HLA, HCP5 and ZNRD1 in HIV A10 and ZNRD1-C) is used as a reference. (E) Association between HLA-A10/ZNRD1-containing genotypes with virologic-immunologic markers of HIV disease. Group 1 (lacking HLA-A10 and ZNRD1-C) is used as a reference and comparisons are made to HLA-A10/ZNRD1 genotypic groups 2 and 5. {M- W P refers to significance values obtained by Mann-Whitney test. bCD4, nCD4, and cCD4 represent baseline and nadir CD4+ T cell counts (cells/mm 3 , mean (6SE)), and cumulative CD4+ T cell count (x105 cell-days/mm 3 , mean (6SE)), respectively; the latter parameter provides a measure of the change in CD4+ T cell count during disease course as described previously [39]. VL, steady-state viral load (log10 HIV mRNA copies/ml, mean (6SE)). DTH, delayed type hypersensitivity skin test reactivity (an in vivo parameter of cell-mediated immunity) was examined as described previously [39] and data reflect the mean (6SE) number of positive skin tests out of the four applied. doi:10.1371/journal.pone.0003636.g002 WHMC cohort. The results of these analyses conducted at the haplotype level (Figure S2) provide further confirmation that the effects associated with the ZNRD1-C allele occur only when this allele is present on the same haplotype on which alleles that categorize to the HLA-A10 serogroup exists. First, haplotype 2 bears the ZNRD1-C allele but lacks HLA-A10 (Figure 1C, bottom), and in the heterozygous state this haplotype is not associated with disease retardation and in the homozygous state it is associated with disease acceleration (Figure S2B; reference category are those lacking haplotype 2). Second, heterozygosity for the haplotype that bears both ZNRD1-C and HLA-A10 (haplotype 3; Figure 1C, bottom) is associated with a slower rate of disease progression (Figure S2C; reference category are those lacking haplotype 3). Third, those who are heterozygous for the haplotype that bears HLA-A10 but not ZNRD1-C (haplotype 5; Figure 1C, bottom) have a slower disease course than those lacking this haplotype (Figure S2D). Taken together, the analyses conducted thus far at the compound genotype (Figure 2D) and haplotype/haplotype pair levels (Figure S2) together demonstrate that in the cohort we examined, the ZNRD1-C allele does not have independent disease protective effects, and instead, the associations observed for the ZNRD1-C allele occur because of its LD with HLA-A10, i.e, they are attributable to the disease-modifying effects of alleles corresponding to the HLA-A10 serogroup. Further underscoring the importance of HLA-A10 in AIDS pathogenesis in EAs is the observation that the HLA-A10-bearing haplotype that lacks ZNRD1-C, albeit low in frequency, is associated with disease retardation (Figure 2D, group 4, and Figure S2D). This suggests further that HLA-A10 influences AIDS pathogenesis independent of ZNRD1-C. To further confirm that the ZNRD1-C allele does not influence disease independent of its association with HLA-A10, we conducted univariate and multivariate analyses. In univariate analyses, both heterozygosity for ZNRD1-C (RH = 0.69; P = 0.030) and HLA-A10 (RH = 0.46; P = 0.005) associated with a reduced rate of disease progression (Table 1 under the column designated as ‘Time to AIDS’). However, in multivariate models, HLA-A10 (RH = 0.51; P = 0.028) but not the ZNRD1-C (RH = 0.85; P = 0.409) allele was associated with a reduced rate of disease progression (Table 1). The aforementioned genotype-phenotype association analyses were conducted using AIDS (1987 criteria) as a phenotypic endpoint. Therefore, it was plausible that although the ZNRD1-C allele did not have a direct impact on disease progression rates to AIDS it influenced surrogate markers of disease (e.g., viral load, baseline CD4+ T cell count). However, we found that subjects who had one ZNRD1-C allele but lacked an HLA-A10 allele (group 2) were similar to those who lacked both ZNRD1-C and HLA-A10 alleles (group 1) with respect to their baseline, cumulative and nadir CD4+ T cell counts, as well as steady-state viral load and delayed type hypersensitivity skin test reactions (a measure of cell mediated immunity [39]) (Figure 2E). Subjects in group 5 (heterozygosity for the HLA-A10/ZNRD1-C-containing haplotype) were also similar to those in group 1 with respect to most of these laboratory and immunologic characteristics, except those in group 5 had lower cumulative CD4+ T cell counts, a parameter that reflects the amount of CD4+ T cell loss over disease course and is a parameter that is highly correlated with development of AIDS ([39]; Figure 2E). These findings conveyed two points: first, in the cohort examined, the ZNRD1-C allele was unlikely to have an impact on disease independent of its LD with HLA-A10; and second, the influence of the HLA-A10/ZNRD1-C-containing haplotype on disease course occurs independent of an impact on the viral load, and that the influence of this haplotype is restricted mainly to the rate and extent of CD4+ T cell loss (assessed by cumulative CD4+ T cell count; Figure 2E), and by extension, on AIDS progression rates (Figure 2D). To clarify further the relationship between the ZNRD1-C allele and steady-state viral load, we determined the influence of ZNRD1-C and HLA-A10 on viral load using univariate and multivariate linear regression analyses. In these analyses, the alleles were considered as independent variables and the steady-state viral load was considered as the dependent variable. These analyses revealed that the impact of the ZNRD1 allele on viral load was minimal and HLA-A10 had no influence on viral load (as reflected Table 1. HIV disease-influencing effects associated with HLA-A10 status and ZNRD1 heterozygosity in European Americans. Model Covariates Time to AIDS Steady-state viral load 1 2 3 ZNRD1 Heterozygosity HLA-A10 ZNRD1 Heterozygosity HLA-A10 RH 0.69 0.46 0.85 0.51 95% CI 0.49 – 0.96 0.27 – 0.79 0.59 – 1.24 0.28 – 0.93 P 0.030 0.005 0.409 0.028 Coeff 20.14 20.07 20.15 0.04 95% CI 20.29 to 0.02 20.28 to 0.15 20.33 to 0.03 20.21 to 0.29 P 0.077 0.534 0.092 0.746 R2 0.0036 0.0007 0.0055 Results are from multivariate Cox proportional hazards regression (for the outcome of time to AIDS) and linear regression models (for the outcome of log10 steady-state viral load). RH, relative hazards; CI, confidence interval, Coeff, regression coefficient obtained from linear regression model R2, the explained variability in steady-state viral loads was assessed using the R2 estimated by analysis of variance (ANOVA). Analysis of ZNRD1-C homozygosity was not included in this analysis because of the small sample number. doi:10.1371/journal.pone.0003636.t001 PLoS ONE | www.plosone.org 6 November 2008 | Volume 3 | Issue 11 | e3636