This report describes the first GWAS evaluating common and internal cIMT, markers of atherosclerosis, among HIV-positive Caucasian men. The results suggest that the gene ryanodine receptor 3 (RYR3), located on chromosome 15 is linked to atherosclerosis in HIV-infected individuals. Two SNPs, rs2229116 and rs7177922 which are in tight linkage disequilibrium (r2=0.97) are significantly associated with the common cIMT above the Bonferroni correction (p< 3.4 × 10−8 and p < 2.74 × 10−8, respectively). The rs2229116 SNP is a nonsynonymous polymorphism that has a missense function, as there is a residue change of Ile to Val resulting from the “A” to “G” nucleotide substitution in the sequence. As shown in and , individuals with the GG genotype have a higher common cIMT than the individuals with AA or AG genotypes. Further, another SNP rs2291734 (r2=0.64 with rs2229116) in RYR3 gene, is also associated with greater common cIMT (p<2.82×10−6). These three SNPs lie between recombination hotspots, located within genomic regions 31579227–31580636 and 31718354–31725688 bp in chromosome 15 (). Whether the association is due to the direct effects of the missense mutation should be investigated in future studies of the gene in the laboratory. Of note, another SNP, rs12046077 in the RYR2 gene (an isoform of RYR3) appears to be associated with common cIMT (p-value< 1.40×10−5).
Figure 3 A comprehensive distribution of 122 informative SNPs from the Illumina humanCNV370-quad beadchip in the RYR3 gene spanning from 31,390,469 to 31,945,595 bp genomic location in chromosome 15 and the −log (p-values) (left y-axis) of genomic association (more ...)
The sequenced RYR3
cDNA spans 15,564 bp and contains an open reading frame of 14,613 bp. RYR3,
along with its two isoforms, RYR1
], are tetramers consisting of 4 identical subunits. The corresponding proteins have a molecular mass around 2200 kDa making these the largest ion-channel proteins that specifically release Ca+2
from intracellular stores following transduction of various extracellular stimuli [38
]. RYR3 is found in human arterial endothelial cells and plays a role in endothelial vasodilation [39
]; endothelial vasodilation is compromised in atherosclerosis. It is of interest that HIV-1 Tat, an important factor in viral pathogenesis, has been shown to cause endothelial dysfunction [40
]. HIV-1 Tat can induce rapid loss of endoplasmic reticulum Ca+2
through the mediation of RYR
]. RYR has been shown to be up-regulated in the atherosclerotic aorta of atherosclerosis-prone mice compared to atherosclerosis-resistant mice [42
]. In a study of mice on western diet, oxidized LDL induced a dose-dependent rise in intracellular calcium in aortic endothelial cells and increased monocyte chemoattractant protein-1 production. Chemically inhibiting intracellular calcium alleviated the effect of oxidized LDL on monocyte chemoattractant protein-1 production [42
]. Though future laboratory studies are needed to determine the functional relationship of these RYR3 SNPS with the pathology of atherosclerosis among ART treated HIV-infected patients, evidence in the literature creates a foundation for a role of RYR calcium signaling in the inflammatory process of atherogenesis in the artery.
Similarly, a deletion of the CNV region within the TCRA
gene in chromosome 14 suggests an association with common cIMT. TCRA
encodes one of two polypeptide subunits that make up immunoglobulin-like integral membrane glycoproteins on the receptors of T cells, which are responsible for recognizing antigens bound to MHC molecules. In one study, genomic region around TCRA
has been localized for familiar hypertrophic cardiomyopathy [43
]. Other studies have shown the presence of T-lymphocytes, mostly bearing alpha/beta TCR, in atherosclerotic lesions [44
]. T-lymphocytes are a key target of HIV itself.
The present study involves a relatively homogenous population of white men receiving HAART therapy. It is known that HIV and antiretroviral therapy both have a cumulative effect on risk factors for CV diseases, including atherosclerosis [1
]. Most patients in industrialized nations are now treated with antiretroviral drugs. Like many cohorts, more than 94% of the FRAM subjects who had IMT performed had been on HAART and >97% had received antiretroviral drugs. Given these data, we cannot assess whether the genetic associations are unique to HAART. Although there was an adjustment for the differential duration of HAART, the cross-sectional measurements of IMTs is also a limitation of the study. Common cIMT can be measured with greater accuracy than the internal carotid artery [46
]. Since, the cIMT measure is reproducible, and valid [47
], the observed association of SNPs in RYR3
gene with common cIMT is convincing.
Another limitation with this study is the small sample size. Based on the simulation-based power calculation, assuming a standard deviation of approximately 0.15 units (in log-scale), as for common cIMT, with the sample size of this present study, there is 80% power to detect differences in the log-transformed common cIMT log(cIMT) of at least 0.08 for SNPs with MAF of 10% and at least 0.06 for MAF 25%. But theoretically, there would be no power to detect significant difference considering multiple comparisons. Of note however, the minor allele frequency (MAF) for the strongest result was 18% and the observed difference in log(cIMT) between highest and lowest groups (two homozygotes) was about 0.33, which would theoretically correspond to an additive model difference of 0.17. Thus, the finding from this study, which passes even stringent Bonferroni adjustment, suggests a true association and needs to be evaluated further.
Continued research is needed to understand the effects of HIV infection and antiretroviral therapy on CVD risk. Large-scale, long-term longitudinal studies are necessary to resolve the conflicting findings regarding accelerated atherosclerosis in HIV-infected individuals treated with HAART, but such studies may not be feasible. Therefore, further research on the underlying genetic influences may illuminate the molecular mechanisms involved in atherosclerosis within and outside the context of HIV and its treatment. Although the resulting polymorphisms seen in this study need to be validated in larger cohorts within similar and other ethnic populations, these genetic variants should lead to identification of biological pathways for atherosclerosis in the context of HIV/HAART. This information could be used to develop innovative therapeutic or preventive interventions to reduce the burden of atherosclerosis in this high-risk population. While the results from this study are preliminary, they show the potential for identification of atherosclerosis genes, particularly in the context of HIV and HAART treatment.