With the implemented quality control-criteria for CNVs, 1565 CNVs (180 double deletions, 649 single deletions, 674 individual insertion and 62 double insertions) were characterized for 164 patients of the study population. The numbers of CNVs ranged from 3 to 28 per individual. The sizes of the CNVs ranged from 5739 to 1 131 066 base pairs and were tagged by 10–204 SNPs. As none of the CNVs passed the genome-wide significance threshold of 3.19 × 10−5 for both the common and internal IMT, the most notable CNV was found on chromosome 14 for common cIMT. The common CNV region of 51 632 base pairs ranging from SNPs rs11157552 to rs2141988 (genomic position 21 921 670 to 21 973 302 in chromosome 14) is within the T-cell Receptor Alpha (TCRA) gene. There are six individuals with a single deletion in this region; they had the log common cIMT residual measurements of 0.193 ± 0.132 versus −0.007 ± 0.150 mm in those without the deletion (P = 0.0005).
This report describes the first GWAS evaluating common and internal cIMT, markers of atherosclerosis, among HIV-positive white 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 (r 2 = 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 Fig. 3 and Table 1, individuals with the GG genotype have a higher common cIMT than the individuals with AA or AG genotypes. Further, another SNP rs2291734 (r 2 = 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 base pairs in chromosome 15 (Fig. 3). 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).
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 cardiovascular diseases, including atherosclerosis [1,4,5]. Most patients in industrialized nations are now treated with antiretroviral drugs. Like many cohorts, more than 94% of the FRAM individuals who had IMT performed had been on HAART and more than 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 are also a limitation of the study. Common cIMT can be measured with greater accuracy than the internal carotid artery . As, the cIMT measure is reproducible, and valid [47,48], the observed association of SNPs in the RYR3 gene with common cIMT is convincing.
Another limitation with this study is the small sample size. On the basis of the simulation-based power calculation, assuming a SD 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. Although 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.
We thank investigators and staff of the Fat Redistribution and Metabolic Change in HIV Infection (FRAM) Study. We would also like to thank the following individuals for their valuable consultation and input: Dr Hemant K. Tiwari with GWAS analyses, Dr Rebecca Scherzer with FRAM data, Dr Daniel O'Leary with the IMT data, and Dr Jerome I. Rotter with the study design. The parent study and this sub-study conformed to the procedures for informed consent approved by institutional review boards at all sponsoring organizations and to human-experimentation guidelines set forth by the United States Department of Health and Human Services. The FRAM study was supported by grants from the NIH (R01-DK57508, HL74814, and HL53359, K23 AI66943, and NIH center grants M01-RR00036, RR00051, RR00052, RR00054, RR00083, RR00636, RR00865 and UL1 RR024131), the Albert L. and Janet A. Schultz Supporting Foundation, and and with resources and the use of facilities of the Veterans Affairs Medical Center, San Francisco, California. The funding agencies had no role in the collection or analysis of the data. The genotyping efforts were supported by NCRR grant M01-RR00425 (GCRC), DERC grant DK063491, and the Cedars-Sinai Board of Governors Chair in Medical Genetics (JIR).
Sites and investigators: University Hospitals of Cleveland (Barbara Gripshover, MD); Tufts University (Abby Shevitz, MD (deceased) and Christine Wanke, MD); Stanford University (Andrew Zolopa, MD); University of Alabama at Birmingham (Michael Saag, MD); John Hopkins University (Joseph Cofrancesco, MD and Adrian Dobs, MD); University of Colorado Heath Sciences Center (Lisa Kosmiski, MD and Constance Benson, MD); University of North Carolina at Chapel Hill (David Wohl, MD and Charles van der Horst, MD); University of California at San Diego (Daniel Lee, MD and W. Christopher Mathews, MD); Washington University (E. Turner Overton, MD and William Powderly, MD); VA Medical Center, Atlanta (David Rimland, MD); University of California at Los Angeles (Judith Currier, MD); VA Medical Center, New York (Michael Simberkoff, MD); VA Medical Center, Washington DC (Cynthia Gibert, MD); St Luke's-Roosevelt Hospital Center (Donald Kotler, MD and Ellen Engelson, PhD); Kaiser Permanente, Oakland (Stephen Sidney, MD); University of Alabama at Birmingham (Cora E. Lewis, MD).
FRAM 2 Data Coordinating Center: University of Washington, Seattle (Richard A. Kronmal, PhD, Mary Louise Biggs, PhD, J. A. Christopher Delaney, Ph.D., and John Pearce).
Image reading centers: St Luke's-Roosevelt Hospital Center: (Steven Heymsfield, MD, Jack Wang, MS and Mark Punyanitya). Tufts New England Medical Center, Boston: (Daniel H. O'Leary, MD, Joseph Polak, MD, Anita P. Harrington).
Office of the principal investigator: University of California, San Francisco, Veterans Affairs Medical Center and the Northern California Institute for Research and Development: (Carl Grunfeld, MD, PhD, Phyllis Tien, MD, Peter Bacchetti, PhD, Michael Shlipak, MD, Rebecca Scherzer, PhD, Mae Pang, RN, MSN, Heather Southwell, MS, RD)
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