Introduction
The human multidrug resistance (MDR1 ) gene encodes for P-glycoprotein (P-gp, ABCB1), an integral membrane protein of 170 kDa and member of the ATP-binding cassette transporters superfamily (ABC). It actively pumps xenobiotics out of the cell, including lipophilic, positively charged molecules such as digoxin [1] [2] [3] [4] [5,6]
P-gp was originally identified in tumor cells by its ability to confer multidrug resistance, decreasing the intracellular concentration of anticancer agents. However, it is expressed in key organs such as intestine, liver, kidney, pancreas and adrenal glands, where it contributes to the elimination of xenobiotics or limits drug absorption from the gastrointestinal tract [7–9] + and CD4+ – the primary receptor of HIV [10–14]
MDR1 is a well conserved gene on chromosome 7 (7p21–21.1) with 28 exons [15] MDR1 , more than 50 different single nucleotide polymorphisms (SNPs) were reported at the SNP database (NCBI ), and the list is expanding [16,17]
Few SNPs were found to significantly affect human P-gp expression and, therefore, may alter drug response. Among the synonymous polymorphisms, the C3435T on exon 26 is the most widely studied and has been associated with low P-gp expression in enterocytes [18] [19] [18]
Several studies have evaluated the influence of C3435T polymorphism in response to antiretroviral treatment in HIV-1-infected patients. Some results suggest that the C3435T SNP has an important role in the immunologic [20] [21] [22–24]
The C3435T polymorphism presents a strong linkage disequilibrium with the C1236T synonymous SNP on exon 12 [25] + T-cell counts after initiation of HAART, with no differences in the rate of viral suppression [26] MDR1 haplotypes on the expression and activity of P-gp [17] MDR1 haplotypes effect on HAART response and HIV-1 infection [24,27]
In-vitro assays have suggested a decrease in HIV infectivity in cells overexpressing P-gp by affecting viral fusion and possibly viral release [28–30] MDR1 mRNA expression and CD4+ T-cell permissiveness [27] [31] [32]
The aim of this study was to explore whether the MDR1 C1236T (exon 12) and C3435T (exon 26) polymorphisms affect HIV-1 vertical transmission and progression to AIDS in a large pediatric cohort.
Patients and methods
Study population
The study was performed in a white-Hispanic population of Argentina and included stored genomic DNA obtained from 231 HIV-seronegative randomly selected blood donors (reference group) and 347 children perinatally exposed to HIV-1 (pediatric cohort) from the Hospital de Pediatría ‘Juan P. Garrahan’ (Buenos Aires, Argentina). The pediatric cohort included 128 exposed uninfected (64 women and 64 men) and 219 HIV-1-infected children (114 women and 105 men) born to HIV-seropositive mothers between 1987 and 2006. The higher proportion of infected than of uninfected children is not indicative of transmission rate because ascertainment was skewed toward infected children. HIV-1 infection status and AIDS definition were established according to the 1994 criteria of the US Centers for Disease Control and Prevention (CDC) classification for children [33] [34]
Genotyping analysis
MDR1 genotypes were identified by a PCR-restriction fragment length polymorphism (RFLP) assay based on the previous report of Cascorbi et al. [35] HaeIII and MboI , respectively. HaeIII has two restriction sites in the amplicon, one corresponding to the C1236T polymorphism. This enzyme cleaves the amplicon in the position 1236 into three fragments of 269, 62 and 35 bp when a cytosine is present and into two fragments of 269 and 97 bp when a thymine is present. MboI cleaves the PCR product into two fragments of 158 and 39 bp when a cytosine is present in the position 3435 but not when a thymine is present. Finally, digested products were separated on 4.5% (exon 12) or 3.5% (exon 26) agarose gel and visualized by ethidium bromide staining under UV light with the El Logic 200 imaging system (Kodak, Rochester, New York, USA). Examples of PCR-RFLP profiles of the MDR1 C3435T and C1236T genotypes are shown in Fig. 1 .
Fig. 1: Restriction fragment length polymorphism profiles of the MDR1 C3435T and C1236T genotypes. (a) Heterozygote 1236CT, wild-type homozygote 1236CC, homozygote 1236TT and the undigested PCR fragment are shown. Digestion was performed with HaeIII restriction enzyme. (b) Wild-type homozygote 3435CC, heterozygote 3435CT, homozygote 3435TT and the undigested PCR fragment are shown. Digestion was performed with MboI restriction enzyme.
The CCR5-Δ32 and SDF1-3′ A alleles were determined with a PCR and PCR-RFLP, respectively as previously described [36]
Statistical analysis
Genotype frequencies were estimated by direct allele counting. Fit to Hardy–Weinberg equilibrium was tested with Pearson's χ 2 . The Fisher–Freeman–Halton test was used to evaluate marginal independence on contingency tables and the P value was estimated with the Monte Carlo method. Haplotype frequencies were estimated using the Expectation Maximization Algorithm (www.bioinfo.iconcologia.net/snpstats D ′ statistic and association of the genotypes was tested with Pearson's χ 2 . The Kaplan–Meier method was used to estimate disease progression and survival curves. Relative hazards were calculated for a Cox proportional hazards model. It alternatively included the exposition or not to HAART as a time-dependent stratum (HAART adjustment). We also analyzed the CCR5-Δ32 and SDF1–3′ A alleles as potential confounders in a stratified model. The Andersen–Gill formulation of the proportional hazards model as a counting process was applied [37]
Results
Role of MDR1 genotypes in HIV-1 vertical transmission
We investigated whether MDR1 polymorphisms might be associated with perinatally HIV-1 transmission. The distribution of MDR1 genotypes was analyzed in 219 HIV-1 infected children, 128 exposed uninfected children born to HIV-seropositive mothers, that represented a high-risk control group, and 231 HIV-seronegative adults, which was a reference group (Table 1 ). Both C1236T and C3435T polymorphisms were highly frequent in the Argentinean population, with frequency of 0.44. MDR1 genotypes distribution did not significantly differ among the three groups, even when the analysis was restricted to children who did not receive antiretroviral prophylaxis to prevent mother-to-child transmission in the subgroups of HIV-infected (n = 166) and exposed uninfected (n = 55). Moreover, no association was found when the analysis was adjusted to zidovudine prophylaxis (Table 1 ). All the genotypes studied fit the Hardy–Weinberg Equilibrium, indicating that there was no population bias in the analyzed groups.
Table 1: Genotype and allele frequencies of MDR1 C3435T and C1236T single nucleotide polymorphisms in HIV-seronegative blood donors and perinatally HIV-1 exposed children.
As previously described, we also found in our population a strong linkage disequilibrium between C1236T and C3435T polymorphisms (D = 0.155, D ′ = 0.633, r = 0.630, P < 0.05). The 3435C allele is linked to the 1236C allele and the 3435T is linked to the 1236T allele, defining nine haplotype pairs. The haplotype frequencies in the three studied groups are shown in Fig. 2 . As expected, the haplotype pairs 3435CC/1236CC, 3435TT/1236TT and 3435CT/1236CT were the most common with frequencies higher than 12%. There was no significant difference observed in the haplotype frequencies among the studied groups.
Fig. 2: Frequencies of the MDR1 haplotype pairs (C3435T and C1236T) in HIV-seronegative blood donors and perinatally HIV-1 exposed children. EU, exposed uninfected.
Collectively, these findings suggest that neither C3435T nor C1236T SNP seem to influence natural HIV-1 infection through vertical transmission in the Argentinean pediatric cohort.
Influence of MDR1 genotypes on progression to pediatric AIDS
We further explored the influence of MDR1 genotypes on progression to pediatric AIDS in 171 HIV-1-infected children with a median follow-up time of 108 months (4–232 months). Kaplan–Meier plots for time to AIDS and modeling by Cox proportional hazards regression showed that children carrying the heterozygote genotype 3435CT progressed to AIDS significantly slower, with an AIDS-free median of 63 months [P = 0.005, relative hazard = 0.537, confidence interval (CI) = 0.348–0.829; Fig. 3 a]. Similarly, children with heterozygotes 1236CT and homozygotes 1236TT had a slower disease progression, with AIDS-free time median of 52 and 39 months, respectively (P = 0.024, relative hazard = 0.599, CI = 0.385–0.934; P = 0.026, relative hazard = 0.553, CI = 0.328–0.933; Fig. 3 b). When analysis was restricted to the period before the initiation of HAART, the same trends were observed for 3435CT (P = 0.011, relative hazard = 0.529, CI = 0.324–0.865), 1236CT (P = 0.063, relative hazard = 0.617, CI = 0.372–1.103) and 1236TT (P = 0.069, relative hazard = 0.574, CI = 0.316–1.040). The number of AIDS events in the post-HAART period was very small (n = 26) to perform an independent analysis. We additionally evaluated a potential confounding effect of CCR5-Δ32 and SDF1–3′ A alleles and no change was observed for the protective role of MDR1 variants in a stratified Cox proportional hazards model (data not shown). As only six patients died during the follow-up period, the number of events was not enough to evaluate the influence of MDR1 variants in terms of survival time.
Fig. 3: Survival curves for time to AIDS in HIV-1-infected children. The Kaplan–Meier method was used to estimate fraction AIDS-free and survival curves. Overall fit was evaluated with the likelihood ratio test and genotype and haplotype pair contrasts were analyzed by a Wald test.
P values and RH were calculated under a Cox proportional hazards model. *, reference group; CI, confidence interval; RH, relative hazard. (a)
MDR1 C3435T: 3435CC (
), 3435CT (
), 3435TT (
). (b)
MDR1 C1236T: 1236CC (
), 1236CT (
), 1236TT (
). (c) 3435CC/1236CC (
), 3435CT/1236CC (
), 3435CT/1236CT (thick
), 3435CT/1236TT (thick
), 3435TT/1236TT (
).
Next, we examined the disease-influencing effect of MDR1 haplotype pairs in disease progression. Only in the context of a 3435CT-containing haplotype, the 1236T allele had an independent contribution in a dose-dependent way (1236TT > 1236CT). Children who carried the haplotype pairs 3435CT/1236TT and 3435CT/1236CT showed a slower progression to AIDS, with an AIDS-free time median of 188 and 64 months, respectively (P = 0.007, relative hazard = 0.282, CI = 0.111–0.714 and P = 0.019, relative hazard = 0.507, CI = 0.287–0.895; Fig. 3 c). No significant difference was observed for the haplotype pairs 3435CC/1236CT, 3435CC/1236TT and 3435TT/1236CT in progression to AIDS (data not shown).
Due to the known impact of HAART on disease course, a potential role of the age at HAART initiation and HAART duration could not be ruled out beforehand and should be adjusted as confounding factors. Thus, Kaplan–Meier analysis was alternatively adjusted including HAART duration as a dichotomic time-dependent covariate. Table 2 shows the adjusted analysis for a Cox proportional hazards model. Noteworthy, similar trends were observed in the adjusted (Table 2 ) and in the unadjusted models (Fig. 3 ), confirming an independent protective effect of the MDR1 haplotype pairs 3435CT/1236CT and 3435CT/1236TT on progression to pediatric AIDS.
Table 2: Progression to AIDS adjusted to age at HAART initiation and HAART duration.
Discussion
In this study, we explored the role of MDR1 C3435T and C1236T genetic variants on HIV-1 susceptibility and disease progression in a well established pediatric cohort. We found that heterozygosity for both polymorphisms and homozygosity for 1236TT significantly delay the onset of pediatric AIDS, whereas no effect was observed on HIV-1 vertical transmission .
In addition, the allelic frequency of the MDR1 C1236T and C3435T polymorphisms was estimated in an adult Argentinean healthy group. Both SNPs are highly frequent, reaching 44% and present strong linkage disequilibrium. Marked variations in allelic frequencies of the MDR1 SNPs have been described in different ethnic populations. The 3435T allele is equally frequent among whites (∼47%) [18,38,39] [40,41] [40] [18,38,39] P < 0.05). No difference in the distribution of MDR1 variants between our population and populations from Asia was observed (China and Japan) [40,41] [18] [41] P < 0.05).
Few studies address the role of MDR1 variants on HIV-1 susceptibility and the majority explored adult cohorts. We found no significant differences either in the genotype distribution or in the allelic frequencies of the C1236T and C3435T polymorphisms between HIV-1-infected and exposed uninfected children, demonstrating that these SNPs do not influence the susceptibility to natural HIV-1 infection acquired by vertical transmission . Our findings are in agreement with those of Ifergan et al. [32] et al. [31]
We showed that MDR1 polymorphisms have a protective disease-influencing effect against AIDS. Heterozygosity for 3435CT and 1236CT and homozygosity for 1236TT significantly delayed the onset of pediatric AIDS. We could not demonstrate a protective effect associated with the homozygosity for 3435TT. In agreement with this finding, Saitoh et al. [21] MDR1 3435CT predicts an advantage (heterosis) in relation to HIV infection. The heterosis phenomenon is common in humans and may occur in up to 50% of all genes associations [42] et al. [43] MBL2 ) alleles was significantly associated with improved survival in intensive care patients compared with the wild-type and mutant homozygous in response to both severe infections and acute inflammation.
The protective effect revealed by 3435CT heterozygotes was even more pronounced in the context of haplotype pairs containing the 1236T allele, haplotype pair 3435CT/1236TT being more protective than 3435CT/1236CT. Similar trends were observed when analysis was conducted before the initiation of HAART or adjusted to the age at HAART initiation and HAART duration, indicating that the impact of the C3435T and the C1236T SNPs on the course of the HIV infection is not confounded by antiretroviral therapy. In contrast to our findings, two reports on adult cohorts did not find any association between C3435T genotypes and disease progression [27,44] + T cells to decline from 500 to 200 cells/μl before treatment [27] [44] MDR1 SNPs on post-HAART AIDS progression because of the restricted number of events during the study period (AIDS events before HAART = 84, AIDS events under HAART = 26). Differences in the study populations, including ethnicity, sample size and study design in adult and pediatric cohorts such as different clinical endpoints of disease progression and antiretroviral regimens, may account for the discrepancies observed and make difficult the comparison between studies. As the influence of CCR5-Δ32 and SDF1–3′ A alleles in progression to pediatric AIDS has been controversial [36,45,46] MDR1 association to AIDS in this studied group.
Different hypotheses have tried to explain a possible relationship between P-gp and HIV-1, including the interaction of P-gp with other cellular membrane proteins such as CXCR4 and caveolin 1 (CAV-1). A strong positive correlation between CXCR4 and P-gp expression level and the co-localization of these proteins within the lymphocyte membrane were reported [19] [47–49] MDR1 genotypes might modulate the clinical course of HIV infection by P-gp. Further research is needed to test this hypothesis.
In conclusion, our results indicate that MDR1 C1236T and C3435T polymorphisms act as a genetic modifier of pediatric AIDS but do not seem to influence HIV-1 vertical transmission , suggesting that different molecular mechanisms may be involved, which need to be elucidated.
Acknowledgements
We gratefully thank Dr Gabriel Catano for critically reviewing the manuscript, Mrs Carmen Gálvez and Ms Natalia Beltramone for technical assistance and Mrs Silvia Marino for technical and scientific support in this study.
This work was partially supported by Fondo Nacional para Ciencia y Tecnología (FONCYT, PICT 25830) and Consejo Nacional de Investigación Científica y Tecnológica (CONICET, PIP 6057).
Part of the data was presented at the XVII International AIDS Conference organized by the International AIDS Society, Mexico City, Mexico, 3–8 August 2008.
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