Association of <i>IL10</i> Polymorphisms and Leprosy: A Meta-Analysis

<div><p>Leprosy is a chronic infectious disease that depends on the interplay of several factors. Single nucleotide polymorphisms (SNPs) in host immune related genes have been consistently suggested as participants in susceptibility towards disease. Interleukin-10 (IL-10) is a crucial immunomodulatory cytokine in mycobacterial pathogenesis and especially the -819C>T SNP (rs1800871) has been tested in several case-control studies indicating association with leprosy risk, although a recent consensus estimate is still missing. In this study, we evaluated the association of the -819C>T SNP and leprosy in two new Brazilian family-based populations. Then, we performed meta-analysis for this polymorphism summarizing published studies including these Brazilian family-based groups. Finally, we also retrieved published studies for other distal and proximal <i>IL10</i> polymorphisms: -3575 T>A (rs1800890), -2849 G>A (rs6703630), -2763 C>A (rs6693899), -1082 G>A (rs1800896) and -592 C>A (rs1800872). Results from meta-analysis supported a significant susceptibility association for the -819T allele, with pooled Odds Ratio of 1.22 (CI = 1.11–1.34) and <i>P</i>-value = 3x10^–5 confirming previous data. This result remained unaltered after inclusion of the Brazilian family-based groups (OR = 1.2, CI = 1.10–1.31, <i>P-</i>value = 2x10^–5). Also, meta-analysis confirmed association of -592 A allele and leprosy outcome (OR = 1.24, CI = 1.03–1.50, <i>P</i>-value = 0.02). In support of this, linkage disequilibrium analysis in 1000 genomes AFR, EUR, ASN and AMR populations pointed to r² = 1.0 between the -592C>A and -819C>T SNPs. We found no evidence of association for the other <i>IL10</i> polymorphisms analyzed for leprosy outcome. Our results reinforce the role of the -819C>T as a tag SNP (rs1800871) and its association with leprosy susceptibility.</p></div

Characteristics for the Brazilian family-based studies.

<p>Abbreviations: SD, standard deviation; WHO, World Health Organization.</p><p>^a Data is presented as total counts (frequency). The number of subject counts in ethnicity and WHO classification can differ from total individuals due to missing information.</p><p>Characteristics for the Brazilian family-based studies.</p

Forest plots summarizing association of <i>IL10</i> promoter polymorphisms and leprosy.

<p><b>(A)</b> Forest plot for -592 C>A (rs1800872). Five case-control studies were evaluated under random-effects model. Bars represent 95% confidence interval and boxes represent OR values. <b>(B)</b> Forest plot for -819 C>T (rs1800871). Nine case-control studies were evaluated under random-effects model. Bars represent 95% confidence interval and boxes represent OR values.</p

Forest plot for -819 C>T (rs1800871) including Brazilian Family-based data (MG and RJ).

<p>Bars represent 95% confidence interval and boxes represent OR values.</p

Flow diagram of the process of identification of eligible studies.

<p>Flow diagram of the process of identification of eligible studies.</p

Genetic association of the miRSNP-196a-2 and miRSNP-146a in the Rio de Janeiro sample: a case-control study of leprosy <i>per se</i>.

<p>Population counts are shown as N (frequency).</p><p>*Adjusted for sex, ethnicity and age.</p>b<p>Genotype or allele used as baseline.</p><p>Global p-value to miRSNP-146a p = 0.002. Trend test: miRSNP-146a <i>C</i>-allele, χ² = 96.6; p = 2.2⁻¹⁶/case-control comparison. HWE of control population: miRSNP-196a-2; χ² = 0.2; p = 0.65 and miRSNP-146a; χ² = 0.95; p = 0.34.</p><p>Genetic association of the miRSNP-196a-2 and miRSNP-146a in the Rio de Janeiro sample: a case-control study of leprosy <i>per se</i>.</p

MiR-146a expression in nerve and skin biopsies, and according to patients' genotype (rs2910164).

<p>MiR-146a mature form expression was analyzed in nerve (A, B) and skin (C, D) specimens by real-time stem-loop RT-PCR after total RNA extraction. RNU48 expression was used to normalization. (A) Analysis was performed comparing the levels of miR-146a expression in nerve biopsies (NL = not leprosy, n = 7/Leprosy, n = 12). (B) MiR-146a expression stratified by rs2910164/G>C genotype, <i>GG</i> (n = 8) and <i>GC</i> (n = 7). The data show mean ± SEM (*p = 0.0099; and **p = 0.0401) (C) Results show neither difference of miR-146a expression in skin biopsies according to clinical forms (PB = 37 and MB = 17), nor by genotype stratification (<i>GG</i> = 10; <i>GC</i> = 22; <i>CC</i> = 4) (D).</p

MiR-146a expression in cells exposed to <i>M. leprae</i>.

<p>Macrophage-like THP-1 cells (5×10⁵) were infected with live <i>M. leprae</i> (MOI 10∶1, 100∶1) for 3, 24 and 48 h at 33°C. RNA was extracted and a real-time stem-loop RT-PCR was performed using RNU48 to normalize. Data show mean ± SEM (*p<0.05 relative to 24 h control, **p<0.05 relative to 48 h control). Results represent four independent experiments.</p

TNF expression and secretion according to the host genotypes (rs2910164).

<p>(A) TNF expression from nerve biopsies was assessed by qRT-PCR after RNA isolation. Standardized expression values are shown. Control genotype (<i>GG</i>) = 12 and risk genotype (<i>GC</i>+<i>CC</i>) = 15. Data show the mean ± SEM (*p = 0.0453). (B) To assess TNF secretion, PBMC (3×10⁶ cells) were infected with BCG Moreau (BCGm) at a MOI 10∶1 for 24 h at 37°C. Supernatant was collected and TNF concentration (pg/mL) was estimated by ELISA. TNF levels produced by infected cells were significantly lower in <i>C-</i>allele carriers (risk variant) when compared with infected cells with <i>GG</i> genotype. Lines represent the median of each group and the dots represent individual PBMC donors. (**p = 0.0352).</p

Family Based Association test of 97 family cores, n = 426 individuals from Duque de Caxias city.

<p>Family Based Association test of 97 family cores, n = 426 individuals from Duque de Caxias city.</p