Infection with HIV-1 is generally characterized by rapid viral mutation and evolution, 1–5 accompanied by hyperactivation of CD4+ and CD8+ T lymphocytes, 6–9 an imbalanced TH1 and 10–12 and progressive destruction of TH2 cytokine profile, CD4+ T cells, especially the HIV-1–specific subsets. 13 While cytokines and chemokines can mediate any of these virologic and immunologic features during HIV-1 infection, substantial interindividual differences in both early and later manifestations of HIV-1 disease 14 clearly imply the involvement of host genetic mediation of HIV-1 infection and pathogenesis. 15–17
The genes encoding various cytokines, chemokines, and their receptors often carry promoter and occasionally coding sequence variations that are functionally important. 18,19 Associations of genetic polymorphisms with varying clinical outcomes during HIV-1 infection have been repeatedly studied for the chemokine receptor and ligand genes CCR2, CCR5, CCL2 (MCP1), CCL3 (MIP1A), CCL5 (RANTES), and CXCL12 (SDF1). 16,17,20 Other immunogenetic studies of HIV/AIDS cohorts have examined single nucleotide polymorphisms (SNPs) in various cytokine genes like IFNG, 21,22 IL4, 23–26 IL6, 27 IL10, 26,28 IL12B, 29 and TNF. 29,30 Consistent findings have often been lacking in cross-cohort comparisons, most likely because the allelic distribution and haplotypic relationships of SNP variants can differ starkly between ethnic groups. 31–33 Such ethnic/racial specificity has been documented repeatedly in earlier studies. 20,34–40 Our genotyping of cytokine and chemokine gene variants in ethnically diverse North Americans now further supports such phenomena.
MATERIALS AND METHODS
Subjects and DNA Samples
The 579 patients, mostly African-American and Hispanic (Latino), were derived from the Reaching for Excellence in Adolescent Care and Health (REACH) project and the HIV-1 Epidemiology Research on Outcome (HERO) study, which targeted adolescents and adults, respectively. 41–43 For REACH participants, analyses were performed for all 183 HIV-1–seronegative (HIV−) participants and for the 227 HIV-1–seropositive (HIV+) participants with repeated virologic and immunologic outcome measures at various visit intervals when antiretroviral therapy (ART) was not taken. 44,45 The remaining 173 (Hispanics of Puerto Rican descent) were HERO participants living in Bronx, New York City. HIV-1− participants from both cohorts were considered at high risk of infection due to self-reported sexual behavior, injection drug use, or both. 41–43 Genomic DNA for each individual was extracted from 2 × 10 6 peripheral blood mononuclear cells using the QIAamp blood kit (QIAGEN, Chatsworth, CA). All DNA samples were diluted to 200 ng/μL and stored at 4°C in TE buffer (10 mM of Tris-HCl [pH 8.0] and 2 mM of EDTA) before use.
HIV-1–Related Outcome Measures
HIV-1 seropositivity could be assessed in both the REACH and HERO cohorts despite their differences in selection criteria, age, ethnic distributions, risk behaviors, treatment protocols, and follow-up strategy. For HIV+ REACH subjects with quarterly follow-up visits, CD4+ T lymphocytes were quantified by flow cytometry in National Institute of Allergy and Infectious Disease (NIAID)–certified laboratories at each clinical site. 46 Plasma HIV-1 RNA concentration (viral load) was measured in a centralized laboratory using either nucleic acid sequence–based amplification (NASBA) or NucliSens assays (Organon Teknika, Durham, NC) as previously described. 47 The lower limits of detection for the NASBA and NucliSens assays were 400 and 80 copies/mL, respectively. Multiple HIV-1 RNA measurements (transformed to log10 ) and absolute CD4+ cell counts were retained for 207 AIDS-free REACH adolescents for a 1-year period (up to 4 visits) when ART was not taken. 44 Analyses of immunologic outcome based on CD4+ T-cell counts were omitted in the HERO cohort because estimated duration of HIV-1 infection at enrollment was unreliable.
Polymerase Chain Reaction–Based Single Nucleotide Polymorphism Genotyping
Common SNPs for 13 genes (IFNG, IL1A, IL1B, IL1R1, IL1RN, IL2, IL4, IL4R, IL6, IL10, IL12B, TGFB1, and TNF;Table 1) in the cytokine system were typed by polymerase chain reaction (PCR) with sequence-specific primers (SSPs; Department of Transplantation Immunology, University of Heidelberg, Heidelberg, Germany) following procedures recommended by the 13th International Histocompatibility Workshop (IWHG) Cytokine Polymorphisms Component (CPC). 33 Reliability of genotyping results was verified in an initial analysis of 50 reference DNA samples distributed by the 13th IWHG. PCR SSP kits were also purchased from another supplier (Pel-Freez Clinical Systems, Brown Deer, WI) to achieve identical resolution (specificity). SNP typing for 3 chemokine genes (CCL2, CCL5, and CXCL12; see Table 1) relied on PCR SSP procedures developed in our own laboratory (J. Tang et al, unpublished data). The strategies were similar to those adopted for the typing of CCR2 and CCR5 SNPs and haplotypes as described elsewhere for the REACH and HERO cohorts. 37,44 In brief, representative genotypes (homozygous and heterozygous) were identified by sequencing and/or restriction fragment length polymorphisms. PCR SSP protocols were optimized using these reference DNA samples before being applied to test samples. Two SNPs (T-3575A and C-2763A) in the distal IL10 promoter were classified by the PCR SSP technique used earlier in a white (European American) cohort. 48
General Population Genetic Analyses
For each study population defined by ethnicity, the allele and carriage (population) frequencies of individual SNP variants were established by direct counting using SAS (Statistical Analysis Software, version 8.5; SAS Institute, Cary, NC), with the numbers of chromosomes (2N) and individuals (n) serving as the denominators, respectively. Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were assessed using the PopGene v1.32 statistical package. 49 Deviation (P < 0.05) from HWE was deemed indicative of sample selection bias or evolutionary advantage for particular SNP genotypes in a given population. Adjacent SNP alleles in strong LD form the basis for assigning local haplotypes; LD analyses were unnecessary for haplotypic combinations directly revealed by PCR SSP.
Genetic Association Analyses
Genetic associations with HIV-related outcome measures were evaluated in 4 steps. First, the overall allelic and/or genotypic heterogeneity between HIV+ and HIV− groups was measured by exact tests based on metropolis algorithms 50 and row by column (R×C) contingency tables. A statistical P value of <0.05 was taken to suggest overall genetic effect. 51 Second, carriage of individual alleles and haplotypes was compared between HIV+ and HIV− groups using χ2 or Fisher exact tests. Third, relationships between genetic variation and log10 HIV-1 virus load and CD4+ T-cell counts in HIV+ patients were defined by generalized linear regression model (GLM) statistics in SAS. Mixed models were also applied to the analyses of repeated measures at multiple (2–4) follow-up intervals when ART was not taken. Fourth, SNP variants showing marginal (P < 0.10) associations with HIV-1 infection and virologic/immunologic outcomes were further tested in multivariable regression and mixed model analyses, with statistical adjustments for race, gender, prior exposure to ART, visit interval (duration of follow-up), and the effects of known genetic factors (HLA, CCR2, and CCR5) in each cohort. 37,45
Polymerase chain reaction with SSPs successfully defined 24 common SNPs for 11 cytokine and chemokine genes (IFNG, IL2, IL4, IL4R, IL6, IL10, IL12B, TNF, CCL2, CCL5, and CXCL12) for most subjects. Only one DNA sample from an HIV− REACH subject had to be eliminated from analyses, and success in typing the 176 HERO Hispanics ranged from 96% to 100% for all loci except IFNG (56%;Table 2). Additional SNP typing for the IL1A, IL1B, IL1R1, IL1RN, and TGFB1 loci was available for about 50% of REACH and HERO samples due to frequent problems with several PCR reactions. Allele assignments for DNA from 6 chimpanzees (Pan troglodytes) were obtained for all but 3 SNPs (IL4R- 551 and the codon 10 and codon 25 SNPs in TGFB1). Homozygous sequences were detected in all chimpanzee samples (see Table 1); those defined at CCL2 and CCL5 loci confirmed several earlier reports. 52,53
Ethnic Differences, Hardy-Weinberg Equilibrium, and Haplotypes
For all but 3 (IL4–1098, IL10–3575, and IL12B 3′ untranslated region [UTR]) SNPs, the distribution of variant alleles differed widely by ethnicity, as did the contrasts in genotypic frequencies (P < 0.01 or P < 0.001) between African-Americans and Hispanics (see Table 2). For those 3 possible genotypes (2 homozygous and 1 heterozygous) defined for the IL10–1082 SNP, the expected and observed frequencies deviated from HWE in all racial groups, as documented by 21 laboratories participating in the 13th IWHG CPC. 33 Other more sporadic deviations from HWE varied from each ethnic group to another, without apparent correlation with sample size or HIV-1 serostatus (see Table 2).
The presence of multiple SNP haplotypes in IL4, IL6, IL10, TNF, CCL2 (MCP1), and CCL5 (RANTES) genes (Table 3) closely matched those reported elsewhere. 26,33,39,52–54 The exclusive LD between IL10–819T and IL10–592A was also confirmed. For SNPs at IL6 and IL10 loci not linked through PCR SSP, uniformly strong LD (Δ = 0.04–0.09; P < 0.001) justified their analysis as distinct haplotypes (see Table 3).
Cytokine and Chemokine Gene Variants in Relation to HIV-1 Serostatus
When the distribution of SNP variants (alleles as well as haplotypes) was compared between HIV+ and HIV− individuals within each ethnic group (see Tables 2, 3); differences with a nominal P < 0.05 (by univariate analyses) were found mostly in a race-specific manner. For example, the increased presence of IL4–33C allele (P = 0.03) in HIV+ versus HIV− African-Americans from REACH derived entirely from the association of −33C/C homozygosity (relative odds [RO] = 2.13, 95% confidence interval (CI): 1.20–3.70, unadjusted P = 0.01), but data from the other REACH and HERO Hispanic subsets did not display these relationships. Likewise, a disproportional decrease of CXCL12 801A (also known as SDF1–3′A) in HIV+ compared with HIV− HERO Hispanics (P = 0.02) could not be replicated in REACH patients.
In multivariable analyses of individual cohorts (Table 4), IL4R 551A/A was associated with the absence of HIV-1 infection in REACH participants (RO = 0.53; P = 0.009), whereas IL10–1082A/A, CCL5 ACT, and CXCL12 801A were independently associated with HIV-1 serostatus in HERO participants (RO = 0.13–0.32; P = 0.001–0.04). Carriage of 2 haplotypes, IL4 GTC and IL10 TCGCC, differed between HIV+ and HIV− groups regardless of ethnicity or cohort (see Table 3), but only the latter remained an unfavorable contributing factor (see Table 4; RO = 2.25; P < 0.001) after adjustment for gender, ethnicity, cohort, and CCR5–Δ32 (a 32–base pair [bp] deletion mutation defined earlier in both cohorts). 37,44
Cytokine and Chemokine Gene Variants in Relation to Early Virologic and Immunologic Outcomes in HIV-1–Infected Adolescents
In analyses restricted to 207 HIV-infected and AIDS-free REACH adolescents, the only SNP genotypes associated with contrasting immunologic outcomes (CD4+ T-cell count) were those defined by the −590 SNP in IL4 promoter (Fig. 1). More specifically, participants with the IL4–590T/T genotype consistently had higher (+87–131 cells/μL) CD4+ T-cell counts during 4 ART-free visit intervals when compared with others without IL4–590T/T (adjusted P = 0.004; see Fig. 1). Despite a clear linear correlation between CD4+ T-cell counts and virologic outcomes (HIV-1 viral load) in subjects who were IL4–590T/T–positive (Pearson r = −0.552; P < 0.0001) and IL4–590T/T–negative (Pearson r = −0.523; P < 0.0001), differences in viral load between the IL4–590T/T–positive and IL4–590T/T–negative subjects were quite modest (<0.10 log10 copies/mL; P > 0.50 by mixed model analyses) during the first 3 visit intervals; the difference at the fourth visit approached 0.25 log10 copies/mL (P = 0.19). Additional analyses of SNP haplotypes at IL4 and at other loci revealed no other appreciable effect on HIV-1 viral load or CD4+ T-cell counts in univariate or multivariable models (data available on request).
Analyses of Partial Data for Additional Loci
Polymerase chain reaction with SSP-based SNP typing for the IL1A, IL1B, IL1R1, IL1RN, and TNFB1 loci was not as informative due to technical difficulties. Use of alternative genotyping techniques, including DNA sequencing, appeared necessary. Nonetheless, partial data from these loci showed no trend for association with either HIV-1 infection (P > 0.25) or immunologic/virologic outcomes (P > 0.50). Meanwhile, there are no known associations of IL1A, IL1B, IL1R1, IL1RN, or TNFB1 variants with HIV-1–related outcomes.
Racial/ethnic specificity (population heterogeneity) poses a major obstacle to both epidemiologic and experimental analyses of HIV-1 infection and pathogenesis. Our demonstration that cytokine and chemokine gene variants differ markedly in their allelic and haplotypic frequencies between ethnic groups is consistent with earlier documentation of population-specific genetic associations with HIV-1–related outcomes. 24,25,28,36,53 More specifically, similarities between positive findings observed in the REACH and HERO cohorts and others reported elsewhere varied from one locus to another. CXCL12 (SDF1) 801A has been seen less frequently in HIV+ than in HIV− Thai sex workers exposed to HIV-1 clade E viruses, 55 and the association between IL4–590T/T with delayed immunodeficiency in REACH cohort mirrors the reportedly slower progression to AIDS in French HIV-1+ subjects carrying the IL4–590T (also known as −589T or −549T). 24,26 On the other hand, our findings on the ACT haplotype of CCL5 (RANTES) contradicted the earlier suggestion that the AC haplotype (at −403 and −28 relative to the transcription start site or −471 and −96 relative to the translation start site) was more common in HIV+ than HIV− European Americans. 53 In 2 other reports, variants found on the CCL5 ACT haplotype have demonstrated unfavorable effects on HIV-1 seroconversion and disease progression (relative hazards of AIDS = 1.57–1.89; P = 0.002–0.08) in US white (European American) populations. 39,56 The mechanisms underlying these seemingly conflicting genetic relationships remain to be defined.
In a separate analysis of individual T-lymphocyte responses to HIV-1–specific 20mer peptides, the IL4 promoter −590T/T genotype in a subset of REACH participants was further associated with increased magnitude of interferon-γ (INFγ)–secreting T cells, as enumerated by ELISpot assay (J. Tang and P.A. Goepfert et al, unpublished data). Thus, delayed immunodeficiency associated with IL4–590T/T in the REACH cohort (see Fig. 1) might reflect sustained HIV-specific immunity as a possible mechanism. A somewhat puzzling aspect was the lack of association between IL4–590T/T with cell-free HIV-1 viral load, which has been widely considered as a reliable predictor of HIV-1 pathogenesis and clinical course of disease progression. 57,58 A modest inverse correlation between viral load and CD4+ T-cell count (Pearson r = −0.52 to −0.55) was confirmed in the REACH subjects, suggesting that 27% to 30% (ie, r 2) of the variability in CD4+ T-cell counts could be captured by measuring differences in viral load. The origins of this dissociation between 2 related outcome measures could be 2-fold. First, viral load was universally lower in REACH adolescents (mostly female) than usually observed in adult male populations such that the power to reveal a statistically significant association across a narrower range of values was reduced in the REACH adolescent cohort. Second, there is preliminary evidence that IL4 genotypes might confer greater influence on HIV-1 coreceptor usage than on virus-host equilibration, as seen in HIV-infected Japanese. 23
Our study of male and female adolescents and adults from distinct ethnic groups was intended to yield more generalizable findings. Major risk factors for HIV-1 infection also differed between these cohorts, with sexual exposure accounting for most of the risk in the REACH project and injection drug use in the HERO study. 41–43 Relatively consistent associations of 2 haplotypes at the IL4 and IL10 loci with HIV-1 infection supported the belief that immunogenetic relationships can be independent of age, gender, ethnicity, and risk behavior. Similarly, robust observations have been reported for chemokine receptor gene (CCR2 and CCR5) variants on chromosome 3, including CCR5- Δ32 on the HHG*2 haplotype. 40,59–61 Nevertheless, CCR5–Δ32 was too rare in the HERO study and REACH project 37,44 to confound the observed relationships of cytokine gene polymorphisms (see Table 4).
On average, an SNP occurs at least once in every 500- to 1000-bp region of genomic sequences. 31,62 The few SNPs being studied here are expected to tag some but not all common clusters of genetic variants at the candidate loci. For example, 2 other IL4 SNPs (G–1136A and G+45A) described more recently contribute to several haplotypes. 63 Several intronic SNPs have been found in a French population. 26 Likewise, new IL10 SNPs have been documented in populations of African, European, or mixed ancestries. 54,64 Continuing discovery of potential IL4 and IL10 effects encourages more systematic evaluation of polymorphisms at these loci. Data from other ongoing HIV/AIDS cohorts differing in study design, research foci, experimental methodologies, and cohort characteristics should also become valuable in testing the validity of other less consistent immunogenetic findings.
The authors thank investigators and staff [listed in J Adolesc Health. 2001;29(Suppl):5–6] of the Adolescent Medicine HIV/AIDS Research Network (1994–2001) as well as others participating in the HERO study for their valuable contributions. We are further indebted to patients in the respective cohorts for their cooperation, to C.A. Rivers and A. Myracle for technical assistance, to A. Moore and D. Buono for data management, and to P.N. Fultz, PhD, for use of chimpanzee DNA samples.
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