This study assesses the impact of the GNB3 825 SNP on HIV-1 acquisition and disease progression in two Kenyan cohorts: a cohort of female commercial sex workers at high risk of sexual transmission of HIV (Pumwani CSW cohort), and a low-risk perinatal HIV transmission cohort (PHT cohort). Given the data indicating population-specific effects of the 825T allele and the high T allele frequency in African compared to Caucasian populations (80% versus 20%, respectively), it is important to understand the effect of the SNP in a population of high HIV-1 prevalence . The effect of the SNP in the CSW cohort is of particular interest, as we have previously reported that a SNP in the CD4 gene, CD4 C868T, is associated with increased risk of HIV-1 acquisition in this cohort. The CD4 and GNB3 genes are located in close proximity on chromosome 12, and may contain SNPs in linkage disequilibrium. We, therefore, wondered whether the GNB3 825T allele might contribute to the epidemiological effects attributed to the CD4 SNP, but no significant linkage was found between the GNB3 825T and CD4 868T alleles.
Given recent evidence suggesting an important role for G protein signaling in promoting HIV-1 replication and entry into resting T cells, and the fact that the GNB3 C825T SNP is one of the most commonly described polymorphisms in human G protein subunit genes [1, 3, 23], it is important to attempt to replicate previously reported associations of the 825T allele with accelerated HIV-1 disease progression and HAART responses [7, 8]. The association of the 825TT genotype with increased risk of HIV-1 progression, however, is somewhat at odds with other reports suggesting a protective effect of the T allele in infection. GNB3 825T is associated with improved cellular responses to Hepatitis B vaccination, and the 825CC genotype may confer a poor response to interferon α/ribavirin therapy in Hepatitis C infection as well as increased risk of infant death due to infection [19, 24, 25]. Given that epidemiological studies linking the 825T allele to non-infectious diseases have found evidence of population-specific effects with little functional data to demonstrate the mechanisms of action of the T allele, it is important to confirm the associations between GNB3 825T and HIV-1 progression, as well as to better understand the impact of the SNP on immune function during chronic disease.
Surprisingly, we were unable to demonstrate any effect of the GNB3 825T allele on HIV-1 acquisition or disease progression in either cohort. Although there was a trend toward increased risk of infant HIV-1 acquisition via breastmilk transmission (p = 0.06 adjusted for maternal viral load), this reflected a total of 16 transmission events (the least frequent mode of mother-to-child transmission observed in this cohort) and was not accompanied by a difference in maternal breastmilk viral load. The lack of association with either sexual or vertical HIV-1 transmission did not appear to be due to any confounding effect of the CD4 868 SNP. To date, there have been no reports of increased HIV-1 acquisition associated with GNB3 genotype in any population. Our analysis also failed to confirm the previously reported observation that the GNB3 825TT genotype is associated with increased HIV-1 disease progression as measured by CD4 decline . Regardless of whether analysis was limited to patients followed from the time of seroconversion, or included HIV-1-positive participants with unknown seroconversion dates (with adjustment for baseline CD4 count), and whether disease progression endpoint was defined as CD4 < 350, CD4 < 250 and/or death, rate of CD4 decline or viral load increase over time, there was no association between GNB3 genotype and HIV-1 disease progression. A small subset of HIV-1-positive GNB3 genotyped patients from the CSW cohort for whom ARV initiation and CD4 count data were available were included in a survival analysis of time to CD4 rebound to > 300 cells/uL following ARV initiation. Among this group (n = 12 CC/CT, 17 TT), there was also no difference in response to ARV therapy (data not shown).
Given the high 825T allele frequency among African populations, it was important to determine whether GNB3 genotype would affect disease progression similarly to the reported effect in a study of Caucasian participants. The absence of a GNB3 effect on disease progression in this study does not necessarily imply that GNB3 genotype does not affect HIV progression in other ethnic populations; rather, it underscores the importance of replicating genetic association studies in populations with disparate allele frequencies. Furthermore, it cannot be ruled out that difference of cohort gender composition between this and previous studies contributed to the contrasting results. Studies of the GNB3 825 SNP in African populations are lacking in the literature, but our results, along with others , suggest that phenotypes associated with the 825T allele may not be detected in African populations, and reinforce the observation that more diverse studies are required. Another gap within the body of GNB3 literature lies in the lack of functional studies to accompany epidemiological analyses. Although the C to T mutation does not affect the amino acid sequence of the Gβ3 protein, it is associated with the production of truncated splice variants, Gβ3s and Gβ3s2 [5, 17]. Biochemical characterization of these transcripts suggests that they are biologically active, and promote increased signaling activity following G protein coupled receptor activation. However, data demonstrating Gβ3s/s2 expression at the protein level in vivo are still lacking [5, 27].
Given the documented effect of enhanced lymphocyte chemotaxis in healthy Caucasian 825T allele carriers  and the potential impact of the splice variants on immune signaling pathways, we assessed the impact of GNB3 genotype on cellular immune activation and plasma cytokine/chemokine levels in healthy and HIV-1-positive CSW cohort participants. No differences in acute or chronic activation marker expression (CD69, HLA-DR and CD38) on either CD4+ or CD8+ T cells were observed in HIV-1-negative participants, confirming and extending a previous report that HLA-DR expression was not altered across GNB3 genotypes in healthy Caucasians . The same study also indicated that healthy Caucasian 825T allele carriers have increased CD4+ T cell counts (with no differences in B cell or CD8+ T cell counts), but did not assess the proportion of any CD4+ T cell subpopulations such as Tregs . In the current study, we did not observe any differences in Treg proportion between GNB3 825CC/CT and TT participants.
Analyses among HIV-1-positive patients also failed to identify any differences in ex vivo immune activation as measured by CD69, HLA-DR, or CD38. To our knowledge, this is the first study to quantify activation markers among HIV-1-positive patients with respect to GNB3 genotype. We also measured expression of IL-7Rα (CD127) and Fas (CD95), two known correlates of disease progression. During HIV infection, IL-7Rα inversely correlates with immune activation and apoptosis, and positively correlates with CD4 count , while Fas expression positively correlates with disease progression [21, 28]. Neither marker was differentially expressed on CD4+ or CD8+ T cells between GNB3 genotypes. Given the limitations of cross-sectional studies in assessing differences between markers correlated with disease progression, we analysed all surface marker expression with adjustment for CD4 count, but still observed no differences between GNB3 genotypes. GNB3 825T allele carriers demonstrate increased SDF-1α-mediated lymphocyte chemotaxis  (a process mediated by G protein signaling); therefore, we wondered whether CXCR4 ligand SDF-1α or CCR5 ligand MIP-1β would be differently expressed between GNB3 genotypes, particularly during HIV-1 infection. Plasma TRAIL levels were also assessed due to the previously reported observation of decreased lymphocyte apoptosis among 825TT individuals . Consistent with the cell surface marker data, SDF-1α, MIP-1β and TRAIL levels were also similar between CC/CT and TT genotypes. While these results do not assess the impact of the GNB3 splice variants on intracellular signaling pathways nor imply that there is no impact of the 825 SNP on signal transduction, our data do suggest that GNB3 genotype does not have a substantial impact on co-receptor expression or the activation of bulk or CD4+CCR5+ T cells. Investigation of these markers in cohorts that do exhibit an impact of GNB3 genotype on HIV-1 progression may provide further information about the mediators of the rate of progression.