High producer haplotype (CAG) of -863C/A, -308G/A and -238G/A polymorphisms in the promoter region of TNF-α gene associate with enhanced apoptosis of lymphocytes in HIV-1 subtype C infected individuals from North India

Abstract

Introduction: The natural history of HIV-1 infection and its progression towards AIDS vary considerably among individuals. Host genetic factors may be one of the possible reasons for variable HIV-1 disease progression. Single nucleotide polymorphisms (SNPs) in the promoter region of TNF-α gene can influence its production. The aim of the present study was to determine the association of functional TNF-α SNPs and its associated parameters related to apoptosis that may influence the rate of HIV-1 disease progression.

Methods: Therapy naive, 100 HIV slow progressors (SPs), 100 HIV fast progressors (FPs), 50 HIV exposed but seronegative individuals (ESNs) and 260 healthy controls from same ethnic origin were recruited. Genotyping of TNF-α variants (-863C/A, -308G/A and -238G/A) was done using PCR-RFLP. CD4 counts were determined by flow cytometry. Plasma viral load was estimated by COBAS AMPLICOR HIV-1 monitor test. Plasma TNF-α concentration was estimated by Human CBA Th1/Th2 cytokine kit. The lymphocyte mitochondrial membrane potential was measured by JC-1 dye by flow cytometry.

Results: Genotype and allele frequency of TNF-α -238G/A and -863C/A was not significantly different in HIV-1-infected patients when compared to controls, while that of TNF-α -308G/A variant (high TNF-α producer) was significantly higher in FPs compared to SPs (p<0.01, OR = 3.43). Haplotype analyses also showed that carriers of high TNF-α producing haplotype CAG was significantly more common among FPs compared to SPs (p<0.01, OR = 3). The circulating TNF-α levels in blood also correlated well with genotypes. The lymphocyte mitochondrial membrane potential of FPs having CAG haplotype was significantly low as compared to wild type (CGG) haplotype (417±22 vs 571±28, p<0.01).

Conclusion: High producer haplotype, CAG of TNF-α gene associates with enhanced apoptosis of lymphocytes in HIV-1 infected individuals, hence faster progression to AIDS. However, further functional studies are needed to confirm this association and this knowledge may help clinicians to better understand the disease outcome.

Figure 1. Restriction fragment length polymorphism (RFLP) analysis of PCR products.

(A) Representative ethidium bromide stained (15%) non-denaturing polyacrylamide gel (PAGE) showing amplified gene product of TNF-α (−238G/A) 118 bp uncut and RFLP pattern observed after digestion with BglII. (Lane M -20 bp ladder, Lane 2 and 8-GA genotype, Lane 3 to 7-GG Genotype. (B) Representative non-denaturing PAGE (15%) showing amplified gene segment of TNF-α (−308G/A) 118 bp uncut and RFLP pattern observed after digestion with NcoI. Lane 1-uncut 118 bp product, Lane 2 and 6-GG Genotype, Lane 3 and 5-GA genotype, Lane 4-AA Genotype, Lane M-20 bp Ladder. (C) Representative non-denaturing PAGE (15%) showing amplified gene segment of TNF-α (−863C/A) 126 bp uncut and RFLP pattern observed after digestion with BsaAI. Lane 1-Negative Control, Lane 2-uncut 126 bp, Lane 3-AA Genotype, Lane 4-CA Genotype, Lane 5, 8 and 9-CC Genotype, Lane 6 and 7-CA Genotype, Lane M-50 bp ladder.

Figure 2. Plasma concentrations of TNF-α in various study groups.

Plasma concentrations of TNF-α (Mean±SEM) in FPs, SPs and ESNs. High TNF-α producing haplotype CAG (dark bar), medium TNF-α producer haplotype CGG (light gray bar) and low TNF-α producing haplotype AGG (gray bar).

Figure 3. Plasma viral load and CD4 counts in different TNF-α haplotypes (CGG, CAG and AGG).

(A) Plasma Log₁₀ RNA copies/mL (Mean±Range) in TNF-α haplotypes, CGG (dark bar), CAG (light gray bar) and AGG (gray bar). (B) CD4 counts at different time intervals in TNF-α haplotypes, CGG (dark bar), CAG (light gray bar) and AGG (gray bar). Values shown as (Median±SD). Significance levels: **p<0.01; *p<0.05.

Figure 4. Gating strategy for the analysis of JC-1 stained PBMCs.

Lymphocytes were gated according to morphological parameters (not shown in figure), JC-1 aggregates and monomers were analyzed on FL2 (PE) and FL1 (FITC) channel respectively, (a) showing JC-1 stained lymphocytes of HCs (P2 gate, 96.4%), (b) protonophore FCCP treated lymphocytes as a positive control showing 84.2% cells having reduced mitochondrial membrane potential. (c) and (d) JC-1 stained lymphocytes in a representative samples from individuals in ‘fast progressors’ group having CGG and CAG haplotypes showing 26% and 37% lymphocytes dying respectively. Change in mitochondrial membrane potential (Δψm) was expressed as percentage of median fluorescence intensity (FL2: FL1/FL2 FCCP: FL1 FCCP) x100.

Figure 5. Median fluorescence intensities of total lymphocyte mitochondrial membrane potential (Δψm).

Bar diagram showing median fluorescence intensity (MFI) of total lymphocyte mitochondrial membrane potential (Δψm) in FPs and SPs having CAG (n = 5) (dark bar) and CGG (n = 5) (light gray bar) haplotypes (shown as Mean±SEM).