas cardiomyopathy [13, 14].

Given the important role played by IL-18 in Chagas disease, in the present study we analyzed the association of three IL18 genetic variants with the predisposition to T. cruzi infection and the development of Chagas cardiomyopathy in different Latin America populations.

Materials and methods

Study design and patient population

A candidate-gene case-control study was performed in Colombian, Bolivian and Argentinian cohorts in order to replicate previous findings [13,14]. Additionally, a meta-analysis was performed combining these cohorts.

A total of 3,608 individuals from Latin American countries (Colombia, Bolivia, Argentina and Brazil) were studied. In all cohorts, patients were classified as seropositive for T. cruzi antigens (n = 2,890) and seronegative (n = 718) based on results of at least 2 of 3 independent tests. Based on clinical evaluation, an electrocardiogram and echocardiogram were recorded to detect any conduction and structural alterations. Subsequently, the seropositive patients who presented cardiac alterations were classified as chronic Chagas cardiomyopathy (CCC, n = 1,707) and asymptomatic (ASY, n = 1,183). The sex distribution for the entire Latin American population studied was 61.4% female and 38.6% male.

Colombian cohort

A total of 406 Colombian individuals from the same population as the study by Leon Rodriguez et al. (2016) [14] 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). 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.

Bolivian cohort

A total of 630 Bolivian individuals residents in Barcelona, Spain were recruited from the Infectious Diseases Department of the Vall d’Hebron University Hospital. In this cohort, only seropositive patients were classified as CCC = 100 and ASY = 530 based on complementary tests and clinical findings. The mean age of the participants was CCC = 50.71 ± 9.41 and ASY = 46.93 ± 9.49. The sex distribution was 69% female and 31% male.

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 through the enzyme-linked immunosorbent assay (ELISA) that uses recombinant antigen and a commercial indirect hemagglutination test. According to the results of these tests, 272 individuals were classified as seropositive for T. cruzi antigens and 78 were classified as seronegative. Based on the results of complementary tests and clinical findings, seropositive patients were classified as CCC = 182 and ASY = 90. The mean age of participants was 53.82 ± 16.53 years for seronegative individuals, CCC = 60.14 ± 10.16 and ASY = 49.30 ± 13.65. The sex distribution was 71% female and 29% male.

Brazilian cohort [13]

A total of 1,051 Brazilian seropositive patients for antibodies against T. cruzi were included in the meta-analysis. From this, 849 individuals were classified as CCC and 202 ASY. The sex distribution was 52% female and 48% male.

Ethics statement

The study was accepted by the Ethics Committees from the Industrial University of Santander and Cardiovascular Foundation, Colombia; the Vall d’Hebron University Hospital, Barcelona, Spain and the National Hospital of Clinics, National University of Cordoba, Argentina. Written informed consent was obtained from all subjects prior to participation. The research protocols followed the principles of the Declaration of Helsinki and informed consent was obtained from all individual participants included in the study.

Selected polymorphisms and genotyping

The gene encoding IL-18 is located on chromosome 11q22.2-q22.3 [15] and consists of six exons and five introns [16] (). Three SNPs previously studied in Chagas disease were selected: rs2043055, rs1946518 and rs360719 for this study [13, 14]. Linkage disequilibrium (LD, R2 and D’) was estimated using an expectation–maximization algorithm implemented in Haploview V4.2 [17] for the studied cohorts: Colombian, Bolivian, Argentinian, and from the American sub-populations genotype data from the 1000 Genomes Project phase III (http://www.1000genomes.org) [18].

Genomic DNA from blood samples was isolated following standard procedures and the genotyping was performed using TaqMan assays (Applied Biosystems, Foster City, California, USA) on a real-time PCR system (7900HT Fast Real-Time PCR System), SNPs were determined by TaqMan 5´ allelic discrimination assay method performed by Applied Biosystems.

Statistical analysis

For the candidate gene study, the statistical analyses were performed with the software Plink V1.9 (http://zzz.bwh.harvard.edu/plink/plink2.shtml) [19]. Deviance from Hardy-Weinberg equilibrium was determined at the 1% significance level in all groups of individuals. Individuals that have not achieved an SNP completion rate of 95% have been filtered out. To test for possible allelic, logistic regression model and Fisher's exact test were assessed in seronegative vs. seropositive individuals and asymptomatic vs. chronic Chagas cardiomyopathy individuals. The Benjamini & Hochberg step-up false discovery rate (FDR) correction was used in all analyses to control for multiple testing. The covariates sex and age were adjusted in logistic regression models. 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 Cochran's Q statistic (Q test P< 0.05) and I heterogeneity index [20]. 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) [21].

The statistical power of the studies was estimated with the Power Calculator for Genetic Studies 2006 (CaTS) software () (http://www.sph.umich.edu/csg/abecasis/CaTS/) [22].

Evaluation of functionality of the three SNPs analyzed was performed with the online software HaploReg v4.1 [23] (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 and T cells lines in the American population. For regulatory features, Ensembl Browser (https://www.ensembl.org) [18] and ReMap 2018 v1.2. (http://tagc.univ-mrs.fr/remap/) [24] were used.

Results

The three IL18 SNPs were in Hardy-Weinberg equilibrium in all the analyzed cohorts (P> 0.01). The genotyping success rate was over 90% and the allele frequencies in all cases were similar to those described for the Americans sub-populations of the 1000 Genomes Project phase III [18] (). The SNPs were in moderate pairwise linkage disequilibrium in the studied cohorts and in the American sub-populations from the 1000 Genomes Project phase III [18] ().

T. cruzi infection susceptibility

The allelic and genotypic frequencies of seronegative and seropositive individuals from Colombia were compared in . The allelic frequencies of the three SNPs were statistically significant even after multiple testing correction. The frequency of the minor allele, G, in rs2043055 was significantly reduced in the seronegative compared to seropositive individuals suggesting an association with higher infection risk, while the frequencies of rs1946518*T and rs360719*G alleles were significantly increased in seronegative compared to the seropositive individuals, suggesting an association with the protection against the infection by T. cruzi.

The allelic and genotypic frequencies of seronegative and seropositive individuals from Argentina are shown in . No associations between IL18 genetic variants were found. However, the rs2043055 remained borderline significant for protection against infection by T. cruzi [P = 0.061, odds ratio (OR) = 0.71, 95% confidence interval (CI) = 0.49–1.02].

In addition, a meta-analysis combining data from Colombian and Argentinean cohorts was performed (). The IL18 rs360719 showed consistent effects among the two meta-analyzed populations with a statistically significant association (CI: 0.66–0.89, P = 0.001, under a fixed-effects meta-analysis) with an OR for the G allele of 0.76, which was statistically significant after a Bonferroni correction (P< 0.017). For this comparison, the sample size attained a statistical power of over 80% for this OR (). In both cohorts, the allele effects size were in concordance and this result indicates an association to the protection against T. cruzi infection in these cohorts.

Chronic Chagas cardiomyopathy susceptibility

The allelic and genotypic frequencies of chronic Chagas cardiomyopathy and asymptomatic patients from Colombia were compared in . The allelic frequencies of IL18 rs2043055 was statistically significant even after multiple testing correction (P = 0.037, OR = 0.79, CI = 0.64–0.99). The frequency of the of the rs2043055* G allele was significantly incremented in asymptomatic patients, suggesting an association with the protection against the development of Chagas cardiomyopathy. However, no significant differences in allelic frequencies were observed for rs1946518 and rs360719.

The SNP IL18 rs2043055 was studied in 1,051 seropositive Brazilian patients (CCC = 849 and ASY = 202) [13]. The frequency of the G allele, in rs2043055, was increased in chronic Chagas cardiomyopathy patients compared to asymptomatic in the Brazilian, Bolivian and Argentinian cohorts, but no significant differences were found in these cohorts. Nevertheless, in the Bolivian cohort (), a trend of association can be observed for this SNP (P = 0.088, OR = 1.39, CI = 0.95–2.02). In the Argentinian cohort (), the frequency of the T allele, in IL18 rs1946518, was increased in asymptomatic compared to chronic Chagas cardiomyopathy patients, and remained borderline significant for suggesting an association with the protection against the development of Chagas cardiomyopathy (P = 0.078, OR = 0.67, CI = 0.44–1.04).

Further, a meta-analysis combining these results were performed (). The results of the available SNPs showed no significant associations.

In silico functional characterization of IL18 gene variants

We further explored the functional annotations of the three variants studied in this work using HaploReg v4.1. The annotation indicated that the SNPs of IL18 are located in a regulatory region of the genome (). The annotation based on the epigenomic information of rs2043055 indicates that this SNP maps in an enhancer region, which is correlated with active gene expression in primary mononuclear cells and in T cells from peripheral blood. The rs1946518 and rs360719 variants mapped in a region enriched in histone marks: H3K4me3, H3K9ac, a hallmark of active promoter region, and H3K27ac in enhancer region in mononuclear cells and T cells (). Furthermore, according to ReMap 2018 v1.2 these three SNPs mapped in regulatory regions of the human genome and it has been described as transcription factors ().

Discussion

Genetic factors and immunologic response may determine the susceptibility against the infection and development of Chagas disease [6-9]. In the present study, three IL18 genetic variants were analyzed in four Latin American populations, being the largest genetic study conducted to date in Chagas disease. Concerning genetic control of the infection, our results evidenced the implication of the IL18 rs360719 polymorphism, in our populations. However, when comparing cardiomyopathy and asymptomatic patients, no significant associations were detected.

Addressing the question of genetic susceptibility to T. cruzi infection through comparison of seropositive with seronegative individuals is not an easy task. The recruitment of an adequate number of subjects from endemic areas exposed to T. cruzi is often challenging, and that is the reason for including only two cohorts in this comparison. The previous study in a Colombian cohort was the first to report an association for rs2043055, rs1946518 and rs360719 with T. cruzi infection and suggested that this association was mainly driven by the polymorphism rs360719 [14]. After the enlargement of this cohort, the association remained, showing consistent results in a well-powered cohort. Replication of these variants was performed in an Argentinian cohort and only rs2043055, showed a borderline genetic association but in the opposite direction compared with the Colombian cohort. These differences could be due to the complex genetic structure of Latin American individuals, reflected by the recent admixture among Native American, European, and West African source populations [25]. Also, the lack of replication may occur if the assessed polymorphism is not the causal variant but is rather in LD with it, i.e., variants correlated with each other more often than expected by chance. LD patterns depend on the genetic background of the founder population and population history [26]. The rs360719 in the Argentinian cohort showed no association with T. cruzi infection, which could be a consequence of an insufficient statistical power (). Interestingly, the meta-analysis showed that the variant IL18 rs360719 presented a consistent effect among the two cohorts, indicating protection against T. cruzi infection. The SNP rs360719 is located in the promoter region of the IL18 gene. The functional annotation of this SNP with empirical data from the ENCODE project revealed that is located in histone marks in primary mononuclear cells and T helper naive cells from peripheral blood, and has been described as transcription factor (S2 Table). These modifications are critically involved in the regulation of gene expression [27]. Also, it has been described that IL18 rs360719 polymorphism leads to loss of the octamer (OCT)-1 transcription factor binding site. OCT-1 is known to be a ubiquitously expressed factor involved in the regulation of certain cytokines, like IL-18 [28]. Thus, the rs360719 would be associated with IL-18 expression in peripheral blood mononuclear cells and may play a role in the susceptibility or resistance to T. cruzi infection.

Chronic chagasic cardiomyopathy, the most frequent clinical outcome of Chagas disease, has been associated with cytokine enriched heart tissue inflammation [29]. Furthermore, local expression of IL-18 in chronic chagasic cardiomyopathy heart tissue has been described and would be associated with mononuclear inflammatory infiltrates, cardiomyocyte destruction and fibrosis [30]. In our study, we analyzed IL18 gene variants in four Latin American populations with chronic Chagas cardiomyopathy. The IL18 genetic variant, rs2043055, studied in the Brazilian cohort, showed nominal significant differences in the genotypic frequencies among moderate and severe chagasic cardiomyopathy patients [13]. When comparing chronic chagasic cardiomyopathy with asymptomatic patients, this variant showed a significant association in the Colombian cohort. However, these results were not validated in the Bolivian and Argentinian cohorts. These discrepancies in the results could be due to the genetic heterogeneity among the study cohorts [25]. The impact of European colonization and slave trade from western Africa has altered the genomes of Native Americans in multiple and dynamic ways. Approximately, 9–9.6 generations have passed since admixture and ancestry-enriched SNPs in Latin American populations may have a substantial effect on health and disease related phenotypes [31, 32]. For instance, Norris et al. reported SNPs with excess African or European ancestry, which are associated with ancestry-specific gene expression patterns and play crucial roles in the immune system and infectious disease responses [33]. In addition, an interesting report by Lima-Costa et al. showed that the prevalence of T. cruzi infection is strongly and independently associated with higher levels of African and Native American ancestry in a Brazilian population [34]. This heterogeneity in ancestry proportions across geographic regions, and also within countries themselves, are challenging in association studies in order to find generalizable results across populations [35-37]. All this, suggests that a fine-scale genomics perspective might represent a powerful tool to understand the role of genetics in this neglected disease diagnosis and prognosis.

As was mentioned, IL-18 plays an important role in the regulation of IFN-G production and development of Th1 response. This interleukin is produced by a wide variety of cells, including dendritic cells, macrophages, keratinocytes, intestinal epithelial cells, and osteoblasts, suggesting a key pathophysiological role in health and disease [12]. Several studies have highlighted the implication of IL-18 in the acute and chronic phase of Chagas disease [38-PLoS pathogens. 2019 ">41]. Considering that infectious diseases exert significant selective genetic pressure, it has been postulated two genetic mechanisms to explain the pathogenesis of Chagas disease [8]. First, pathogen resistance genes (PRG) would be involved in inhibits infection by directly reducing pathogen burden and secondly, disease tolerance genes (DTG) will operate to minimize tissue damaging effects of the pathogen [42-44]. Consequently, polymorphisms in PRG and DTG will be associated with differential disease progression. One of the most relevant disease tolerance genes identified was related to directly or indirectly inhibit IFN-G production or Th1 differentiation [8], and therefore, IL-18 could be implicated in this regulation.

In conclusion, our results validated the previous work suggesting that IL18 rs360719 plays an important role in the susceptibility to T. cruzi infection [14]. On the other hand, no evidence of association was found between the IL18 genetic variants and chronic Chagas cardiomyopathy in the Latin American population. Even though, meta-analyses offers a powerful approach to identify genetic variants that influence susceptibility of common diseases [45, 46], in the context of Chagas disease is necessary to contemplate the challenges of studying such an heterogeneous populations like Latin Americans with recent admixture, where fine-scale genomic assessments may be necessary [25]. Finally, further studies are needed to reach more conclusive results concerning the genetic basis of Chagas disease.

Location of IL18 rs2043055, rs1946518 and rs360719 within the gen (A). R2 (B) and Linkage disequilibrium D’ (C) plots estimated by using expectation maximization algorithmin Haploview V4.2. in Americans (AMR) from 1000 Genomes Project Phase III and in Colombian, Bolivian and Argentinian cohorts.

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Statistical power calculation of the candidate gene study (A) and the meta-analysis (B) considering the allele frequencies for each SNP, the prevalence of the disease in each country with three different OR.

(DOCX)

Click here for additional data file.

In silico functional characterization of IL18 gene variants (A) Regulatory feature consequences for IL18 gene variants (B).

(DOCX)

Click here for additional data file.

Membership of the Chagas Genetics CYTED Network.

(DOCX)

Click here for additional data file.

14 Aug 2019

Dear Dr Strauss:

Thank you very much for submitting your manuscript "Association of IL18 genetic polymorphisms with Chagas disease in Latin American populations" (#PNTD-D-19-01126) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Strauss and colleagues performed a replication study to confirm whether IL18 variation was associated with predisposition to Trypanosoma cruzi infection and the development of chronic Chagas cardiomyopathy in Latin American populations. With that aim, they recruited a considerable large case-control study group from different countries, which allowed them to conduct the analyses with an adequate statistical power. An appropriate ethical statement was included, and I do not have concerns about regulatory requirements being met, as the study was performed by an international consortium with a proven experience on this matter. However, I have some considerations to make in relation to the methods.

- Although it is understood that the SNP selection criteria was based on a previously published study, it would be interesting to include additional supplementary information about the gene structure and specific location of the analysed genetic variants within the region.

- The demographic and clinical data of the analysed Brazilian cohort was missing. The authors should indicate the number of individuals showing each phenotype and whether the sex and age information was available, as mentioned for the other cohorts.

Reviewer #2: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

YES

-Is the study design appropriate to address the stated objectives?

YES

-Is the population clearly described and appropriate for the hypothesis being tested?

YES

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

YES

-Were correct statistical analysis used to support conclusions?

YES but the authors can go further with additional ones.

-Are there concerns about ethical or regulatory requirements being met?

YES

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results are consistent and I must say that they have been brilliantly organised and discussed. The workflow was appropriate and the tables clearly presented and with a good quality. I have just one comment related to this section:

- An evaluation of functionality of the three studied SNPs was performed. However, no description of the findings was included in the results section.

Reviewer #2: -Does the analysis presented match the analysis plan?

YES

-Are the results clearly and completely presented?

YES

-Are the figures (Tables, Images) of sufficient quality for clarity?

YES

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: In general, this is a well structured and nicely written manuscript in which the conclusions are clear and supported by the data obtained. This type of studies is of high relevance considering the incidence of Chagas disease in the studied areas and the lack of knowledge regarding the genetic influence in the development of its most severe phenotypes. In this regard, the relevance for the public health is addressed in the manuscript. However, although the authors acknowledge the limitations of the study, I have a specific concern regarding the following:

- The authors speculated that the discrepancies observed in their results could be due to genetic heterogeneity amongst the study populations. I may agree on this, but a more detailed discussion of this idea (using more recent citations) should be added to the manuscript.

Reviewer #2: -Are the conclusions supported by the data presented?

YES

-Are the limitations of analysis clearly described?

+/-

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

+/-

-Is public health relevance addressed?

YES

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: The supplementary tables are differently called in the text and in the supporting information (e.g. Table S1 / Table A).

Reviewer #2: Association of IL18 genetic polymorphisms with Chagas disease in Latin American populations.

Reference PNTD-D-19-01126

Given the influence of interleukin 18 in the development of chagas disease, the authors analyzed three IL18 variants regarding the predisposition to Trypanosoma cruzi infection and the development of chronic Chagas cardiomyopathy (CCC), in several Latin America populations.

1) Genetic data were obtained for 3,608 patients from Colombia, Bolivia, Argentina, and Brazil. Authors performed meta-analysis to validate previous findings with increased statistical power.

2) Among the Colombian and Argentinean cohorts, rs36071927 showed a significant genetic effect.

3) rs2043055 showed an association with protection from cardiomyopathy in the Colombian cohort.

4) The meta-analysis of the CCC vs asymptomatic patients from the four cohorts showed no evidence of association.

According to the authors their results validated the association found previously in the Colombian cohort suggesting that IL18 rs360719 plays an important role in the susceptibility to T. cruzi infection and no evidence of association was found between the IL18 genetic variants and CCC in the Latin American population studied.

Major corrections:

Page 6 126-127: Three single nucleotide polymorphisms (SNPs) of IL18 gene, rs2043055, rs1946518 and rs360719 were selected. The selected SNPs were in moderate pairwise linkage disequilibrium (LD, r2 < 0.5) in the American population; values were estimated using LDlink website tool

The authors need to provide in supplementary data the D’ and R2 values between each marker in their various study populations and in the reference population. Moreover, there is not enough information on the American population that they used as reference.

Page 5 686-87: The seropositive patients who presented cardiac alterations were classified as chronic Chagas cardiomyopathy (CCC, n= 1,707) and asymptomatic (ASY, n= 1,183). The sex distribution for the entire Latin American population studied was 61.4% female and 38.6% male.

Does the sex distribution is significantly different between the two groups on the whole cohort and in each population.

Page 7 153-154: Evaluation of functionality of the three SNPs analyzed was performed with the online software HaploReg v4.1.

The authors need to use some tools that are more appropriate as remap which is also using the Encode library. However, these data were cured before to be included in Remap.

Page 9: Regarding infection levels, three polymorphisms were associated in the Colombian population and none of them in the Argentinian population. Then authors performed a meta-analysis on these two populations. At this level authors indicated that the polymorphism IL18 rs360719 is associated.

In the same way the authors do not underlined the results obtained in their meta-analysis for rs1946518. It is not consistent.

I will really appreciate if the authors can compare the frequencies of these SNPs between the seronegative individuals extracted from the Colombian population vs seronegative individuals extracted from the Argentinian population. It may provide arguments to better understand their meta-analysis data.

Page 10: for chronic disease the authors detect one association in the Columbian population that is not confirmed in the Argentinian population or in the Bolivian population. In the previous study done on a Brazilian cohort no association was detected too. On the Brazilian population significant association was detected when the authors compared the moderate CCC vs the severe CCC.

I invite the authors to compare severe CCC vs moderate CCC in their Colombian, Argentinian and Bolivian population.

Without this analysis the sentence “Regarding genetic control of the chronic Chagas cardiomyopathy, no association was detected in a well-powered cohort” is too strong.

Page 11-12:

Authors wrote sentences in their discussion

These differences could be due to the complex genetic structure of Latin American individuals, reflected by the recent admixture among Native American, European, and West African source populations

However, these results were not validated in the Bolivian and Argentinian cohorts. These discrepancies in the results could be due to the genetic heterogeneity among the study cohorts (Bryc, et 277 al. 2010).

These two sentences describe a very important point of this manuscript. I fully agree. I therefore invite the authors to go further in this reflection. Do we have information on the origin of these population in terms of origin and migration. Moreover, the reference needs to be properly formatted.

Minor corrections:

Page 4 64-65: Polymorphism in genes encoding cytokines may influence the level of cytokines production and, consequently, cause different immunological responses [10, 11].

The selected references are not appropriate it‘s is better to indicate reference on Chagas disease

Page 4 68-70: Previous genetic studies performed in a Colombian and Brazilian cohort found associations between variants of IL18 gene and the predisposition to T. cruzi infection and chronic Chagas cardiomyopathy [13, 14].

The authors need to respect the time of publication. Brazil study came out first.

Finally, the authors need to correct the manuscript that includes several typing errors

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The manuscript presented by the authors may definitively help to continue advancing towards a better understanding of the genetic causes leading to cardiomyopathy in Chagasic patients. Due to the power limitations of the studies performed to date, it is crucial to confirm the available genetic associations in larger and independent populations. Because of this, collaborative efforts like the one reported here are required. If the authors could address properly the concerns describe above, I have no doubt that this could be a highly valuable manuscript.

Reviewer #2: (No Response)

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Reviewer #2: No

21 Oct 2019

Dear Dr Strauss,

We are pleased to inform you that your manuscript, "Association of IL18 genetic polymorphisms with Chagas disease in Latin American populations", has been editorially accepted for publication at PLOS Neglected Tropical Diseases.

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: No additional comments.

Reviewer #2: Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

YES

-Is the study design appropriate to address the stated objectives?

YES

-Is the population clearly described and appropriate for the hypothesis being tested?

YES

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

YES

-Were correct statistical analysis used to support conclusions?

YES

-Are there concerns about ethical or regulatory requirements being met?

YES

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: No additional comments.

Reviewer #2: -Does the analysis presented match the analysis plan?

YES

-Are the results clearly and completely presented?

YES

-Are the figures (Tables, Images) of sufficient quality for clarity?

YES

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: No additional comments.

Reviewer #2: -Are the conclusions supported by the data presented?

YES

-Are the limitations of analysis clearly described?

YES

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

YES

-Is public health relevance addressed?

YES

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: No additional comments.

Reviewer #2: I rewiewed the original submission and did several comments (major and minor ones)

the authors took in considerations all the remarks I made

they answered to all these queris and their responses were appropriates

In this way, the quality of the manuscript has been seriously improved

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The manuscript has been substantially improved. All my concerns have been solved.

Reviewer #2: NA

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Reviewer #1: No

Reviewer #2: No

13 Nov 2019

Dear Dr Strauss,

We are delighted to inform you that your manuscript, "Association of IL18 genetic polymorphisms with Chagas disease in Latin American populations," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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PLOS Neglected Tropical Diseases

Table 1

Colombian cohort. Genotype and allele distribution for IL18 variants in seronegative and seropositive individuals.

SNP		A1| A2	Genotype. N (%)			MAF	Allele test
			1|1	1|2	2|2		OR	(L95-U95)	P LogstReg	P FDR_BH
rs2043055	Seronegative (631)	G|A	82 (13.00)	300 (47.54)	249 (39.46)	36.77	1.30	(1.10–1.53)	0.002	0.004
	Seropositive (927)		164 (17.69)	450 (48.54)	313 (33.76)	41.96
rs1946518	Seronegative (631)	T|G	163 (25.83)	334 (52.93)	134 (21.24)	52.3	0.79	(0.67–0.92)	0.003	0.004
	Seropositive (927)		214 (23.09)	448 (48.33)	265 (28.59)	47.25
rs360719	Seronegative (631)	G|A	103 (16.32)	299 (47.39)	229 (36.29)	40.02	0.75	(0.63–0.89)	0.001	0.004
	Seropositive (927)		107 (11.54)	426 (45.95)	394 (42.50)	34.52

1: minor allele | 2: major allele; alleles are showed in forward strand. MAF: minor allele frequency. OR: odds ratios, L95-U95: confidence intervals of 95% L: lower limit; U: upper limit. Values adjusted by sex and age. Significant P value is shown in bold.

Table 2

Argentinian cohort.

Genotype and allele distribution for IL18 variants in seronegative and seropositive individuals.

SNP		A1| A2	Genotype. N (%)			MAF	Allele test
			1|1	1|2	2|2		OR	(L95-U95)	P LogstReg
rs2043055	Seronegative (77)	G|A	20 (25.97)	29 (37.66)	28 (36.36)	44.81	0.71	(0.49–1.02)	0.061
	Seropositive (270)		33 (12.23)	126 (46.67)	111 (41.11)	35.56
rs1946518	Seronegative (77)	T|G	19 (24.67)	35 (45.46)	23 (29.87)	47.4	1.03	(0.71–1.49)	0.883
	Seropositive (270)		54 (20)	151 (55.92)	65 (24.08)	47.96
rs360719	Seronegative (77)	G|A	11 (14.28)	33 (42.85)	33 (42.85)	35.71	0.87	(0.60–1.31)	0.552
	Seropositive (270)		25 (9.26)	128 (47.40)	117 (43.33)	32.96

1: minor allele | 2: major allele; alleles are showed in forward strand. MAF: minor allele frequency. OR: odds ratios, L95-U95: confidence intervals of 95% L: lower limit; U: upper limit. Values adjusted by sex and age.

Table 3

Meta-analysis of IL18 variants, Argentinian and Colombian cohorts for T. cruzi infection susceptibility.

	Colombian cohort			Argentinian cohort			Meta-analysis
SNP	OR	(L95-U95)	P	OR	(L95-U95)	P	OR	(L95-U95)	P
rs2043055	1.30	(1.10–1.53)	0.002	0.71	(0.49–1.02)	0.061	1.17	(1.01–1.36)	0.035
rs1946518	0.79	(0.67–0.92)	0.003	1.03	(0.71–1.49)	0.883	0.82	(0.71–0.94)	0.006
rs360719	0.75	(0.63–0.89)	0.001	0.87	(0.60–1.31)	0.552	0.76	(0.66–0.89)	0.001

Total number of individuals: seropositive n = 1,209 and seronegative n = 718

OR: odds ratios, L95-U95: confidence intervals of 95% L: lower limit; U: upper limit. Marked in bold the P value ≤ than the individual cohorts. Significant association based on the Bonferroni correction P< 0.017.

Table 4

Colombian cohort.

Genotype and allele distribution for IL18 variants in asymptomatic and chronic Chagas cardiomyopathy (CCC) individuals.

SNP		A1| A2	Genotype. N (%)			MAF	Allele test
			1|1	1|2	2|2		OR	(L95-U95)	P LogstReg	P FDR_BH
rs2043055	Asymptomatic (358)	G|A	83 (23.18)	159 (44.41)	116 (32.40)	45.39	0.79	(0.64–0.99)	0.037	0.046
	CCC (569)		81 (14.24)	291 (51.14)	197 (34.62)	39.81
rs1946518	Asymptomatic (358)	T|G	82 (22.91)	160 (44.69)	116 (32.40)	45.25	1.14	(0.92–1.41)	0.225	0.229
	CCC (569)		132 (23.20)	288 (50.62)	149 (26.19)	48.51
rs360719	Asymptomatic (358)	G|A	45 (12.57)	155 (43.30)	158 (44.13)	34.22	0.99	(0.79–1.26)	0.994	0.765
	CCC (569)		62 (10.90)	271 (47.63)	236 (41.48)	34.71

1: minor allele | 2: major allele; alleles are showed in forward strand. MAF: minor allele frequency. OR: odds ratios, L95-U95: confidence intervals of 95% L: lower limit; U: upper limit. Values adjusted by sex and age. Significant P value is shown in bold.

Table 5

Bolivian cohort.

Genotype and allele distribution for IL18 variants in asymptomatic and chronic Chagas cardiomyopathy (CCC) individuals.

SNP		A1| A2	Genotype. N (%)			MAF	Allele test
			1|1	1|2	2|2		OR	(L95-U95)	P LogstReg
rs2043055	Asymptomatic (528)	G|A	72 (13.64)	260 (49.24)	196 (37.12)	38.26	1.39	(0.95–2.02)	0.088
	CCC (100)		16 (16)	50 (50)	34 (34)	41
rs1946518	Asymptomatic (528)	G|T	101 (19.13)	268 (50.76)	159 (30.11)	44.51	1.24	(0.85–1.80)	0.260
	CCC (100)		19 (19)	55 (55)	26 (26)	46.5
rs360719	Asymptomatic (528)	G|A	54 (10.23)	237 (44.89)	237 (44.89)	32.67	0.98	(0.66–1.45)	0.934
	CCC (100)		10 (10)	50 (50)	40 (40)	35

1: minor allele | 2: major allele; alleles are showed in forward strand. MAF: minor allele frequency. OR: odds ratios, L95-U95: confidence intervals of 95% L: lower limit; U: upper limit. Values adjusted by sex and age.

Table 6

Argentinian cohort.

Genotype and allele distribution for IL18 variants in asymptomatic and chronic Chagas cardiomyopathy (CCC) individuals.

SNP		A1| A2	Genotype. N (%)			MAF	Allele test
			1|1	1|2	2|2		OR	(L95-U95)	P LogstReg
rs2043055	Asymptomatic (89)	G|A	9 (10.11)	40 (44.94)	40 (44.94)	32.58	1.26	(0.82–1.95)	0.291
	CCC (181)		24 (13.26)	86 (47.51)	71 (39.22)	37.02
rs1946518	Asymptomatic (89)	T|G	21 (23.59)	51 (57.30)	17 (19.10)	52.25	0.67	(0.44–1.04)	0.078
	CCC (181)		33 (18.23)	100 (55.25)	48 (26.51)	45.86
rs360719	Asymptomatic (89)	G|A	8 (8.99)	47 (52.81)	34 (38.20)	35.39	0.81	(0.52–1.27)	0.364
	CCC (181)		17 (9.32)	81 (44.75)	83 (45.86)	31.77

1: minor allele | 2: major allele; alleles are showed in forward strand. MAF: minor allele frequency. OR: odds ratios, L95-U95: confidence intervals of 95% L: lower limit; U: upper limit. Values adjusted by sex and age.

Table 7

Meta-analysis of IL18 variants, Latin American cohorts for Chagas cardiomyopathy susceptibility.

	Colombian cohort		Bolivian cohort		Argentinian cohort		Brazilian cohort		Meta-analysis
SNP	OR (L95-U95)	P	OR (L95-U95)	P	OR (L95-U95)	P	OR (L95-U95)	P	OR (L95-U95)	P
rs2043055	0.79 (0.64–0.99)	0.037	1.39 (0.95–2.02)	0.088	1.26 (0.82–1.95)	0.291	1.06 (0.85–1.32)	0.598	1.05 (0.82–1.35)	0.259
rs1946518	1.14 (0.92–1.41)	0.225	1.24 (0.85–1.80)	0.260	0.67 (0.44–1.04)	0.078	-	-	1.07 (0.90–1.26)	0.426
rs360719	0.99 (0.79–1.26)	0.994	0.98 (0.66–1.45)	0.934	0.81 (0.52–1.27)	0.364	-	-	0.95 (0.79–1.15)	0.629

Total number of individuals: rs2043055 CCC n = 1,707 and asymptomatic n = 1,183; rs1946518 and rs360719: CCC n = 858 and asymptomatic n = 981

OR: odds ratios, L95-U95: confidence intervals of 95% L: lower limit; U: upper limit.