Genetic association of Toll-like-receptor 4 and tumor necrosis factor-α polymorphisms with Plasmodium falciparum blood infection levels

Dysregulated innate immune responses due to inappropriate signaling by Toll-like receptors (TLRs) and aberrant production of pro-inflammatory cytokines are implicated in the immunopathology and disease outcome in Plasmodium falciparum malaria. This study investigates the relationship between polymorphic variability of candidate genes including TLR-2, -4, -9, tumor necrosis factor-α and lymphotoxin-α and blood infection level in Indian mild malaria patients. Genotyping was carried out by PCR-RFLP and sequencing. Association of parasite load with genotypes was examined using model based and model free approaches. Allele and haplotype based risk assessment for disease severity was performed by stratifying the patients into high and low parasitemic groups on the basis of a threshold value derived by employing a two-component mixture model and expectation-maximization algorithm. The mean parasitemia was significantly increased for variant homozygous genotype (C/C) at TNF-α promoter −1031 and major homozygous genotypes encoding Asp/Asp and Thr/Thr at codons 299 and 399, respectively, on TLR4 polypeptide. Individuals harboring combined genotype C/C-Asp/Asp-Thr/Thr on TNF-α and TLR4 presented the highest parasite load. The frequencies of variant allele C in TNF-1031 (OR = 1.91 with 95% CI = 1.24–2.94) and TNF-α promoter haplotypes C-C-G-G (OR = 1.99 with 95% CI = 1.21–3.27) and C-C-G-A (OR = 2.96 with 95% CI = 1.19–7.37) pertaining to loci TNF-1031/-857/-308/-238 were significantly elevated in the high parasitemic group. On the contrary, the frequencies of variant allele encoding Ile at 399 (OR = 0.55 with 95% CI = 0.32–0.94) and haplotype corresponding to Gly-Ile (299–399) (OR = 0.51 with 95% CI = 0.28–0.9) in TLR4 were higher in low parasitemic group. In silico analysis indicate differential binding of transcription factors to TNF-α promoter haplotypes and alteration in the surface charge distribution of the TLR4 variant proteins. Our results support a genetic role of TLR4 and TNF-α in controlling the blood infection level in mild malaria.