Genes and Immunity (2009), 1–5 & 2009 Macmillan Publishers Limited All rights reserved 1466-4879/09 $32.00 www.nature.com/gene 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.