Long-term nonprogressive HIV-1-infected subjects (LTNPs) are patients who remain clinically, virologically, and immunologically stabilized for 10 to 15 years after infection with HIV-1 without the intervention of any antiretroviral drug.1,2 They represent 1% to 5% of HIV-1-infected patients.2 The existence of this subset of patients suggests that some individuals have the ability to contain HIV-1 multiplication, thus limiting expansion of the viral burden. The factors that lead to the LTNP state are not known, but it is believed that there is a complex interaction between the viral and host factors that determines the clinical evolution of the infection.2
Among host-dependent factors, attention has recently been paid to the genetic polymorphism of the cellular coreceptors used by HIV-13 and their ligands, the chemokines,4-10 given their critical role in the process by which HIV-1 enters the cell. Several single nucleotide polymorphisms (SNPs) in the chemokine receptors and chemokine gene promoters have been shown to alter the transcriptional activity of the gene and may significantly influence the pathogenesis and progression of the disease. This is well established for the genetic variants of CCR5-the main coreceptor that HIV-1 uses in the early phases of infection-particularly for the CCR5Δ32 allele.5-10 RANTES, which is the main natural ligand that binds to CCR5,11 is one of the candidate chemokines to be investigated in this respect, but data reported to date are scarce and often controversial.12-20 Moreover, RANTES genetic variants in individuals who show a true LTNP evolution have not been assessed. We therefore carried out a study to evaluate the role of allelic variants in the gene promoter of the chemokine RANTES on the natural history of HIV-1 disease, particularly in the state of long-term nonprogression, in a cohort of white Spanish patients and controls. We also assessed the distribution of the CCR5Δ32 allele.
PATIENTS AND METHODS
We performed a cross-sectional multicenter genetic association case-control study. We recruited patients at the HIV outpatient clinics of the 5 hospitals participating in the project. Many of the HIV-1-infected subjects we care for at these clinics are injecting drug users. Two subsets of HIV-1-infected patients were studied: LTNPs and usual progressors (UPs). The criteria for LTNPs were (1) asymptomatic HIV-1 infection of more than 16 years' duration; (2) in the absence of antiretroviral treatment, a CD4+ cell count that was stable and persistently greater than 500 cells/μL; and (3) a plasma HIV-1 viral load value that was repeatedly less than 5000 copies/mL.2 Patients were considered to be UPs if HIV-1 infection had progressed to advanced disease, namely, the appearance of class C HIV-1 disease according to the 1993 criteria of the Centers for Disease Control and Prevention (CDC),21 and had a plasma viral load of greater than 35,000 copies/mL and a progressively declining CD4+ cell count over time that was lower than 350 cells/μL at least once during the first 10 years of infection. For a few patients whose date of infection was unknown, we assumed this to be the midpoint between the date of the last negative and first positive HIV-1 blood tests.13 As a control group, we also studied a sample of healthy subjects recruited from blood donors. All subjects studied were white Spaniards. Immigrants from other countries, including those from other European countries, and their descendants were excluded. Informed consent was obtained from each participant. The project was approved by the local ethical research committees.
Blood, DNA, and Plasma
After 12 hours of fasting, blood samples with ethylenediaminetetraacetic acid (EDTA) were obtained from an antecubital vein. Five milliliters of whole blood was sent to determine CD4+ lymphocyte cell count, and 500 μL was sent for DNA isolation using a MagNa Pure LC Instrument (Roche Molecular Biochemicals, Basel, Switzerland). Plasma for determining HIV-1 viral load was obtained by centrifugation at 3500 g for 15 minutes at 4°C.
HIV-1 infection was diagnosed by means of a positive enzyme immunoassay and was confirmed by a positive Western blot test.
Plasma HIV-1 Viral Load
Plasma HIV-1 viral load was determined by the COBAS AMPLICOR HIV-1 Monitor Test, version 1.5 (Roche Diagnostics, Basel, Switzerland). The cutoff for undetectable viral load was 50 copies/μL.
Assessment of Blood CD4+ Lymphocyte Cell Count
Samples were analyzed by flow cytometer FACScan (Becton Dickinson Immunocytometry Systems, San José, CA). Data acquired were analyzed using the Multiset program.
RANTES −28 C>G, −109 T>C, and −403 G>A Single Nucleotide Polymorphisms
To date, 3 SNPs have been reported in the RANTES gene promoter.22-24 The positions indicate the nucleotide where the mutation is located at the beginning of the transcription. The SNPs are as follows: position −28, transversion C>G; position −109, transition T>C; and position −403, transition G>A. These were analyzed by a polymerase chain reaction (PCR) assay and automatic sequencing in an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA). Because of their proximity, SNPs at positions −28 and −109 were assessed together by sequencing a unique fragment. The mutation −403 was analyzed separately.
Analysis of the Single Nucleotide Polymorphisms −28C>G and −109T>C
By means of a PCR assay, a fragment of 196 base pairs (bp) was amplified using the following specific primers: a primer with sense 5′ TGA GAG AGC AGT GAG G 3′ and a primer with antisense 5′ GTC CAC GTG CTG TCT T 3′. The correct amplification of the desired fragment was evaluated in 2% agarose gel tinged with ethidium bromure and Marker VIII (Roche Molecular Biochemicals), a marker of known molecular size. The PCR products were subsequently purified using the QIAquick PCR Purification Kit (Qiagen). After purification, a sequencing PCR assay was performed with the primer with sense using the BigDye Terminator sequencing kit, version 3.0, Cycle Sequencing Ready reaction (PE Applied Biosystems) in accordance with the manufacturer's instructions. If needed, the sequencing products were purified with the DyeEx Spin kit (Qiagen). After denaturalization at 95°C for 3 minutes, the samples were analyzed in an ABI PRISM 310 sequencer using the DNA sequencing analysis program, version 3.3 (PE Applied Biosystems), which assigns a specific color to each nucleotide base. Heterozygote individuals were detected by the presence of 2 peaks, whereas homozygote individuals have only 1 peak.
Analysis of the SNP −403G>A
Analysis of the SNP −403G>A was assessed using the previously described method. The following primers were used for the PCR assay: a primer with sense 5′ AGT GTG CTC ACC TCC TTT 3′ and a primer with antisense 5′ GAT CAG AAG TCA CTG AGT 3′, which generates a 174-bp fragment. For the PCR sequencing, we used the primer with sense. The Genbank accession number for the 3 RANTES SNPs was S64885.
The PCR primers were as follows: 5′ CGT CTC TCC CAG GAA TCA TC 3′ and antisense 5′ TTC CCG AGT AGC AGA TGA CC 3′, with annealing 60°C. The Genbank accession number was AF031237.1. The PCR products were visualized on 2.5% agarose gel. The wild-type CCR5 gene led to a 174-bp fragment, whereas the CCR5Δ32 mutant allele led to a 142-bp fragment.
Statistical analysis was performed using the SPSS/PC+ statistical package (version 12.0 for Windows; SPSS, Chicago, IL). Descriptive data were expressed as the mean ± standard deviation (SD) or median (range) for nonparametric distributions. Differences in levels between groups were compared using the Student t test, or Mann-Whitney U test when necessary. Hardy-Weinberg equilibrium was assessed by the χ2 goodness-of-fit test. Genotype and allele frequencies in the different groups were compared by the χ2 test, and the Fisher exact test was used when necessary. A P value of less than 0.05 was considered significant.
Two hundred sixty-seven white Spaniards were included in the study (167 HIV-1-infected patients and 100 healthy controls). Of these patients, 109 were UPs and 58 were LTNPs. The age, gender, and risk factors for acquiring HIV-1 were comparable for the UP and LTNP groups. As expected and because of the epidemiologic pattern of HIV-1 infection in our geographic area, more than two thirds of the HIV-1-infected patients studied were injecting drug users. Most UPs (>90%) were receiving highly active antiretroviral therapy (HAART). The characteristics and main data of the populations analyzed are shown in Table 1.
RANTES Gene Promoter Polymorphisms
Table 2 shows the genotype distribution and allele frequencies in the RANTES gene promoter region at the positions −28, −109, and −403, for the control group, UP group, and LTNP group. The genotype distribution in the population studied fits the expected Hardy-Weinberg equilibrium. No significant differences were detected in the allelic distribution of the variants analyzed for the 3 groups. Also, the distribution of genotypes was similar between groups. Homozygous mutants were observed only at position −403, but the frequency was low. Taking this into account, we split the groups into 2 subsets according to the presence or absence of the mutant allele. Further analyses of RANTES genotype and allele frequencies performed after the 45 individuals who carried the CCR5Δ32 allele had been excluded also showed no differences between the groups. We studied the distribution of the 3 most common RANTES −28 and −403 haplotypes: I (−28C and −403 G), II (−28C and −403A), and III (−28G and −403 A). The distributions of haplotypes I, II, and III for UPs were 81.8%, 17.2%, and 1%; for LTNPs, they were 82.7%, 14.6%, and 2.7%, respectively (P = not significant [NS]). With regard to the genotypes of the RANTES gene promoter region at the positions −28, −109, and −403, there was no relation between viral load and CD4+ cell count in the LTNP group.
There were no significant differences for CCR5 genotype or allele distributions among the different groups (see Table 2). Homozygosity was not observed for the CCR5Δ32 allele.
This study shows that there is no relation between the SNPs −28C>G, −109T>C, and −403G>A of the chemokine RANTES gene promoter and long-term nonprogressive HIV-1 infection in a cohort of white Spaniards who acquired HIV-1 mainly by injecting illicit drugs. Also, there are no differences in genotype or allelic distribution between HIV-1-infected subjects and uninfected subjects, and no particular genotype or haplotype was found to protect or predispose to HIV-1 infection. This is independent of the presence of the CCR5Δ32 allele.
Three polymorphic sites have been described to date in the RANTES gene promoter.22-24 An increase in promoter activity and RANTES messenger RNA (mRNA) expression and transcription has been shown in 2 of these sites (−28 C>G and −403 G>A),12,13 but no information is yet available regarding the SNP −109 T>C. Two other polymorphic sites have also been detected in other regions of the gene: in1.1C in the first intron and 3′222C in the 3′ untranslated region.14
Previous reports have studied the influence of RANTES gene promoter SNPs on the epidemiology and natural history of HIV-1 infection.12-20 None of these reports has specifically evaluated true long-term nonprogression, however, and the results strongly depend on the population analyzed. Carriage of the rare mutant −403A allele increases susceptibility to HIV-1 infection in white Americans13,14 and Han Chinese15 but not in Japanese,12 Spaniards (present report),16 or other cohorts.17,20 Other authors have even found that the wild-type −403G allele is associated with increased susceptibility to infection in Chinese subjects.19 These findings should be interpreted with great caution, however, because in some of the studies reported as well as in the present study, individuals from the control group were blood donors with no known risk practices for acquiring HIV-1 infection. It is therefore plausible that among subjects infected with HIV-1, risk practices for acquiring HIV-1 infection overshadow the possible protective effect of any particular RANTES genotype or haplotype. Interestingly, however, a recent report in white subjects found no differences in RANTES genotype between HIV-1-infected patients and repeatedly exposed but uninfected subjects.17 This strongly suggests that carriage of allelic variants of RANTES has no predisposing or protective role with regard to HIV-1 infection.
Among infected subjects, and in relation to disease progression, the rare −403A allele has been associated with a slower slope of CD4+ lymphocytes and a slow progression toward AIDS in some multiethnic cohorts.13,14 Information on Japanese subjects is discordant, because some studies found no effect12 and others found slow progression.19 Duggal et al25 found that individuals homozygous for a RANTES haplotype containing the −403G wild-type allele had a lower early plasma HIV-1 viral load compared with other haplotypes in African Americans, thus anticipating slow progression. The influence of the −28G mutant allele on disease progression also depends on ethnicity, ranging from no effect in a multiethnic cohort,13 to slow progression in Japanese,12,19 to rapid progression toward AIDS in Chinese.18 None of the reports analyzed has addressed this issue regarding true long-term nonprogression, and the results presented here indicate that RANTES allelic variants do not modulate this particular subset of HIV-1-infected patients. Although the −28G mutant allele was more frequent in LTNPs than in UPs (5% vs. 2%, respectively), statistical analysis showed that this difference was not significant; hence, our conclusion. Nevertheless, we recognize that the small numbers studied here may render the statistical analysis underpowered, because the power of the comparison was lower than 40%.
When we compared our data with those reported in the literature, we found that the influence of the allelic variants of RANTES gene promoter on the risk of HIV-1 infection and disease progression varied widely and depended on the origin of the subjects analyzed. In fact, the distribution of RANTES haplotypes in our patients was closely related to data on white subjects13 but was markedly different from those on Asian subjects.12 This makes the hypothesis that the influence of the RANTES genotype might be variable in different ethnic groups plausible.26
Finally, we found no association between the CCR5Δ32 heterozygosity and long-term nonprogression, because the frequency of this allele did not increase in this subset of patients. We have therefore not been able to reproduce the protective effect of CCR5Δ32 on disease progression that has been reported by several authors.5-7,9,10 Conversely, our results agree with those of other studies that reported no association between CCR5Δ32 heterozygosity and delayed immunologic slope in 2 cohorts of HIV-1-infected injection drug users.27,28 Note that most of our patients acquired HIV-1 through the injection of illicit drugs. Because the transmission route is a major determinant of the immune response to HIV-129 and outcome, it is plausible that the influence of host genetic background on disease progression depends on the transmission route.
In conclusion, our results show that, at least in the Spanish white population, SNPs at the RANTES gene promoter and CCR5Δ32 allele are not associated with long-term nonprogressive HIV-1 disease, and thus do not modulate this particular clinical evolution.
The authors thank M. Olona for her help in epidemiologic and statistical assessment.
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The members of the Chemokine and Long-Term Nonprogressive Study Group and coauthors of this paper are as follows: Francesc Vidal, Joaquim Peraire, Consuelo Viladés, Montserrat Broch, Cristina Gutiérrez, Sergi Veloso, Maria Saumoy, Miguel López-Dupla, Montserrat Olona, Joan Josep Sirvent, and Cristóbal Richart (Hospital Universitari de Tarragona Joan XXIII and Universitat Rovira i Virgili); Pere Domingo, Ma Antonia Sambeat, Àngels Fontanet, Josep Cadafalch, and Montserrat Fuster (Hospital de la Santa Creu i Sant Pau and Universitat Autònoma de Barcelona); Mireia Cairó, David Dalmau, and Anna Ochoa (Hospital Mútua de Terrassa); Enric Pedrol, Elisabeth Deig, and Anna Soler (Unitat de Malaltíes Infeccioses-VIH, Observatori de Salut Carles Vallbona, Hospital General de Granollers); Hernando Knobel, Milagros Montero, and Ana Guelar (Hospital del Mar de Barcelona); and Judit González (Pius Hospital de Valls).