The determinants of HIV susceptibility are complex and reflect both biologic and behavioral issues. In areas where the HIV epidemic is developing, further information concerning both of these areas of risk is needed to plan effective preventive interventions. China is one such place with growing numbers of HIV-positive persons. The projected number of HIV infections in China is predicted to be 10 million by 2010 (1). Rates of infection are higher in certain areas such as the southwestern Yunnan province, where the route of transmission is predominantly through intravenous drug use (1). Epidemiologic evidence suggests that many of the 56 minority populations in China are at particularly high risk of acquiring HIV infection (2). Although some of these patterns reflect behavioral factors, further understanding of the biologic susceptibility to HIV in the different Chinese ethnicities, especially those at high risk, is needed.
One important component of biologic susceptibility to HIV is now recognized to be chemokine receptors. Certain chemokines such as RANTES, MIP-1α, and MIP-1β inhibit the entry of HIV-1 into cells (3,4). Further work has showed that some selected chemokine receptors function as HIV-1 coreceptors essential for viral entry into macrophages and T lymphocytes (5–9). One of these chemokine receptors is CCR5, which represents the major coreceptor for primary M-tropic HIV-1 strains. M-tropic HIV-1 strains predominate during the asymptomatic phase of the disease in infected individuals and are thought to play a large role in HIV-1 transmission. The CCR5 gene product is a member of the seven-transmembrane, G-protein-coupled receptor family, and the genomic sequence of CCR5 has been characterized (10,11). A mutant allele of the gene CCR5 with a 32-base pair deletion (denoted by Δccr5) that prevents HIV entry has also been found (12–14). Homozygotes for the mutation are highly, although not absolutely, resistant to HIV infection (12–14). Heterozygotes have a delay in progression to AIDS (14–17). This mutant allele is mainly found in members of the white population. We undertook this study to determine the role of the CCR5 receptor in the risk for HIV infection in the Han majority and six Chinese ethnic minorities.
The samples studied were obtained from the Yunnan province, Xinjiang Uygur autonomous region, and Inner Mongolia autonomous region in China. Each individual represents at least three generations of pure ethnic background (the donor, the donor's parents, and the donor's four grandparents were of the same ethnic origin), and all samples were from unrelated individuals. After obtaining written informed consent, demographic information and blood samples were obtained from adults belonging to the Han majority and the Meng, Zang, Weiwuer, Zhuang, Yi, and Dai minorities. Samples from 10 members of each group were examined.
BD (Becton Dickinson, USA) Vacutainer systems were used for collection of blood samples. Two 5-mL blood samples were obtained from each individual. One sample was collected in K3 EDTA anticoagulant and used for genotyping, and the other was collected in heparin anticoagulant and used for flow cytometry.
Genotyping of Individuals by PCR Analysis
Polymerase chain reaction (PCR) analysis was performed on genomic DNA samples, using 5´-CCTGGCTGTCGTCCATGCTG-3´ and 5´-CTGATCTAGAGCCATGTGCACAACTCT-3´ as forward and reverse primers, respectively. Reaction mixtures consisted of 40.82 μL of distilled water, 1 μL of 50× buffer (50mM KCl, 10mM Tris-Cl, and 0.1% Triton X-100, pH 9.0, at 25°C), 1.2mM MgCl2, 0.1mM dACG, 0.1mM dT, 2% formamide, 5 μL of target DNA, 5.8 pmol of each of the primers, and 2 U of Taq polymerase. Conditions for PCR analysis were as follows: 94°C for 5 minutes; 35 cycles of 94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 60 seconds; and 72°C for 5 minutes. After the PCR reaction, the PCR products were precipitated and dried for Eco RI cleavage. The cleavage solution consisted of 33 μL of distilled water, 4 μL of 10× buffer H (90mM Tris-HCl, 10mM MgCl2, and 50mM NaCl, pH 7.5, at 37°C), and 3 μL of Eco RI (20 U/μL). The PCR products were incubated with the cleavage solution for 3 hours at 37°C. Both the PCR product and the cleavage product for one individual were applied to 12% polyacrylamide gel (Figure 1).
Flow Cytometry Analysis of Expression of CCR5
The heparinized sample was used for flow cytometry. One 100-μL aliquot of blood sample mentioned above was incubated with 20 μL of R-phycoerythrin–conjugated mouse IgG2a (Becton Dickinson) for 30 minutes at room temperature in a dark place and served as the matching isotype control tube. Another 100-μL aliquot was incubated with 20 μL of mouse antibody to CCR5 R-phycoerythrin (PharMingen, USA) (IgG2a), antibody to CD4 FITC (Becton Dickinson), and antibody to CD3 CY-chrome (Becton Dickinson) for 30 minutes at room temperature in a dark place and served as the test tube. After incubation, the cells were incubated with 1:10 diluted lysing buffer (Becton Dickinson) for 10 minutes at room temperature in a dark place to remove red blood cells. Then, the cells were centrifuged for 5 minutes at 300 g, and the supernatant was discarded. The precipitate was centrifuged for 5 minutes at 300 g in 2 L of PBS supplemented with 1% BSA and 0.1% sodium azide, and the supernatant was discarded. Finally, the cells resuspended in 500 μL of PBS were analyzed on a Calibur flow cytometer (Becton Dickinson) for three-color fluorescence immediately (Figure 2). Scatter gates were chosen to acquire only small cells with low granularity, which were >95% lymphocytes.
Genotyping of Individuals by PCR Analysis
PCR analysis of samples from 10 members of each minority was performed to genotype individuals. There was a Δ32-CCR5 heterozygote in the Weiwuer minority (Figure 1), but no mutations were found in the other 69 samples investigated.
Flow Cytometry Analysis of Expression of CCR5
The mean percentages of CCR5 expression in the seven ethnic groups in China are shown in Table 1. Populations expressing CD3 (as a measure of T lymphocytes) and CD4 (found on T helper lymphocytes and some macrophages) were analyzed for the percentage expressing CCR5. The mean percentage of cells expressing CCR5 in the Dai minority was significantly different from the those of cells expressing CCR5 in all other minorities for both CD3+CCR5+ and CD4+CCR5+ sets (p < .01, one-way ANOVA). For the Weiwuer heterozygote subject, the percentage of lymphocytes expressing CD3+CCR5+ was 31.47%, and the percentage of lymphocytes expressing CD4+CCR5+ was 9.94% (data not shown), which were not significantly different than findings for the homozygotes examined.
Samples from 10 adults from seven different Chinese ethnicities (Han, Meng, Zang, Weiwuer, Zhuang, Yi, and Dai) were examined. There was only one CCR5 heterozygote in the Weiwuer minority, and no mutations were found in the other 69 samples investigated. The indigenous Chinese individuals of the Han majority and the Meng, Zang, Weiwuer, Zhuang, Yi, and Dai minorities have a significantly lower frequency of CCR5 mutation (Δccr-5; overall frequency, 0.007) than do the persons of Northern European descent (gene frequency, ≈10%) (17) and thus might have a higher susceptibility to HIV infection. The frequency of the CCR5 allele in Han Chinese was previously was found to be 0.00119 (18). The sole Δ32-CCR5 mutation in our study was found in a person of Weiwuer ethnicity. Most individuals of the Weiwuer minority live in the Xinjiang Uygur autonomous region in northwest China that is contiguous to Russia, and the finding of a heterozygote in this pilot study seems to be consistent with the CCR5 mutation presence in Russia (17), reflecting gene flow from Russia into Xinjiang. The one individual in the study found to be a Δ32 heterozygote had CCR5 expression measured by flow cytometry similar to those in wild-type individuals. CCR5 expression in heterozygotes is usually lower on the whole than in wild-type individuals; however, individual CCR5 expression is highly variable, and expression at levels similar to those in wild-type individuals in heterozygotes has been described (19).
We found that the mean CCR5 expression on cells harvested from the Dai minority was significantly higher than that on cells from other ethnic groups examined. Higher CCR5 expression was seen both in the total lymphocyte population and in the CD4+ cells, which would be the main target of HIV infection. Augmentation of CCR5 expression has been seen in the immune activation (20) associated with tuberculosis (21,22) and in malaria (23). CCR5 expression has been correlated with macrophage susceptibility to HIV in vitro (24,25). Higher CCR5 expression has been found in individuals whose HIV infections progress to AIDS compared with individuals whose HIV infections do not progress, even in CCR5 wild-type homozygotes (26). CCR5 cell density can be correlated with viral load (27) and CD4 cell decline (28). The elevated CCR5 expression in the Dai minority, whether due to increased immune activation or other factors, may give the Dai minority a greater CCR5-dependent susceptibility to acquisition of HIV infection. Indeed, most HIV-positive cases in the Yunnan province have been found in the Dai minority (2). Further studies to better understand the regulation of CCR5 in the Dai population seem warranted. In addition, consideration of biologic factors that might influence the transmission of HIV is critical for development of country-specific HIV prevention strategies.
The authors are grateful to Drs. Qiuling, Luyan, and Zhuanping and other local officers for their work in the sample collection and preparation.
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