GWAS loci associated with Chagas cardiomyopathy influences DNA methylation levels

Cardiomyopathies; Genomics; Chagas diseaseCardiomiopatías; Genómica; Enfermedad de ChagasMiocardiopaties; Genòmica; Malaltia de ChagasA recent genome-wide association study (GWAS) identified a locus in chromosome 11 associated with the chronic cardiac form of Chagas disease. Here we aimed to elucidate the potential functional mechanism underlying this genetic association by analyzing the correlation among single nucleotide polymorphisms (SNPs) and DNA methylation (DNAm) levels as cis methylation quantitative trait loci (cis-mQTL) within this region. A total of 2,611 SNPs were tested against 2,647 DNAm sites, in a subset of 37 chronic Chagas cardiomyopathy patients and 20 asymptomatic individuals from the GWAS. We identified 6,958 significant cis-mQTLs (False Discovery Rate [FDR]<0.05) at 1 Mb each side of the GWAS leading variant, where six of them potentially modulate the expression of the SAC3D1 gene, the reported gene in the previous GWAS. In addition, a total of 268 cis-mQTLs showed differential methylation between chronic Chagas cardiomyopathy patients and asymptomatic individuals. The most significant cis-mQTLs mapped in the gene bodies of POLA2 (FDR = 1.04x10-11), PLAAT3 (FDR = 7.22x10-03), and CCDC88B (FDR = 1.89x10-02) that have been associated with cardiovascular and hematological traits in previous studies. One of the most relevant interactions correlated with hypermethylation of CCDC88B. This gene is involved in the inflammatory response, and its methylation and expression levels have been previously reported in Chagas cardiomyopathy. Our findings support the functional relevance of the previously associated genomic region, highlighting the regulation of novel genes that could play a role in the chronic cardiac form of the disease.This research was supported by grants from Programa Iberoamericano de ciencia y tecnología para el desarrollo (RIMGECH - 217RT0524) to Chagas Genetics CYTED Network. MAH was supported by Ministerio de Ciencia e Innovación-Juan de la Cierva fellowship (IJC2018-035131-I). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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.Fil: Strauss, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Palma-Vega, Miriam. Instituto de Parasitología y Biomedicina López Neyra; EspañaFil: Casares Marfil, Desiré. Universidad Autónoma de Barcelona. Hospital Vall D' Hebron; EspañaFil: Pau Bosch, Nicolau. Universidad Autónoma de Barcelona. Hospital Vall D' Hebron; EspañaFil: Lo Presti, Maria Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Molina, Israel. Universidad Autónoma de Barcelona. Hospital Vall D' Hebron; EspañaFil: Gonzáles, Clara Isabel. Universidad Industrial de Santander; ColombiaFil: Martin, Javier. Instituto de Parasitología y Biomedicina López Neyra; EspañaFil: Acosta Herrera, Marialbert. Instituto de Parasitología y Biomedicina López Neyra; Españ

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.Fil: Strauss, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Acosta Herrera, Marialbert. Consejo Superior de Investigaciones Científicas; EspañaFil: Alcaraz, Alexia. Consejo Superior de Investigaciones Científicas; EspañaFil: Casares Marfil, Desiré. Consejo Superior de Investigaciones Científicas; EspañaFil: Bosch Nicolau, Pau. Internacional Hospital Universitari Vall d’Hebron; EspañaFil: Lo Presti, Maria Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Molina, Israel. Internacional Hospital Universitari Vall d’Hebron; EspañaFil: González, Clara Isabel. Universidad Industrial Santander; ColombiaFil: Martín, Javier. Consejo Superior de Investigaciones Científicas; EspañaFil: Chagas Genetics CYTED Network. No especifíca

HLA-B*08 identified as the most associated MHC locus for anti-carbamylated protein antibody-positive/anti-CCP-negative rheumatoid arthritis

Objective: Previously, only the HLA-DRB1 alleles have been assessed in rheumatoid arthritis (RA). The aim of the present study was to identify the key major histocompatibility complex (MHC) susceptibility factors showing a significant association with anti-carbamylated protein antibody-positive (anti-CarP+) RA. Methods: Analyses were restricted to RA patients who were anti-cyclic citrullinated peptide antibody negative (anti-CCP-), because the anti-CCP status dominated the results otherwise. Therefore, we studied samples from 1,821 anti-CCP- RA patients and 6,821 population controls from Spain, Sweden, and the Netherlands. The genotypes for ~8,000 MHC biallelic variants were assessed by dense genotyping and imputation. Their association with the anti-CarP status in RA patients was tested with logistic regression and combined with inverse-variance meta-analysis. Significance of the associations was assessed according to a study-specific threshold of P < 2.0 × 10-5 . Results: The HLA-B*08 allele and its correlated amino acid variant Asp-9 showed a significant association with anti-CarP+/anti-CCP- RA (P < 3.78 × 10-7 ; I2 = 0). This association was specific when assessed relative to 3 comparator groups: population controls, anti-CarP-/anti-CCP- RA patients, and anti-CCP- RA patients who were positive for other anti-citrullinated protein antibodies. Based on these findings, anti-CarP+/anti-CCP- RA patients could be separated from other antibody-defined subsets of RA patients in whom an association with the HLA-B*08 allele has been previously demonstrated. No other MHC variant remained associated with anti-CarP+/anti-CCP- RA after accounting for the presence of the HLA-B*08 allele. Specifically, the reported association of HLA-DRB1*03 was observed at a level comparable to that reported previously, but it was attributable to linkage disequilibrium. Conclusion: These results identify HLA-B*08 carrying Asp-9 as the MHC locus showing the strongest association with anti-CarP+/anti-CCP- RA. This knowledge may help clarify the role of the HLA in susceptibility to specific subsets of RA, by shaping the spectrum of RA autoantibodies. © 2020, American College of Rheumatology

Genomic approaches to unravel the pathogenesis of Chagas disease

La enfermedad de Chagas es una enfermedad infecciosa causada por el parásito Trypanosoma cruzi. Esta enfermedad es endémica de Latinoamerica donde el principal vector de transmisión son insectos hematófagos. Se estima que afecta a unas 6-7 millones de personas en todo el mundo ya que, debido a procesos como la migración y globalización, se ha extendido a zonas no endémicas. La enfermedad cursa con una fase aguda seguida de una fase indeterminada en la que la mayoría de individuos permanecen asintomáticos de por vida. Sin embargo, algunos pacientes pueden desarrollar la forma crónica de la enfermedad años después de la infección, manifestándose con sintomatología digestiva y/o cardíaca, siendo esta última la más frecuente y grave conocida como cardiomiopatía chagásica crónica. Dada las diferencias en el desarrollo de la infección en zonas endémicas así como la evolución diferencial a la forma crónica cardíaca, se ha sugerido la influencia de la variación genética del hospedador en la patogénesis de la enfermedad. La presente tesis doctoral se centra en el estudio del componente genético del hospedador mediante diferentes estrategias de análisis genético, para dilucidar su papel tanto en la infección como en el desarrollo de la cardiomiopatía chagásica crónica. Para ello y con el objetivo de confirmar asociaciones previamente descritas, se realizaron estudios de genes candidatos para evaluar la relación de variantes genéticas localizadas en los genes IL6, IL17A e IL18, que codifican citoquinas implicadas en la respuesta inmune contra Trypanosoma cruzi. Dichas asociaciones se confirmaron para los genes IL17A e IL18, mientras que la relación de la variante del gen IL6 fue descartada. Sin embargo, la evaluación de variantes genéticas a lo largo de todo el genoma mejora la capacidad de identificación de variación asociada con la enfermedad de una manera no sesgada, para lo cual un estudio de asociación del genoma completo (GWAS, del inglés “genome-wide association studies”) supuso la herramienta de elección. Así, se realizó un GWAS en individuos procedentes de Colombia, Bolivia y Argentina que, junto con la información de población brasileña previamente publicada, se meta-analizó tanto para la susceptibilidad a la infección como para el desarrollo de la cardiomiopatía chagásica crónica. Este análisis nos permitió identificar una asociación a nivel de significación genómica con la forma cardíaca cercana al gen SAC3D1, siendo la primera asociación genómica identificada en esta enfermedad. Por otro lado, dada la mezcla de las poblaciones ancestrales Nativo Americana, Europea y Africana presente en las poblaciones latinas, se evaluó la relación de estos bloques de ascendencia con la susceptibilidad a la enfermedad de Chagas. Para esto se realizó un análisis de mapeo por mezcla en la cohorte colombiana. Se identificó una asociación de protección de la ascendencia Nativo Americana con la susceptibilidad a la infección en la región del complejo mayor de histocompatibilidad, en contraposición de la ascendencia Europea que resultó de riesgo, poniendo de manifiesto el componente evolutivo en la relación parásitohospedador de estas poblaciones. En esta región genética se localizan numerosos genes implicados en la respuesta inmunológica, entre los que cabría destacar los genes HLA, que tienen una gran transcendencia en el reconocimiento antigénico, y algunos miembros de la familia de los TRIM, conocidos por su papel en la respuesta inmune frente a patógenos. Finalmente, se llevó a cabo un análisis de rasgos cuantitativos de metilación (mQTLs, del inglés “methylation quantitative trait loci”) en la región genómica identificada en el GWAS. Con esta estrategia se analizó el efecto de la variación genética en los patrones de metilación de un subconjunto de pacientes procedentes de Bolivia. Entre los resultados más importantes destacamos aquellos mQTLs en genes que se han relacionado previamente con caracteres hematológicos y cardiovasculares. Además, identificamos mQTLs del gen CCDC88B, que tiene un papel relevante en el proceso inflamatorio. Este gen había sido previamente identificado como diferencialmente metilado y expresado en tejido cardiaco de pacientes con cardiomiopatía chagásica crónica, lo cual pone de manifiesto su importancia en la patogénesis de la enfermedad. Nuestros resultados confirmaron la influencia de los genes de esta región, destacando la funcionalidad de los mismos más allá de la variación en la secuencia del ADN. Adicionalmente, la identificación de genes previamente identificados en tejido cardiaco, pone de manifiesto la importancia del uso de tejidos más accesibles en la identificación de biomarcadores. Los resultados presentados en esta tesis doctoral destacaron el papel de la genética del hospedador en el desarrollo de la enfermedad de Chagas. Esto, junto con los análisis de metilación y expresión global que se están realizando en estas muestras, representan un avance en el conocimiento de la patogénesis de esta enfermedad tropical desatendida.Chagas disease is an infection caused by the parasite Trypanosoma cruzi. This disease is endemic form Latin America, where the main transmission vectors are hematophagous bugs. It is estimated that the infection affects 6-7 million people around the world, where it has been extended to non-endemic areas given migratory movements and globalization. Chagas disease has an acute phase followed by an indeterminate phase, and the majority of individuals remain asymptomatic for life. Nevertheless, some patients could develop the chronic form of the disease even years after the infection, which manifests with digestive and/or cardiac symptomatology. The latter the most frequent and severe form of the disease is known as chronic Chagas cardiomyopathy. Given the inter-individual differences in the infection in endemic regions as well as the differential development of the chronic cardiac form, it has been suggested the influence of the host genetic component in the pathogenesis of the disease. The present doctoral thesis focused in the analysis of the host genetic component through different genetic analysis approaches, in order to elucidate its role in the infection and the progress to the chronic Chagas cardiomyopathy. In order to confirm previously described associations, candidate gene studies were performed to assess genetic variants located in the IL6, IL17A and IL18 genes, which encode cytokines with relevance in the immune response against Trypanosoma cruzi. The associations were confirmed for the IL17A and IL18 variants, while it was discarded for the IL6 gene variant. The assessment of genetic variation throughout the entire genome improves the capacity of identifying disease associations in an unbiased fashion; therefore a genome-wide association study (GWAS) was the strategy of choice. For this, Colombian, Bolivian and Argentinian individuals’ genomic data was meta-analyzed with previously published data from the Brazilian population. This analysis was carried out for the susceptibility to the infection and the development of chronic Chagas cardiomyopathy. Using this strategy, it was possible to identify an association at genomic significance level with the chronic cardiac form in the vicinity of the SAC3D1 gene, being this the first genomic association identified for the disease. On the other hand, given the admixture of Native American, European and African ancestries of the Latin American populations, the relation among ancestry genetic blocks and the susceptibility to Chagas disease was assessed. Thus, an admixture mapping analysis in the Colombian population was performed. This approach revealed a protective association of the Native American ancestry with the susceptibility to the infection located in the major histocompatibility complex region. In contrast, the European ancestry showed a risk association with the disease, highlighting the evolutionary component in the parasite-host relationship of these populations. The HLA genes and the TRIM gene family were highlighted in this locus, which are of great importance for antigenic recognition and their contribution in the immune response against pathogens. Finally, a methylation quantitative trait loci (mQTLs) analysis was performed in the significant genomic region from the GWAS. This strategy allowed us to evaluate the effect of the genetic variation in the methylation patterns in a subset of individuals from Bolivia. Significant mQTLs were identified, including the CCDC88B, which is an important player in the inflammatory process. Interestingly, a previous analysis in chronic Chagas cardiomyopathy patients revealed this gene as differentially methylated and expressed. Our results highlighted the functionality of the genes within this region beyond the variation in the DNA sequence. In addition, the exposure of genes previously reported in cardiac samples, highlighted the importance of using more accessible tissues in the identification of disease biomarkers. The results presented in this doctoral thesis emphasized the role of the host genetics in the development of Chagas disease. This, together with the global methylation and expression analyses being carried out, represents an advance in the knowledge of the pathogenesis of this neglected tropical disease.Tesis Univ. Granada

Genomic opportunities for drug repositioning in systemic seropositive rheumatic diseases

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GWAS loci associated with Chagas cardiomyopathy influences DNA methylation levels.

A recent genome-wide association study (GWAS) identified a locus in chromosome 11 associated with the chronic cardiac form of Chagas disease. Here we aimed to elucidate the potential functional mechanism underlying this genetic association by analyzing the correlation among single nucleotide polymorphisms (SNPs) and DNA methylation (DNAm) levels as cis methylation quantitative trait loci (cis-mQTL) within this region. A total of 2,611 SNPs were tested against 2,647 DNAm sites, in a subset of 37 chronic Chagas cardiomyopathy patients and 20 asymptomatic individuals from the GWAS. We identified 6,958 significant cis-mQTLs (False Discovery Rate [FDR]<0.05) at 1 Mb each side of the GWAS leading variant, where six of them potentially modulate the expression of the SAC3D1 gene, the reported gene in the previous GWAS. In addition, a total of 268 cis-mQTLs showed differential methylation between chronic Chagas cardiomyopathy patients and asymptomatic individuals. The most significant cis-mQTLs mapped in the gene bodies of POLA2 (FDR = 1.04x10-11), PLAAT3 (FDR = 7.22x10-03), and CCDC88B (FDR = 1.89x10-02) that have been associated with cardiovascular and hematological traits in previous studies. One of the most relevant interactions correlated with hypermethylation of CCDC88B. This gene is involved in the inflammatory response, and its methylation and expression levels have been previously reported in Chagas cardiomyopathy. Our findings support the functional relevance of the previously associated genomic region, highlighting the regulation of novel genes that could play a role in the chronic cardiac form of the disease.We thank all the patients who participated in this study and the Medical team from Barcelona, Spain, for the sample recruitment. This research is part of the doctoral degree awarded to D.C.M., within the Biomedicine program from the University of Granada entitled “Genomic approaches to unravel the pathogenesis of Chagas disease”

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

Malaltia de Chagas; IL18Enfermedad de Chagas; IL18Chagas disease; IL18Host 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.This research was supported by grants from Ministerio de Ciencia y Tecnología de Córdoba (GRFT 2017, https://mincyt.cba.gov.ar/), Secretaría de Ciencia y Tecnología, Universidad Nacional de Córdoba, Argentina (https://www.unc.edu.ar/ciencia-y-tecnología/) and Red Iberoamericana de medicina genómica en enfermedad de Chagas - CYTED (http://www.cyted.org). Mariana Strauss performed the experimental work in this article during an internship at the Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, Granada, España. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Lack of Association of IL6 polymorphism with the susceptibility to Chagas disease in Latin American cohorts

The aim of the present study was to analyze IL6 rs1800795 genetic variant in the susceptibility to Trypanosoma cruzi infection and in the development of chronic Chagas cardiomyopathy (CCC), in four independent Latin America cohorts. A total of 3,087 individuals from Latin American countries (Colombia, Bolivia, Argentina, and Peru) were studied. In all cohorts, patients were classified as seropositive for T. cruzi antigens (n= 1,963) and seronegative (n=1,124). Based on clinical evaluation, the seropositive patients, were classified as CCC (n= 900) and asymptomatic (n=1,063). No statistically significant differences in the frequency of IL6 rs1800795 between seropositive and seronegative, or between CCC and asymptomatic patients, were found. Furthermore, after the meta-analysis of these cohorts, no statistically significant differences were observed. Our results do not support a contribution of IL6 rs1800795 genetic variant in the susceptibility to the infection and the development of chronic Chagas cardiomyopathy, in the studied cohorts.Fil: Strauss, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Casares-Marfil, Desiré. Instituto de Parasitología y Biomedicina López-Neyra, Granada; EspañaFil: Alcaráz, Alexia. Instituto de Parasitología y Biomedicina López-Neyra, Granada; EspañaFil: Palma Vega, Miriam. Instituto de Parasitología y Biomedicina López-Neyra, Granada; EspañaFil: Pau Bosch, Nicolau. Unidad de Medicina Tropical y Salud Internacional Hospital Universitari Valld'Hebron; EspañaFil: Lo Presti, Maria Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Molina, Israel. Unidad de Medicina Tropical y Salud Internacional Hospital Universitari Valld'Hebron; EspañaFil: González, Clara Isabel. Universidad Industrial de Santander, Bucaramanga; ColombiaFil: Martín, Javier. Instituto de Parasitología y Biomedicina López-Neyra, Granada; EspañaFil: Acosta-Herrera, Marialbert. Instituto de Parasitología y Biomedicina López-Neyra, Granada; Españ