Full-exon resequencing reveals toll-like receptor variants contribute to human susceptibility to tuberculosis disease

Abstract

Tuberculosis (TB) is the leading cause of death worldwide due to an infectious agent. Data have accumulated over decades suggesting that variability in human susceptibility to TB disease has a genetic component. Toll-like receptors (TLRs) play a critical role in initiating the innate immune response to many pathogens in mouse models, but little is known about their role in human infections. Human TLRs have been reported to recognize mycobacterial antigens and initiate an immune response. We tested the hypothesis that amino acid-altering polymorphisms in five TLRs were associated with susceptibility to TB disease using a population-based case-control study with 1,312 adult TB patients and controls. Full-coding region sequencing of the five TLR genes in all 1,312 subjects yielded a data set in excess of 16 Mb. Rare nonsynonymous polymorphisms in TLR6-TLR1-TLR10 were significantly overrepresented among African-American TB cases compared with ethnically-matched control subjects. Common nonsynonymous polymorphisms in TLR6-TLR1-TLR10 also were significantly associated with TB disease in certain ethnic groups. Among African Americans, homozygotes for the common-variant haplotype TLR1-248S, TLR1-602I, and TLR6-249S had a significantly increased TB disease risk. A transmission/disequilibrium test on an independent sample found that the TLR1-248S variant was preferentially transmitted to diseased children, thereby confirming disease association. These results are consistent with recent reports implicating TLR1 variants, including TLR1-602, in significantly altered innate immune responses. Also consistent with disease association, rare TLR6 variants were defective in their ability to mediate NF-kappaB signal transduction in transfected human cells. Taken together, the data suggest that variant TLRs contribute to human susceptibility to TB disease. Extensive full-exon resequencing was critical for revealing new information about the role of TLRs in human-pathogen interactions and the genetic basis of innate immune function.

Figure 1. Schematic of the TLR proteins and sequence variants.

(A) Schematic of human chromosome 4 showing location of TLR genes. (B) Summary of variants identified in five TLR proteins. Shown are all variant sites that would be made by the TLR alleles identified in either the TB patients or control subjects. Black, rare variant; red, variants that are common in all three ethnic groups analyzed. SG, signal peptide; TM, transmembrane domain; TIR, Toll/IL-1 receptor domain; NSP, nonsynonymous polymorphisms (includes nonsynonymous single nucleotide polymorphisms, nonsense mutations, and insertion and deletions). Numbers in parentheses denote amino acid residue positions.

Figure 2. Altered NF-κB signal transduction by rare variants of TLR6.

HEK 293 cells were transiently co-transfected with TLR2 and the indicated TLR6 variant expression plasmids, NF-κB reporter (firefly luciferase), and β-actin Renilla luciferase plasmids. NF-κB activation in transfected cells was measured 24 hrs post transfection. The data shown are the mean ± SD from 4 independent experiments expressed as the percent relative firefly luciferase activity (RLU) (normalized to Renilla luciferase activity) induced in cells co-expressing TLR2 and WT TLR6. * P<0.01; ** P<0.001 compared to TLR6 WT NF-κB activity.