A novel genetic association of IL32 with tuberculosis

IL32 is a pleiotropic intracellular cytokine with an emergent role in tuberculosis. The different isoforms of IL32: α, β, γ and δ have varying pro and anti-inflammatory potentials. We studied the role of genetic variants of IL32 and its isoforms in susceptibility to tuberculosis using a case-household contact association study.

Using a targeted sequencing approach, IL32 (+1kb) gene was sequenced in 64 pairs of culture positive TB cases and their culture negative household contacts. Subsequently the identified variants were validated in an independent cohort of cases and household contacts using TaqMan genotyping assay. Regulatory role of the associated variants was assessed using GTExPortal, RegulomeDB score, HaploReg and ENCODE histone ChIP-seq data. Expression of IL32 and its isoforms was evaluated by RT-PCR in PBMC from unexposed healthy controls (N = 25) with different genotype background and stimulated with TB antigens ESAT6 and CFP10. ∼ 200 bp around the associated variant was cloned into pGL3 promoter vector to assess enhancer activity by dual luciferase assay in cell lines.

Intronic variant rs9927163(G/T) was found associated with pulmonary TB, T being the risk allele (OR = 2.3(1.40-3.83, p = 0.03)), while G is the protective allele. This finding was validated in independent set of TB cases and household contacts (p = 0.0435). rs9927163 is an eQTL for the genes IL32 (p = 4.1e-10) and BICDL2 (p = 2.1e-7) in whole blood and interrupts an AP-1 binding site. ENCODE histone ChIP-seq data shows rs9927163 residing within T cell specific H3K4me3 peak. The G allele is associated with greater enhancer activity in a T cell line (2.12 fold, p = 0.0059). The TT genotype showed greater normalized expression of IL32δ, a less proinflammatory isoform compared to the GT and GG genotypes together following ESAT6 (p = 0.02288) and CFP10 (p = 0.04595) treatment. This indicates that greater expression of a potentially less protective IL32 isoform within individuals with the TT genotype might be a risk factor for developing TB.