C Haplotype Block Contains Major Determinants for Host Control of HIV

ORIGINAL ARTICLE The HLA-B/-C haplotype block contains major determinants for host control of HIV

E Trachtenberg1,6, T Bhattacharya2,3,6, M Ladner1,6, J Phair4, H Erlich1,5 and S Wolinsky4 1Center for Genetics, Children’s Hospital Oakland Research Institute, Oakland, CA, USA; 2The Santa Fe Institute, Santa Fe, NM, USA; 3Theoretical Division, Los Alamos National Laboratory, Los Alamos, NM, USA; 4Division of Infectious Diseases, The Feinberg School of Medicine, Northwestern University, Chicago, IL, USA and 5Department of Human Genetics, Roche Molecular Systems, Pleasanton, CA, USA

A genome-wide association study of people with incident human immunodeficiency virus (HIV) infection selected from nine different cohorts identified allelic polymorphisms, which associated with either viral set point (HCP5 and 50 HLA-C) or with HIV disease progression (RNF39 and ZNRD1). To determine the influence of these polymorphisms on host control of HIV, we carried out a population-based association study. The analysis revealed complete linkage disequilibrium between HCP5 and HLA-B*5701/HLA-Cw*06, a modest effect of 50 HLA-C on viral set point in the absence of HLA-B*5701, and no influence of the RNF39 /ZNRD1 extended haplotype on HIV disease progression. No correlation was found between the infection status and any of these genetic variants (P40.1, Fisher’s exact test). These findings suggest a pattern of strong linkage disequilibrium consistent with an HLA-B/-C haplotype block, making identification of a causal variant difficult, and underscore the importance of validating polymorphisms in putative determinants for host control by association analysis of independent populations. Genes and Immunity advance online publication, 20 August 2009; doi:10.1038/gene.2009.58

Keywords: HIV; HLA-B/-C haplotype block; SNP

Introduction cated in the control of the viral set point (the HLA complex, HCP5 and the 50 region of the HLA-C (50 HLA-C) Host proteins have crucial roles in the human immuno- gene) and two in the progression of disease (ring finger deficiency virus (HIV) life cycle and contribute to the protein 39 (RNF39) and zinc ribbon domain-containing 1 diversity of the host response to infection and disease (ZNRD1)).3 However, rare variants, population differ- progression. Genetic association analyses of populations ences in linkage disequilibrium patterns, and epistasis at risk for specific HIV-related outcomes have revealed can complicate the interpretation of whole genome the influence of host allelic polymorphisms on suscept- association studies. Accordingly, observations of deter- ibility to HIV infection, the time to AIDS and death. minants for host control of infection based on single At least 15 allelic variants in genes encoding human nucleotide polymorphisms (SNPs) need to be replicated leukocyte antigen (HLA) antigens, chemokine receptors in relevant populations, checking for patterns of linkage and their ligands, and cytokines have been shown to 1,2 disequilibrium with possible risk-related genes, followed influence HIV/AIDS susceptibility. Nevertheless, each by a deeper genomic and functional analyses of the of the described host factors are relevant to only a small associated region to verify putative causal variants. proportion of people who continue to resist infection or AIDS-defying illness decades after infection with HIV. Genome-wide association studies provide opportu- Results nities to uncover host polymorphisms that influence susceptibility and resistance to HIV infection free of To test for the influence of genetic variation in viral set mechanistic hypotheses. Of the allelic polymorphisms point and time to HIV disease progression, we analyzed 0 identified in people with incident HIV infection by the SNPs in HCP5 (rs2395029), 5 HLA-C (rs9264942), genome-wide association studies, significant associations ZNRD1 (rs9261129, rs9261174, rs3869068) and RNF39 between five host proteins and HIV pathogenesis were (rs2301753, rs2074480, rs2074479) using data from ma- reported.3 Three of the identified proteins were impli- trix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer assays. Genomic DNA samples were collected from men enrolled in the Correspondence: Dr E Trachtenberg, Center for Genetics, Children’s Chicago component of the Multicenter AIDS Cohort Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Study, a natural history study of men who have sex with Way, Oakland, CA 94609, USA. E-mail: [email protected] men. Of the 1351 men enrolled in the study, 998 had 6These authors contributed equally to this work. peripheral blood samples available for SNP genotyping, Received 24 March 2009; revised and accepted 09 July 2009 562 were infected with HIV and 436 were at risk for The HLA-B/-C haplotype block E Trachtenberg et al 2 Table 1 The frequency of HCP5, 50 HLA-C, RNF39 and ZNRD1 SNPs in men enrolled in the Chicago component of the MACS

Locus rs number SNP Frequency HWE P-value LD (r2) value: B*5701 Subjects

HCP5 rs2395029 TT 0.929 GT 0.069 0.38 0.86 997 GG 0.002 50 HLA-C rs9264942 TT 0.383 CT 0.482 0.42 0.06 995 CC 0.135 RNF39 rs2074479 TT 0.679 CT 0.283 0.20 0.008 997 CC 0.038 RNF39 rs2074480 AA 0.681 CA 0.282 0.23 0.008 997 CC 0.037 RNF39 rs2301753 CC 0.681 CA 0.282 0.23 0.008 997 AA 0.037 ZNRD1 rs3869068 GG 0.680 AG 0.283 0.28 0.008 998 AA 0.037 ZNRD1 rs9261174 TT 0.681 CT 0.282 0.23 0.008 998 CC 0.037 ZNRD1 rs9261129 TT 0.679 TC 0.284 0.28 0.008 998 CC 0.037

Abbreviations: HLA, human leukocyte antigen; HWE, Hardy–Weinberg equilibrium; MACS, Multicenter AIDS Cohort Study; RNF39, ring finger protein 39; SNP, single nucleotide polymorphism; ZNRD1, zinc ribbon domain-containing 1. SNPs are telomeric (top) to centromeric (bottom). The HWE P-values are for a Fisher’s exact test for HWE. The LD r2 values give the linkage disequilibrium with the B*5701 assuming HWE distribution of the double heterozygotes and ignore 10 patients who had not been HLA typed.

infection. We defined disease outcome either by the level also in linkage disequilibrium with HLA-C*0602, and of of viral RNA in plasma measured after the initial burst of the 180 men who carry HLA-C*0602, 63 men carry the viral replication during acute infection or by the rate of minor G allele. CD4 þ T-cell decline over a minimum period of 2 years, There is a pattern of strong linkage disequilibrium including data from at least four study visits. These between the minor G allele of HCP5 and HLA-B*5701.As measurements were taken before the onset of AIDS and a result, only 5 of 916 men carry both TT and HLA-B*5701 before the start of treatment. High-resolution HLA class I and only 5 of 69 men carry GT and not HLA-B*5701. and class II allelic typing data were available for all Consequently, we do not have sufficient data to attribute subjects.4 Table 1 lists the frequencies of these eight SNPs the effect to the two genes differentially. We also note that in these men. the predominant B*57 allele in African-Americans, HLA- We first compared our data with the previously B*5703, is not in linkage disequilibrium with the HCP5 identified association of genetic variation in the HCP5 minor G allele. In a recent study of 16 elite controllers, gene with HIV-related disease progression rates 10 African-Americans carried the HLA-B*5703 allele, 1 (Table 2a). HCP5 is a human endogenous retroviral African-American carried the HLA-B*5702 allele, and all element with sequence homology to the pol gene that 11 of these men carried the major T allele of HCP5.5 These encodes two proteins expressed in lymphocytes. Our observations are consistent with the hypothesis that results confirm that the polymorphism located in the protection from HIV is conferred by b*57 alleles and that HCP5 gene (rs2395029) is in very strong linkage the association of the HCP5 minor G allele with low disequilibrium with HLA-B*5701 (Table 1). In our data levels of plasma HIV RNA is due to linkage disequili- set, 71 men carry HLA-B*5701, 66 of whom (including brium with HLA-B*5701. Testing this hypothesis will both the HLA-B*5701 homozygotes) carry the minor G require the analyses of larger populations, including allele of rs2395029 that is found at a frequency of f ¼ 0.037 those of African descent, in whom B*57 is not in linkage in this sample. The remaining five men who carry disequilibrium with the minor G allele of HCP5. HLA-B*5701 are homozygous for the major T allele We examined the association between the polymorph- of rs2395029 that is found at a frequency of f ¼ 0.963. ism in 50 HLA-C (rs9264942) with variation in the level of The effect of this HCP5 rs2395029/HLA-B*5701 poly- HIV RNA in plasma (Table 2). This polymorphism is morphism on the level of HIV RNA in plasma is large; thought to influence the expression levels of HLA-C.6 the viral set point in men with rs2395029 homozygous In our data set, the frequency of the major T allele for the more common T allele is 18 010 copies per ml at rs9264942 is f ¼ 0.624 and the frequency of the minor (interquartile range (IQR) 4456–41 680), whereas in C allele is f ¼ 0.376. We confirmed that the HLA-C men containing at least one G allele it is 3648 copies polymorphism is in linkage disequilibrium with HCP5/ per ml (IQR 627–11 460), which is highly significant HLA-B*5701. In all, 69 of the 608 people carrying the (Mann–Whitney U-test, P ¼ 7 107). The HCP5 locus is minor C allele at rs9264942 also carry HLA-B*5701 (95%

Genes and Immunity The HLA-B/-C haplotype block E Trachtenberg et al 3 Table 2 (a) HCP5 and 50 HLA-C SNP genotype frequency and association between HIV RNA at set point and HLA-B*5701 and C*0602; (b) 50 HLA-C SNP association with HIV RNA at set point and HLA-B*5701; (c) 50 HLA-C SNP association with HIV RNA at set point and HCP5

Locus rs number SNP Frequency HIV RNA in plasma at Mann–Whitney HLA-B*5701 HLA-C*0602 set point, median and U-test P-value association of given association of (IQR) SNP allele given SNP allele

(a) HCP5 rs2395029 TTa 0.929o 18 010 (4456–41 680) TT vs GT or GG 5 of 916 117 of 916 GTa 0.069 64 of 69 61 of 69 3648 (627–11 701) P ¼ 7 107 GGb 0.002 2 of 2 2 of 2 TTa 0.383 21 980 (5104–49 850) TT vs CT Po0.07 2 of 377 1 of 377 50 HLA-C rs9264942 CTa 0.482 15 480 (3558–37 050) CT vs CC Po0.03 41 of 475 111 of 475 CCb 0.135 7918 (1060–20 780) CC vs TT Po0.002 28 of 133 68 of 133

(b) Locus rs number SNP HLA-B*5701 Frequency of genotype, HIV RNA in plasma Mann–Whitney association frequency with at set point, median U-test P-value (+indicates set point and (IQR) against heterozygous heterozygosity)c

50 HLA-C rs9264942 TT + 2, 1 20320 0.298 CTa + 41, 14 4920 (732, 12 430) CCb + 26, 12 773 (300–7443) 0.167 TT 375, 137 21 980 (4975, 49 820) 0.328 CTa 434, 182 17 000 (4359, 39 700) CCb 105, 34 11 430 (4464–32 600) 0.341

(c) Locus rs number SNP HCP5 SNP Frequency of genotype, HIV RNA in plasma Mann–Whitney associationd frequency with at set point, median U-test P-value set point and (IQR) against heterozygous

50 HLA-C rs9264942 TT GT 2, 2 17 110 (15 510–18 710) 0.158 CTa GT 41, 22 4920 (720–11 720) CCb GT 26, 11 708 (300–5295) 0.057 TT TT 379, 137 23 200 (4975, 49 890) 0.258 CTa TT 439, 184 17 000 (4346–39 570) CCb TT 106, 36 11 430 (4935–29 490) 0.324

Abbreviations: HIV, human immunodeficiency virus; HLA, human leukocyte antigen; IQR, interquartile range; SNP, single nucleotide polymorphism. aMajor SNP alleles. bMinor SNP alleles. cTwo individuals homozygous for HLA-B*5701 were omitted to avoid confounding. dTwo patients with HCP5 with GG homozygosity were omitted to avoid confounding. Note: analysis was carried out without correction for multiple comparisons suggested by previous studies. confidence interval on prevalence 8.9–14.1%), whereas findings indicate that the key protective effect of 50 HLA- only 2 of the remaining 377 people do (95% confidence C is primarily due to linkage disequilibrium with the interval on prevalence 0.06–1.9%), which, not surpris- polymorphism at HCP5/HLA-B*5701, with possibly a ingly, is associated with low levels of HIV RNA in the modest effect conferred in the absence of these protective plasma (Table 2a). The median viral RNA set point for alleles. men homozygous for the minor C allele (including both Next we compared our data with the identified men also homozygous for HLA-B*5701) is 7918 copies association of genetic variation in the ZNRD1 per ml (IQR 1060–20 780 copies per ml) against 15 480 (rs9261129, rs9261174 and rs3869068) and RNF39 copies per ml (IQR 3558–37 050 copies per ml) in men (rs2301753, rs2074480 and rs2074479) genes with HIV heterozygous at rs9264942, and 21 980 copies per ml (IQR disease progression rates. The ZNRD1, RNF39 and HCP5 5104–49 850 copies per ml) in men homozygous for the genes are in close physical proximity along human major T allele. The difference for heterozygotes measured chromosome 6 (cytogenetic band 6p21.33) within the against the minor C homozygotes is significant (Mann– highly polymorphic HLA class I gene-rich region. The Whitney U-test, Po0.03), and when measured against ZNRD1 gene encodes an RNA polymerase I subunit; the the major T homozygotes, shows a strong trend in the RNF39 gene is poorly characterized. The ZNRD1 and same direction (Mann–Whitney U-test, Po0.07). The RNF39 polymorphisms are in very strong linkage significance of the trend is reduced because of the disequilibrium. In particular, except for one man (who amount of data (P ¼ 0.096–0.34), should we stratify the was homozygous for T at rs9261174 and A at rs2074480, men by whether they carry the HLA-B*5701 allele heterozygous at the other three loci), the pattern of the (Table 2b) or analyze them with a given genotype at six SNPs within ZNRD1 and RNF39 are explainable by the HPC5 (rs2395029) locus (Table 2c) separately. These the presence of two extended haplotypes; a major (82%)

Genes and Immunity The HLA-B/-C haplotype block E Trachtenberg et al 4 TTGCAT haplotype at the respective SNPs, a minor specific populations, a small African elite suppressor (17%) CCAACC haplotype and rare SNP variants study,5 which found that the protective HCP5 minor G differing from the major haplotypes at a single position allele is not in linkage disequilibrium with HLA-B*57 in each (the observed variants were CTGCAT, TTGAAT, that group, is of particular interest herein, as it gives TTGCAC, CCACCC and TCAACC). credence to the hypothesis that B*57 confers the Assuming this resolution for all cases, the TTGCAT protective allele status in the African controllers. Without and CAACC alleles are consistent (Fisher’s exact strong functional data to support a role for HCP5 in the P ¼ 0.24) with Hardy–Weinberg equilibrium (HWE), as control of HIV, the pattern of linkage disequilibrium are all the minor ones except TTGCAC that appears only suggests an association between loci in a HLA-B/-C in the homozygous state and in one man. Therefore, the haplotype block with low levels of HIV RNA in plasma. constituent SNPs are in high linkage disequilibrium These findings underscore the need to locate the causal (r240.97 assuming HWE) and their effects on disease variant in the genomic region first identified by genome- progression cannot be distinguished. These entire hap- wide association studies. lotypes are also in linkage disequilibrium with the HCP5 Unlike the earlier study, we find no association polymorphism (rs2395029). Although all the men homo- between the polymorphisms in ZNRD1 and RNF39 or zygous for the minor CCAACC haplotype (including its their extended haplotypes and AIDS susceptibility, a variations) are homozygous for the major T allele at finding consistent with others.9 Although the early study HCP5 rs2395029, we estimate r2 ¼ 0.008 assuming HWE, focused on the individual polymorphisms in ZNRD1 and so that the effects of the HCP5 polymorphism can be RNF39, we took a more comprehensive approach, separately studied from those of the haplotype. We examining the spatial structure of linkage disequili- analyzed the effect of the ZNRD1, RNF39 and HCP5 brium. The strong degree of linkage disequilibrium that polymorphisms on HIV disease progression rates with- spreads across the HLA-B/-C region and their surround- out taking into account the confounding effects of ings leads to the existence of extended haplotypes. Apart ethnicity, risk factors or HLA. Owing to the high linkage from trivial departures, we find distinct major and minor disequilibrium between the six SNPs within ZNRD1 and haplotypes that accurately describe the linkage disequi- RNF39, we selected one (rs3869068) as a representative librium data. We did not, however, find an association polymorphism. We found no significant effect of the between ZNRD1 and the alleles within the HLA-A10 rs3869068 polymorphism on the RNA set point before serogroup, such as HLA-A*25 and HLA-A*26 in Eur- therapy initiation (P40.7) or on the CD4 slope at that opean-Americans or HLA-A*34 and HLA-A*66 in Afri- time (median 34, IQR (100, 4) in the men carrying can-Americans.4 the homozygous minor allele compared with median Our data do not support claims that the HCP5 gene per 53, IQR (98, 23) in the people carrying the major se explains viral set point, nor that ZNRD1 and RNF39 allele; Mann–Whitney U-test, P ¼ 0.2). explain HIV disease progression. The strong linkage disequilibrium between two genetic loci (HCP5 and 50 HLA-C) plus HLA-B*5701 and between the RNF39 and ZNRD1 polymorphisms, combined with no established Discussion functions for the SNP gene products in AIDS pathogen- Significant genotype associations for four genetic loci esis, provides few clues as to the causal variant that can within a short distance on chromosome 6p21 were influence HIV disease progression. This finding empha- identified for HIV/AIDS susceptibility in an earlier sizes the problem of statistical analyses of loci in regions study,3 with people carrying the minor allele variant of of extended linkage disequilibrium, such as the HLA-B/- HCP5 (rs2395029) and 50 HLA-C (rs9264942) having a C locus, and the importance of validating polymorph- lower viral RNA set point and people carrying six minor isms that explain differences in disease outcome by polymorphisms in ZNRD1 (rs9261129, rs9261174, association analyses of independent populations. rs3869068) and RNF39 (rs2301753, rs2074480, rs2074479) having a faster progression to AIDS. In our population- based study, we find an association between the HCP5 Materials and methods (rs2395029) minor G allele and 50 HLA-C (rs9264942) minor C allele and a lower viral RNA set point; however, Study participants the key protective association of the minor allele variants The participants were men enrolled in the Chicago in 50HLA-C and HCP5 may be largely due to linkage component of the Multicenter AIDS Cohort Study who disequilibrium with HLA-B*5701. Further, the pattern of were followed at 6-month intervals, queried about risk the six polymorphisms within ZNRD1 and RNF39 are behaviors, tested for antibodies to HIV, and had their explainable by the presence of two extended haplotypes CD4 þ and CD8 þ T-cell numbers enumerated. Men that had no influence on HIV disease progression. infected with HIV received antiretroviral therapy and Like the earlier study,3 our data show very strong levels of HIV RNA in plasma measured by quantitative linkage disequilibrium between HCP5 and HLA-B*5701, reverse transcriptase–PCR (Roche Molecular Diagnostic a known marker in the control of HIV disease.4,7 Systems, Branchburg, NJ, USA). All men had equal Although others confirm an association between the access to care. Of the 1351 men originally recruited for minor allele variant of HCP5 (rs2395029) and 50 HLA-C the Chicago component of the Multicenter AIDS Cohort (rs9264942) and a lower viral RNA set point, they did not Study, 998 had peripheral blood samples available for determine whether this was due to linkage disequili- SNP analysis and 562 of them were infected with HIV. brium between HCP5 and 50 HLA-C and a nearby locus, The time from the acquisition of antibodies against HIV such as the HLA-B/-C region.8 Although its limited to AIDS (CD4 þ T cells o200 per mm3) was established numbers will require confirmation in larger ethnically for only 64 men. Accordingly, we defined disease

Genes and Immunity The HLA-B/-C haplotype block E Trachtenberg et al 5 outcome either by the level of viral RNA in plasma Acknowledgements measured after the initial burst of viral replication during acute infection (the viral level at set point), or by rate of We thank Koy Saeteurn, Isabel Nocedal, Sherry Haw- CD4 þ T cell decline over a minimum period of 2 –years, becker, Patricia Otto and Samuel Wu for their technical including data from at least four interval study visits. assistance with sample preparation. E Trachtenberg and These measurements were considered before the onset of S Wolinsky conceived and managed the project. M AIDS and before the start of therapy. All men provided Ladner developed the SNP-based assays and performed written informed consent according to the guidelines of all of the SNP genotyping. T Bhattacharya performed all the human subjects protection committee of North- of the statistical analyses. J Phair is the Director of the western University. MACS and oversees all projects using the MACS cohorts. S Wolinsky, H Erlich, T Bhattacharya, M Ladner and E HLA and SNP genotyping Trachtenberg wrote the paper. All authors have agreed to High-resolution HLA class I and class II allelic typing the content in this paper. This work was supported by was previously carried out for all subjects.4 The HCP5, grants from the National Institutes of Health (AI 65254- 50 HLA-C, RNF39, and ZNRD1 gene fragments were 01A1 and AI035039-17). analyzed using a novel assay developed using iPLEX Gold chemistry (SEQUENOM, San Diego, CA, USA) and analyzed on a MALDI-TOF mass spectrometer. Briefly, samples were amplified by PCR using primers that References captured the region surrounding the SNP. A shrimp alkaline phosphatase treatment was used to neutralize 1 Singh KK, Spector SA. Host genetic determinants of HIV unincorporated dNTPs, and then the reaction mixture infection and disease progression in children. 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