The present study was approved by the Ethics Committee of University of Khartoum, Khartoum, Sudan (5/2018). This study adheres to the Declaration of Helsinki (1964). Written informed consents were obtained from all participants in the study or legally responsible guardians for participants less than 18 years old.
Genomic DNA was isolated from blood samples with the large volume kit for the MagNA Pure system (Roche, Almere, The Netherlands) according to the manufacturer's descriptions. The isolated DNA was stored at -20 C.
Genomic variants of TLR1 (rs5743604, rs5743611, rs5743618, rs76600635, rs5743551, rs5743557, rs5743565, rs5743566, rs5743580, rs5743594 rs4833095, rs5743595, rs5743596); TLR2 (rs1816702, rs5743704 rs5743708, rs7656411, rs11938228, rs893629, rs1898830, rs121917864, rs4696480, rs3804099, rs5743699 rs3804100); TLR4 (rs7869402, rs1927907, rs1927911, rs1927914, rs6478317, rs55912718, rs5030719, rs10759931, rs10759933, rs2770150, rs1554973, rs11536878, rs11536879, rs7873784, rs11536889, rs4986790, rs4986791, rs11536897, rs11536898); TLR6 (rs3796508, rs5743810, rs5743831, rs1039559, rs6531670, rs5743788, rs5743794); TLR8 (rs4830805, rs4830808, rs3747414, rs3761624, rs1548731, rs2109134, rs3788935, rs1013150, rs5744068, rs3764879, rs5744080, rs3764880, rs17256081, rs5741883, rs2407992, rs178998); TLR9 (rs5743836, rs164637, rs352139, rs352140, rs352143, rs352162, rs352165, rs352167, rs187084); and TLR10 (rs4129009, rs7694115, rs10856839, rs11466645, rs4274855, rs11096955, rs11096956, rs11096957, rs7698870, and rs10776483) genes were detected by PCR followed by restriction enzyme fragment analysis (PCR-RFLP) (Figures 1 and 2). All PCR primers are stated in Table 2. Each of the PCRs consisted of a pre-denaturation step of 4 minutes at 94 C and 40 cycles each of 30 seconds denaturation at 94 C, 30 seconds annealing at 55 C and 30 seconds elongation at 72 C. This was followed by a post-elongation step of 7 minutes at 72 C. Restriction endonucleases were selected using the NEBcutter software (http://nc2.neb.com/NEBcutter2/). Restriction endonucleases were obtained from Fermentas (st. Leon-rot, Germany), and Roche (Penzberg, Germany) and were used as described by the manufacturer. Restriction fragments were visualized by electrophoresis on 2% agarose gels (Hispanagar, Sphaero Q, Leiden, The Netherlands).
The criteria for the selection of SNPs for this study were based on their previously reported to change the level or function of corresponding gene products and influence susceptibility/resistance to infections. Also, the selection was based on the publically available information on the polymorphisms of TLR genes available in the 1000 Genomes project for the South Asian population (http://www.internationalgenome.org).
The mean age of the patient population and the control population were compared by the unpaired t-test. Gender, occupation and BCG-vaccination status between the patient and control population were compared with the Fisher exact test. Verification of Hardy-Weinberg equilibrium (HWE) was performed with Pearson's chi2 test. The effect of the TLR1; TLR2; TLR4; TLR6; TLR8; TLR9; and TLR10 polymorphisms on susceptibility to tuberculosis were assessed with the Fisher exact test. P-value of <0.05 was deemed statistically significant. All statistical analyses were performed using SPSS for Windows v11.0 statistical analysis software. This study was in accordance with the principles of the Helsinki Declaration (1964). This study was in accordance with the principles of the Helsinki Declaration (1964) This study was in accordance with the principles of the Helsinki Declaration (1964)