Extended IL10 haplotypes and their association with HIV progression to AIDS

Abstract

Interleukin-10 (IL-10) is a pleiotropic cytokine with both immunosuppressive and immunostimulatory functions. Its roles in infections and autoimmunity may have resulted in selective pressures on polymorphisms within the gene, leading to genomic coexistence of several semi-conserved haplotypes involved with diverse pathogen interactions during genomic evolution. Previous studies focused either exclusively on promoter haplotypes or on individual SNPs. We genotyped 21 single nucleotide polymorphisms in the human IL10 gene and examined this variation compared to other mammalian species sequences. Haplotype heterogeneity in human populations is centered around 'classic' 'proximal' promoter polymorphisms: -592, -819 and -1082. High-producing GCC haplotypes are by far the most numerous and diverse group, the intermediate IL-10 producing ACC-inclusive haplotypes seem to be related most closely to the ancestral haplotype, and the ATA-inclusive haplotypes cluster a separate branch with strong bootstrap support. We looked at associations of corresponding haplotypes with HIV progression. A haplotype trend regression confirmed that individuals carrying the low-producing ATA-inclusive haplotypes in European Americans progress to AIDS faster, and most likely explain the role of IL10. Our findings are consistent with the hypothesis that existing polymorphisms in this gene may reflect a balance of historic adaptive responses to autoimmune, infectious and other disease agents.

Figure 1

Evolutionary conserved regions (ECRs) plot generated by ECR Browser, comparing the genomic region of IL10 between rhesus monkey, dog, mouse, rat and opossum (http://ecrbrowser.dcode.org/). A map of SNPs (corresponding to Table 1) genotyped in the IL10 region (a) is superimposed onto the conservation profile created by aligning the human sequence with five mammalian species (b) in a pairwise fashion. The three ‘proximal’ promoter haplotype SNPs are marked by dark purple dots above the circled SNP numbers that correspond to Table 1. Similarly, ‘distal’ haplotype SNPs are marked by light pink dots above the circled SNP numbers. ECRs are identified as regions of high sequence identity against a neutrally evolving background and indicated as horizontal red bars (minimum sequence identity 77%, minimum length 350 bp). Exons are presented in blue, introns in red, untranslated regions (UTRs) in orange and intergenic sequences in light blue shading. Repetitive elements in humans are vertical light gray bars.

Figure 2

Neighbor-joining tree of IL10 gene haplotypes^† built using distances based on the nucleotide differences between 21 known haplotype segregating sites in humans^‡. ACC is likely to be the ancestral (underlined) since four ACC-inclusive haplotypes are distributed at the base of the tree, separate from the very long derived ATA-inclusive clade. The GCC-inclusive haplotypes form another derived, but more diverse clade, with the exception of GCC-7, whose position on the tree is yet unresolved. ‘Distal’ haplotypes are dispersed among the clades (See Supplementary Table S2). ^†Haplotype names correspond to those used in Table 1. ^‡The haplotype tree was inferred with the neighborhood joining method using 10 000 bootstrap replications, under the assumption of equal base frequency and the number of nucleotide differences as distance measure (MEGA 3.1). The tree was rooted using the entire IL10 homologous sequence from Pan troglodytes and Macaca mulatta (not shown). The two primate sequences were aligned using the sequence of the flanking regions around the haplotype-defined SNPs in the reference sequence of human IL10 (Build 36). Both P. troglodytes and M. mulatta had ACC-inclusive haplotypes in their IL10 promoter region based on the two known sequences.

Figure 3

Relationship between haplotype phylogeny and relative hazards for progression to AIDS-1987^†. Reduced median haplotype network for the IL10 gene haplotypes^‡ was built using 21 SNPs in (a) European Americans and (b) African Americans. In both diagrams ACC1 is used as a reference, as it was the most frequent haplotype and the best approximation to the inferred ancestral haplotype. The ATA1 in European American (AIDS-1987) sero-converters shows higher hazard ratio compared with any other haplotypes (Tables 2 and 3). No haplotype effect is observed among African Americans. ^†The networks are rooted by the inferred ancestral haplotype derived by comparison of haplotypes from Pan troglodytes and Macaca mulatta. The inferred ancestral haplotype was not observed among human haplotypes in this study. Distance is indicated by tick marks on branches that correspond to the number of allelic changes between sequential haplotypes. The colors correspond to the groups based on the existence of ‘proximal’ IL10-promoter haplotypes: GCC (green), ACC (blue) and ATA (orange). Confidence intervals for relative hazards for each of the haplotypes are shown relative to their position in the haplotype network and with size of the circles respective to their population frequency in sero-converters. ^‡Haplotype names correspond to those used in Table 1.

Figure 4

Kaplan–Meier analysis of progression from HIV-1 sero-conversion to clinical AIDS-1987 (AIDS-defining illness) and AIDS-1993 (AIDS-defining illness or CD4⁺ T-cell counts <200 cells/mm³) by IL10 haplotypes among treatment-free European Americans (a–f, n = 649) and African Americans (g, h, n = 339) from the three AIDS cohorts. ATA haplotype carrying HIV-positive European Americans progress to AIDS faster than European-American individuals carrying any other haplotypes regardless if they are analyzed as 21 SNP-extended haplotypes (a and b), collapsed into ‘classical’ ‘proximal’ haplotypes (c and d), or expanded to include pairs of extended haplotypes (that is, haplotypic genotypes) (e and f) (Table 2). (a) Kaplan–Meier survival curves with AIDS-1987 as end point by all haplotype categories (EA). (b) Kaplan–Meier survival curves with AIDS-1993 as end point by all haplotype categories (EA). (c) Kaplan–Meier survival curves with AIDS-1987 as end point by the classical ‘proximal’ ATA, ACC and GCC haplotype categories (EA). (d) Kaplan–Meier survival curves with AIDS-1993 as end point by the ‘proximal’ ATA, ACC and GCC haplotype categories (EA). (e) Kaplan–Meier survival curves with AIDS-1987 as end point by haplotype pairs, ATA/ATA, ATA/ACC, ATA/GCC, ACC/GCC, ACC/ACC and GCC/GCC (EA). (f) Kaplan–Meier survival curves with AIDS-1993 as end point by haplotype pairs, ATA/ATA, ATA/ACC, ATA/GCC, ACC/GCC. ACC/ACC and GCC/GCC. (g) Kaplan–Meier survival curves with AIDS-1987 as end point by all haplotype categories in African Americans. (h) Kaplan–Meier survival curves with AIDS-1987 as end point by the classical ATA, ACC and GCC haplotype categories in African Americans.