by the low R2 values and non-significant P values in Table 1 under column designated as ‘Steady-state viral load’). Effects of HLA-C and HCP5 on disease progression Because of (i) their proximity to HLA-B*57, (ii) the previously reported dominant protective role of B*57 alleles in virologic control and disease progression, and (iii) the high degree of LD between the HLA-C59-C, HLA-B*57 and HCP5-G alleles (Figure 1B and Figure 1C, top), we determined whether the effects attributable to the SNPs in HLA-C and HCP5 were in part due to these LD patterns and their presence within HLA-B*57- containing genotypes. To this end, we used strategies that were complementary to those we used to clarify the relationship between ZNRD1 and HLA-A10 alleles. We first evaluated the associations and distributions separately for the HLA-B*57, HLA- C59 and HCP5 alleles (Figure 3), and then because of their LD patterns, we determined the associations for B*57-containing genotypes that contained or lacked HLA-C59 and HCP5 alleles (Figure 4). We took this analytical approach of first analyzing the genotype-disease-influencing phenotypes at the level of the haplotype and then haplotype pairs (genotypes), as ultimately the phenotypic effect is conveyed by the genotype. In univariate analyses, possession of a B*57 allele (Figure 3A, and Table 2, model 1), and heterozygosity and homozygosity for the HLA-C59-C allele (Figure 3B and Table 2, model 2) were each associated with significant protective effects on the rate of disease progression to AIDS. The impact of the HLA-C59-C allele on disease progression was greater in EAs than AAs (Figure 3B). In univariate analyses, the HCP5-G allele was associated with protective effects on disease progression, but this trend did not achieve statistical significance and was mostly restricted to EAs (Figure 3C and Table 2, model 3). Consistent with the results of the univariate analyses, with increasing AIDS-free status, there was a statistically significant step-wise increase in the proportion of subjects possessing a HLA- B*57 allele, including the B*5701 allele (Figure 3D). A similar statistically significant trend for a shift in distribution with increasing AIDS-free status was also observed for the HLA-C59- C allele (Figure 3D). Thus, compared to subjects who developed AIDS within 2 years, HIV+ subjects who were AIDS-free for more than 10 years were 12.7, 5.6 and 2.1 times more likely to possess a HLA-B*57, HLA-B*5701 and the HLA-C59-C allele, respectively (Table 3). Increasing AIDS-free status was also associated with a trend for enrichment of the HCP5-G allele although this association was not significant (Figure 3D and Table 3). It was noteworthy that the degree of enrichment of subjects with the HCP5-G allele with increasing AIDS-free status was similar to that observed for the HLA-B*5701 subtype (Figure 3D). This observation is consistent with the LD between HLA-B*5701 subtype and the HCP5-G allele (D’ = 0.91 for EAs and 0.73 for AAs, Figure 1B). When placed in a multivariate model with HLA-B*57, heterozygosity and homozygosity for the HLA-C59-C indepen- dently associated with disease-retardation (Table 2, compare models 2 and 4). However, the statistical significance of the protective effects associated with HLA-C59-C in multivariate models were slightly lower than those observed in univariate models (Table 2, compare models 2 and 4). This was most prominent for homozygosity for HLA-C59-C as the hazard ratios for the rate of progression to AIDS were 0.66 (P = 0.025) and 0.72 (P = 0.067) in univariate and multivariate analyses, respectively (compare models 2 and 4, Table 2). These results indicated that possibly because of its LD with HLA-B*57, the strength of the HLA, HCP5 and ZNRD1 in HIV disease-retarding effects associated with HLA-C59-C may in part be due to the effects conferred by the HLA-B*57 allele. In univariate analysis, the hazard ratio for the rate of disease progression to AIDS associated with the HCP5-G allele was ,1 (RH = 0.67, 95% CI = 0.39–1.14, P = 0.135), suggesting that this allele might convey weak disease-retarding effects (Table 2, model 3). However, when examined in the context of a multivariate model with B*57, the hazard ratio for the HCP5 allele was .1 (RH = 2.06; 95% CI = 0.98–4.33, P = 0.056; Table 2, model 5). Conversely, in the univariate analyses, the hazard ratio for rate of disease progression associated with the B*57 allele was 0.38 (P = 0.0002, Table 2, model 1), whereas when examined in the context of a multivariate model with HCP5-G, the hazard ratio was lower (RH = 0.25) and the strength of the association increased (P = 0.0001 Table 2, model 5). When HLA-C59-C heterozygosity and homozygosity were also added to the multivariate model (Table 2, model 6), the strength of the observed detrimental and beneficial effects associated with the HCP5-G and B*57 alleles, respectively, on disease progression became even further accen- tuated (Table 2, compare models 5 and 6). We inferred that the opposing direction of the disease- influencing effects associated with the HCP5-G allele in the univariate (RH,1, protective effects) and multivariate (RH .1, detrimental effects) analyses, and the accentuation of the protective effects of the B*57 in the multivariate models, convey the following: when examined at the haplotype level, those haplotypes which contain both B*57 and HCP5-G alleles are associated with a faster rate of disease progression compared to those B*57- containing haplotypes that bear the HCP5-T allele. Consequently, in multivariate models when the overall beneficial effects of B*57 are partitioned out or accounted for, the HCP5-G allele, which is in nearly 100% LD with a subset of HLA-B*57 alleles, associates with disease-acceleration. Conversely, in multivariate models when the effects of B*57/HCP5-G haplotypes are partitioned out, the overall beneficial effects associated with B*57 are accentuated. Concor- dant results (and inferences) were obtained when we studied the association of the HLA-C59–HLA-B–HCP5 haplotypes shown in Figure 1C with rates of disease progression (Figure S3). Influence of HLA-C and HCP5 on viral load We next examined whether the direction and the relative strength of the effects observed for the HLA-B*57, HCP5 and HLA- C59 alleles on rates of disease progression are similar to those observed when the steady-state viral load is used as a phenotypic endpoint. The hierarchal order of the extent to which the B*57, HLA-C and HCP5 alleles explained the variability in the steady- state viral load in the WHMC HIV+ cohort (R2 estimates as percentages are in parenthesis; Table 2) was: B*57 (3.2%; model 1).HLA-C (0.72%; model 2).HCP5 (0.38%; model 3). These findings indicated that at least within the context of a natural history cohort, the SNPs in HLA-C and HCP5 explained only one- third to one-tenth of the variability in steady-state viral load than the B*57 allele, and several fold less than the reported estimates in the GWAS [27]. To further evaluate the influence of these alleles on viral load, univariate and multivariate linear regression analyses were conducted in which these alleles were considered as independent variables and the steady-state viral load was considered as the dependent variable. Univariate analysis revealed that (i) a HLA- B*57 allele, (ii) one (heterozygosity) or two (homozygosity) copies of the HLA-C59-C allele, and (iii) a HCP5-G allele each associated with a lower steady-state viral load (as reflected by the negative values of the regression coefficients; Table 1, models 1 to 3). However, the viral load-mitigating effects observed for possession of the HCP5-G PLoS ONE | www.plosone.org 7 November 2008 | Volume 3 | Issue 11 | e3636