The spatial proximity between the HLA-A locus and the ZNRD1 SNP (Figure 1A) prompted us to investigate whether the latter SNP was in LD with specific HLA-A alleles. We considered the results of de Bakker et al [32] who found a high LD between HLA- A*2601 and the SNP designated as rs2301751. The latter SNP is 244 bp from rs2301753, which is one of the seven ZNRD1-RFN39 SNPs that we genotyped. Furthermore, rs2301753 is reported to be in very high LD (D’ = 1.0) with rs2301751 by the HapMap project in Utah residents of European descent. Accordingly, we found a very strong LD between the ZNRD1 SNP with not only HLA-A*26, but also with A*25, which along with HLA-A*34 and HLA-A*66 make up the serogroup HLA-A10 [40] (Figure 1B and Figure 1C, bottom). Among the WHMC HIV+ subjects, the HLA-A10 group was composed mainly of HLA-A*25 and HLA- A*26 alleles (89% of the alleles that categorize to the HLA-A10 serogroup) in European Americans (EAs), whereas HLA-A*34 and HLA-A*66 alleles (78% of the alleles that categorize to the HLA- A10 serogroup) were more common in African Americans (AA; Table 1 in Materials S1). This difference in the distribution of alleles that belong to the HLA-A10 serogroup across the two major ethnic groups in the WHMC HIV+ cohort was statistically significant (x2 = 72.7; P = 1.1610215). Consistent with the results of the GWAS [27], a nearly 100% LD was noted between the HCP5 SNP and B*5701 (Figure 1B and Table 2 in Materials S1). Moreover, there was a high degree of LD between the HCP5-G, HLA-C59-C and HLA-B*57 alleles, and the HLA-C59-C/HLA-B*57/HCP5-G-containing hap- lotype was found almost exclusively in European Americans (Figure 1B and Figure 1C, top). No significant differences were found in the frequency of these SNPs among HIV-infected and - uninfected individuals (Table 3 in Materials S1). Because of the observed LD patterns, in the analyses described below, we examined whether the influence on disease course of the SNPs in ZNRD1 or HCP5 and HLA-C were independent of HLA- A10 and HLA-B*57, respectively. Effects of ZNRD1 SNP on HIV disease In the entire cohort of WHMC HIV+ subjects, heterozygosity but not homozygosity for the ZNRD1-C allele was associated with disease-retardation (Figure 2A, left panel). We stratified the cohort based on whether subjects were EAs or African Americans (AAs), and the stratified analysis revealed that the disease-retarding effects associated with the ZNRD1-C allele was restricted mainly to EAs (Figure 2A, middle and right panel). These results are consistent with the observations by Fellay et al [27] who found that the ZNRD1-C allele is associated with a slower disease course in subjects of European descent. However, we also found that this allele does not influence HIV disease course in HIV-positive African Americans in the WHMC cohort. We next determined whether the disease retarding effects associated with the ZNRD1-C allele were due to those associated with alleles categorized to the HLA-A10 serogroup for three reasons. First, the ZNRD1-C allele was in high LD with HLA-A10 (Figure 1B and Figure 1C, bottom) and of the 60 HIV-positive EAs who possessed an allele corresponding to the serogroup A10, 52 subjects also possessed at least one ZNRD1-C allele. Second, the ZNRD1-C allele was associated with a reduced rate of disease progression to AIDS only in HIV+ EAs (Figure 2A). Similarly, only HIV+ EAs who possessed alleles corresponding to the HLA- A10 serogroup had a significantly slower rate of progression to AIDS compared to those who lacked alleles corresponding to the A10 serogroup (Figure 2B). An association between HLA-A10 and disease course was not detected in HIV+ AA (data not shown). Notably, the strength of the disease-retarding effects (relative HLA, HCP5 and ZNRD1 in HIV hazards, RH) associated with HLA-A10 were similar before (RH = 0.62) and after (RH = 0.44) adjustment for explanatory covariates that in previous studies we had found influenced disease progression in this cohort ([39]; Figure 2B). The strength of the protective effects associated with HLA-A10 were also consistent when the analysis was restricted to subjects who had not received highly active antiretroviral therapy (HAART) (RH = 0.52; 95% confidence interval (CI) = 0.028–0.93; P = 0.028 for comparison of those possessing and lacking HLA-A10; Figure S1). Third, we surmised that if both HLA-A10 and ZNRD1-C each had independent effects on disease, then the protective effects of the ZNRD1-C allele that are not because of its LD with HLA-A10 should be evident in those 85 individuals who possessed at least one ZNRD1-C allele but lacked alleles corresponding to A10 serogroup. For these reasons, we stratified HIV+ EAs into six genotypic groups based on their ZNRD1-C allele and/or HLA-A10 status (Figure 2C). Those lacking both ZNRD1-C and HLA-A10 served as the reference category in the statistical analyses (Figure 2C, group 1). Subjects possessing one ZNRD1-C allele but lacking HLA-A10 (Figure 2C, group 2) had a disease course that was indistinguish- able from those who lacked both ZNRD1-C and HLA-A10 alleles (group 1). This suggested that in itself the ZNRD1-C allele was not associated with disease-retarding effects in the HIV+ EAs we examined (Figure 2D, compare survival curves for genotypic groups 1 and 2). By contrast, subjects who possessed both a ZNRD1-C and a HLA-10 allele (group 5) had a slower rate of disease progression (Figure 2D, compare survival curves for group 5 versus group 1 (P = 0.004) or group 2 (P = 0.057)). Additionally, although the number of subjects was small, those who were HLA-A10+ but lacked the ZNRD1-C allele (group 4) also had a slower disease course that was comparable to those in group 5 who were A10+ but possessed a ZNRD1-C allele (Figure 2D). These data indicated that the disease-retarding effect associated with the ZNRD1-C allele is evident only when it resides on a haplotype that also contains HLA-A10. The failure to detect an effect of the ZNRD1-C allele on disease course that is independent of HLA-A10 did not appear to be due to insufficient sample size because there were more HIV-positive EAs who possessed one ZNRD1-C allele but lacked HLA-A10 (n = 83) than those who had one allele each of ZNRD1-C and HLA-A10 (n = 50; Figure 2D). Furthermore, similar to what was observed for HLA-A10, the associations for the six genotypic groups defined by HLA-A10 and ZNRD1-C genotypes remained consistent when the analysis was restricted to subjects who had not received HAART (data not shown). The survival curves associated with homozygosity for ZNRD1-C suggested that it might be associated with a rapid rate of disease progression (Figure 2A, middle; RH = 4.26; 95% CI = 1.36–13.3; P = 0.013). Although there were few subjects who were homozy- gous for the ZNRD1-C allele with (group 6) or without HLA-A10 (group 3), it was interesting to note that subjects assigned to either of these two groups had a progressive disease course (Figure 2D). The small sample sizes preclude firm inferences but the direction of the effects associated with ZNRD1-A10 genotypic groups 3and 6 suggest that homozygosity for the ZNRD1-C allele might associate with disease acceleration even in those instances when these genotypes contain HLA-A10 alleles. The results shown in Figure 2D were derived from the genotype of HLA-A10 and ZNRD1-C alleles. To confirm that the aforementioned inferences derived from the associations of these compound HLA-A10/ZNRD1 genotypes are valid, we determined the disease-influencing effects of the individual A10-ZNRD1 haplotypes shown in Figure 1C in the EA component of the PLoS ONE | www.plosone.org 4 November 2008 | Volume 3 | Issue 11 | e3636