Effect of CCR5-Δ32 Heterozygosity on HIV-1 Susceptibility: A Meta-Analysis

Background So far, many studies have investigated the distribution of CCR5 genotype between HIV-1 infected patients and uninfected people. However, no definite results have been put forward about whether heterozygosity for a 32-basepair deletion in CCR5 gene (CCR5-Δ32) can affect HIV-1 susceptibility. Methods We performed a meta-analysis of 18 studies including more than 12000 subjects for whom the CCR5-Δ32 polymorphism was genotyped. Odds ratio (OR) with 95% confidence interval (CI) were employed to assess the association of CCR5-Δ32 polymorphism with HIV-1 susceptibility. Results Compared with the wild-type CCR5 homozygotes, the pooled OR for CCR5-Δ32 heterozygotes was 1.02 (95%CI, 0.88–1.19) for healthy controls (HC) and 0.95 (95%CI, 0.71–1.26) for exposed uninfected (EU) controls. Similar results were found in stratified analysis by ethnicity, sample size and method of CCR5-Δ32 genotyping. Conclusions The meta-analysis indicated that HIV-1 susceptibility is not significantly affected by heterozygosity for CCR5-Δ32.


Introduction
Inter-individual variability in susceptibility to HIV-1 infection, transmission, disease progression, and response to antiviral therapy has been attributed to host variability in multiple genes [1]. CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) are co-receptors for the entry of human immunodeficiency virus type 1 (HIV-1) into target cells [2]. A natural knockout deletion of 32 bases in CCR5 gene introduces a premature stop codon resulting in truncated protein product [3]. People homozygous for CCR5-D32 are naturally resistant to R5 HIV infection and the heterozygous state is associated with up to 2-4 years delay in disease progression [4] , . Recently, Allers et al reported that they have successfully cured a HIV infected patient through CCR5-D32/D32 stem cell transplantation [5,6]. On the other hand, the evidence for protection from HIV-1 infection among CCR5-D32 heterozygotes is mixed. A meta-analysis of Despina et al suggested that perinatal infection rates are not strongly determined by the number of functional CCR5 receptors in the children [7]. For adults, some studies have reported that CCR5-D32 heterozygotes could be protective against HIV transmission [8][9][10][11][12][13][14][15], whereas others have not confirmed that [16][17][18][19][20][21][22][23][24][25][26][27][28]. Therefore, we performed a metaanalysis of the accumulated data to address this question definitively.

Search Strategy and Study Selection
English database of Google Scholar (GS), PubMed and Chinese database of CNKI were searched till June 2011 using key words: CCR5-D32 and HIV-1. Studies satisfying the following criteria were included: case-control studies reporting the association of CCR5-D32 genotype with HIV-1 susceptibility, distribution of CCR5-D32 genotype between the cohorts was shown, not a prenatal HIV-1 infection study.

Data Extraction and Statistical Analysis
Two reviewers( SiJie Liu, Jie Wu) independently performed data extraction and then checked the results together. The following information was extracted from included studies: authors, year of publication, ethnicity, country, sample size, method of CCR5-D32 genotyping and CCR5-D32 genotype of cohorts.
Odds ratio (OR) and its 95% confidence intervals (CI) were used to evaluate the association of CCR5-D32 heterozygotes with HIV-1 susceptibility. Subgroups were identified by ethnicity, sample size and method of CCR5-D32 genotyping. A chi-squarebased Q-test was carried out to assess heterogeneity across studies [29]. A P value less than 0.10 was used to denote statistical significance. Fixed effects (Mantel and Haenszel) model was employed to pool the effects of studies without heterogeneity, otherwise the random effects (Dersirmonian and Laird) model was used [30,31]. Publication bias was evaluated by Egger's and Begg's test with funnel plots [32,33]. Asymmetry of the funnel plot suggests publication bias. A P value less than 0.05 was used to denote statistical significance. One-way sensitivity analyses were performed to examine the influence of individual studies on metaanalysis's results. Data were analyzed using Stata version 10.0 (StataCorp, College Station, Tex). Figure 1 summarized the selection process of literatures. The electronic search yielded 1232 records, after screening over titles and/or abstracts, 24 articles were selected for further review. Finally, 18 studies involving 6427 cases and 5809 controls were included in the meta-analysis. Study sample size ranged from 140 to 2605 subjects. Study characteristics of the 18 eligible studies were summarized in Table 1. Distribution of CCR5 genotype among subjects was shown in Table 2. Briefly, 9 studies involved Caucasian subjects [8,[10][11][12]17,[22][23][24], 4 studies involved Mongoloid subjects [16,21,25,28], 3 studies involved African subjects [8,12,24], 3 studies involved Latina subjects [12,18,27]. In addition to CCR5-D32 genotype, 8 studies provided the subjects' CCR2-64I genotype [10,16,[19][20][21][26][27][28], 5 studies provided the subjects' SDF-1 genotype [16,20,21,26,27]. All studies were done in subjects of mixed genders except that by Downer et al [24], which only included women.
We also performed stratified analysis by ethnicity, sample size and method of CCR5-D32 genotyping. The results were summarized in Table 3. All the results were consisted with overall analysis and no publication bias were observed.
Sensitive analysis was conducted by deleting one study at a time to examine the influence of individual data-set to the pooled ORs. All of the corresponding pooled ORs were not materially altered (Data not shown).

Discussion
Meta-analysis offers a powerful method to synthesize information of independent studies with similar intention. It has been proved that CCR5-D32 homozygotes are associated with near complete protection to HIV-1 infection. Moreover, published data have demonstrated that a disease-retarding effect of CCR5-D32 heterozygosity in HIV-1 infected individuals [34]. Whereas it   The study demonstrated that CCR5-D32 heterozygosity has little or no protective effect against HIV-1 infection among adults. This result is similar to a previous study of perinatal HIV-1 infection [7]. Several factors might underlie the lack of observed association between CCR5-D32 heterozygosity and HIV-1 susceptibility. First, the expression of CCR5 is influenced by factors other than CCR5 genotype. Even an individual with CCR5-D32 heterozygosity could still express high level of CCR5 [35,36]. Second, susceptibility to HIV-1 infection is affected by a combination of genes besides CCR5. The CCR5-D32 heterozygotes couldn't provide a full resistant to HIV-1 infection as the homozygotes. It is possible that a single D32 allele exerts a protective effect against HIV-1 infection only if it occurs combined with other protective factors [9].
There are a number of limitations to our study. First, although test of publication bias have generated negative results, studies solely in conference or in local journals may have been overlooked. Second, HIV-1 of X4 strain take advantage of CXCR4 as coreceptor. It has been reported that new infections in individuals are primarily established by strains that use R5 [37][38][39]. Currently, it is remains controversial about if HIV could use CXCR4 as co-receptor in primary HIV infection [40]. Primary infection with CXCR4-using HIV-1 strains is believed to be a rare event [41]. Thus, we might believe that in most of the cases HIV-1 R5 strain cause the initial infection, rather than the X4 strain. Although we couldn't exclude the interference of X4 viruses, it is unlikely that virus of X4 strain would significantly affect the results. Third, controls of some studies were solely derived from healthy individuals. For studies concerning disease susceptibility, it'll be more proper to take samples from exposed uninfected people as controls. Fourth, susceptibility to HIV-1 is influenced by multiple factors other than CCR5, they might interfere the precision of analysis.
In conclusion, our study involving more than 12000 subjects suggested that CCR5-D32 heterozygosity has little effect on protecting from HIV-1 infection. Therefore, other chemokine receptors and transmission mechanisms may play a more important role.

Author Contributions
Conceived and designed the experiments: HZZ. Performed the experiments: SJL CJK JW. Analyzed the data: SJL JW. Contributed reagents/ materials/analysis tools: SJL CJK HY. Wrote the paper: SJL. Figure 3. Funnel plots to detect publication bias in the meta-analysis. (a) Healthy controls considered; (b) exposed uninfected controls considered. The horizontal line indicates the pooled log odds ratio (OR) and guidelines to assist in visualizing the funnel are pooled at 95% pseudo confidence limits for this estimate. doi:10.1371/journal.pone.0035020.g003 Table 3. Stratified analysis of CCR5-D32 heterozygotes and susceptibility to HIV-1.