Association of IFN-γ +874 A/T SNP and hypermethylation of the -53 CpG site with tuberculosis susceptibility

Abstract

Introduction: Tuberculosis (TB) is now the 2nd leading infectious killer after COVID-19 and the 13th leading cause of death worldwide. Moreover, TB is a lethal combination for HIV-patients. Th1 responses and particularly IFN-γ are crucial for immune protection against Mycobacterium tuberculosis infection. Many gene variants for IFNG that confer susceptibility to TB have been described in multiple ethnic populations. Likewise, some epigenetic modifications have been evaluated, being CpG methylation the major epigenetic mark that makes chromatin inaccessible to transcription factors, thus avoiding the initiation of IFNG transcription.

Methods: We evaluated both genetic and epigenetic changes involved in IFN-γ production and TB susceptibility in Argentine population. Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was performed for the IFN-γ +874 A/T polymorphism (rs2430561) genotyping in 199 healthy donors (HD) and 173 tuberculosis (TB) patients. IFN-γ levels from M. tuberculosis-stimulated PBMCs were measured by ELISA. The methylation status at the -53 CpG site of the IFNG promoter in individuals with latent infection (LTBI), TB and HD was determine by pyrosequencing.

Results: Using a case-control study, we found that A allele and, consequently, AA genotype were overrepresented in patients with active disease. Moreover, HD carrying T allele (AT or TT genotype) evidenced an augmented IFN-γ secretion compared to TB patients. Codominance was the genetic model that best fits our results according to the Akaike information criterion (AIC). In addition, increased methylation levels at the -53 CpG site in the IFN-γ promoter were observed in whole blood of patients with active TB compared to LTBI individuals.

Discussion: IFN-γ is regulated by genetic variants and epigenetic modifications during TB. Besides, AA genotype of the rs2430561 single nucleotide polymorphism could be considered as a potential TB susceptibility genetic biomarker in Argentina and the methylation of the -53 CpG site could result in a useful predictor of TB reactivation.

Keywords: IFNg; SNPs; methylation; susceptibility; tuberculosis.

Figure 1

Distribution of genotype and allele frequencies of IFN-γ gene (+874 A/T) among patients with tuberculosis (TB) and Healthy Donors (HD).

Figure 2

Production of IFN-γ by PBMCs from HD and TB patients carrying the genotypic variants of the IFN-γ gene (+874 A/T). PBMCs from healthy donors (HD, n = 63) and TB patients (TB, n = 102) carrying the different genotypes of the IFN-γ gene (+874 A/T) SNP were stimulated for five days with M. tuberculosis-Ag, and IFN-γ production was determined by ELISA. P values were calculated by one way ANOVA and Tukey’s multiple comparison test. ** p < 0.01 between HD genotypes. # p < 0.05 between HD and TB patients AT/TT genotypes. ns, not signficant.

Figure 3

Methylation status of the -53 CpG of IFNG promoter in whole blood. Genomic DNA was obtained from whole blood from HD, TB patients and LTBI. Bisulfite converted-DNA was amplified by PCR and pyrosequencing was performed to determine the degree of methylation at the -53 CpG site of the IFNG promoter. (A) P values were calculated by one way ANOVA for non-parametric data and Dunn’s multiple comparison test. (B) A representative pyrogram is shown.

Figure 4

ROC curve analyses for evaluation of the predictive value of -53 CpG methylation on disease status. ROC curve analyses for evaluation of the predictive value of whole blood methylation levels at IFNG -53 CpG for differentiating HD individuals from TB, HD individual from LTBI and TB from LTBI. ROC, receiver operating characteristic; AUC, area under the ROC curve.