Blood Gene Signatures of Chagas Cardiomyopathy With or Without Ventricular Dysfunction

Abstract

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects 7 million people in Latin American areas of endemicity. About 30% of infected patients will develop chronic Chagas cardiomyopathy (CCC), an inflammatory cardiomyopathy characterized by hypertrophy, fibrosis, and myocarditis. Further studies are necessary to understand the molecular mechanisms of disease progression. Transcriptome analysis has been increasingly used to identify molecular changes associated with disease outcomes. We thus assessed the whole-blood transcriptome of patients with Chagas disease. Microarray analysis was performed on blood samples from 150 subjects, of whom 30 were uninfected control patients and 120 had Chagas disease (1 group had asymptomatic disease, and 2 groups had CCC with either a preserved or reduced left ventricular ejection fraction [LVEF]). Each Chagas disease group displayed distinct gene expression and functional pathway profiles. The most different expression patterns were between CCC groups with a preserved or reduced LVEF. A more stringent analysis indicated that 27 differentially expressed genes, particularly those related to natural killer (NK)/CD8+ T-cell cytotoxicity, separated the 2 groups. NK/CD8+ T-cell cytotoxicity could play a role in determining Chagas disease progression. Understanding genes associated with disease may lead to improved insight into CCC pathogenesis and the identification of prognostic factors for CCC progression.

Keywords: Cardiomyopathy; Chagas disease; NK cells.; whole-blood transcriptome.

Figure 1.

Whole-blood microarray analysis of patients with Chagas disease. Whole-blood transcriptome for 150 patients (30 per group) assigned to 4 different clinical/parasitological groups based on Chagas disease and Trypanosoma cruzi polymerase chain reaction (PCR) status, as well as seronegative controls, was assessed by microarray analysis. All possible comparisons between any 2 groups (10 comparisons in total) were performed to identify genes highly expressed in one of the groups. Genes were considered highly expressed in the group if they were upregulated in at least 2 comparisons (P < .05; fold change, >1.25) in the control group, the asymptomatic (ASY), PCR-negative group, and the ASY PCR-positive group. Abbreviations: CCC, chronic Chagas cardiomyopathy; FC, fold change; Mod, moderate; Sev, severe; −, negative; +, positive.

Figure 2.

Findings of gene set enrichment analysis. Corrplot analysis of gene expression results shows blood transcription modules and functional pathways that have increased (red) or reduced activated (blue) in the peripheral blood of each subject group. Abbreviations: CCC, chronic Chagas cardiomyopathy; IFN, interferon; MHC, major histocompatibility complex; Mod, moderate; NES, normalized enrichment score; NK, natural killer; Sev, severe; TLR, Toll-like receptor; −, negative; +, positive.

Figure 3.

Findings of network and pathway analysis. Ingenuity Pathway Analysis was performed with the list containing the 27 differentially expressed genes capable of segregating patients with moderate chronic Chagas cardiomyopathy (CCC) from those with severe CCC on the basis of their respective fold changes and P values. The most significant network generated was related to cell death and survival, hematological disease, immunological disease (P = 10^–39). Genes upregulated in moderate as compared to severe CCC are depicted in red, while downregulated genes are depicted in green.

Figure 4.

Validation of microarray results by real-time quantitative reverse transcription polymerase chain reaction analysis (qRT-PCR). Real-time qRT-PCR assays for 7 genes belonging to the natural killer (NK)/CD8⁺ T-cell cytotoxicity pathways: GZMB (which encodes granzyme B), PRF1 (perforin 1), GLNY (granulysin), KIR2DL1/KIR2DL3 (killer immunoglobulin-like receptor genes), and KLRC2 and KLRF1 (killer cell lectin-like receptor genes). The fold change was calculated as the mean ± SD for each group as individual data points, by the 2^−ΔΔCt method. *P < .05, **P < .01, and ***P < .0001 by the Mann–Whitney rank sum test.