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Male-Specific Region of the Y Chromosome and Cardiovascular Risk Phylogenetic Analysis and Gene Expression Studies Lisa D.S. Bloomer, Christopher P. Nelson, James Eales, Matthew Denniff, Paraskevi Christofidou, Radoslaw Debiec, Jasbir Moore, Cardiogenics Consortium, Ewa Zukowska-Szczechowska, Alison H. Goodall, John Thompson, Nilesh J. Samani, Fadi J. Charchar, Maciej Tomaszewski Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 Objective—Haplogroup I of male-specific region of the human Y chromosome is associated with 50% increased risk of coronary artery disease. It is not clear to what extent conventional cardiovascular risk factors and genes of the malespecific region may explain this association. Approach and Results—A total of 1988 biologically unrelated men from 4 white European populations were genotyped using 11 Y chromosome single nucleotide polymorphisms and classified into 13 most common European haplogroups. Approximately 75% to 93% of the haplotypic variation of the Y chromosome in all cohorts was attributable to I, R1a, and R1b1b2 lineages. None of traditional cardiovascular risk factors, including body mass index, blood pressures, lipids, glucose, C-reactive protein, creatinine, and insulin resistance, was associated with haplogroup I of the Y chromosome in the joint inverse variance meta-analysis. Fourteen of 15 ubiquitous single-copy genes of the male-specific region were expressed in human macrophages. When compared with men with other haplogroups, carriers of haplogroup I had ≈0.61and 0.64-fold lower expression of ubiquitously transcribed tetratricopeptide repeat, Y-linked gene (UTY) and protein kinase, Y-linked, pseudogene (PRKY) in macrophages (P=0.0001 and P=0.002, respectively). Conclusions—Coronary artery disease predisposing haplogroup I of the Y chromosome is associated with downregulation of UTY and PRKY genes in macrophages but not with conventional cardiovascular risk factors. (Arterioscler Thromb Vasc Biol. 2013;33:1722-1727.) Key Words: association ◼ cardiovascular risk ◼ DNA ◼ gene ◼ genetics ◼ mRNA T he human Y chromosome is one of the smallest in the genome. Its major part, the male-specific region (MSY), constitutes ≈95% of its length, does not recombine with the X chromosome during meiosis, and is inherited as an indivisible unit from fathers to sons.1 Because of its haploid nature, the MSY has been routinely excluded from large-scale genomewide association studies and is one of the most underexplored portions of the human DNA. A growing body of evidence suggests that the human MSY may contribute to cardiovascular risk in men. Several studies conducted in the general population revealed associations between single nucleotide polymorphisms of the MSY and blood pressure,2–4 low-density lipoprotein-cholesterol,5 a surrogate of proatherogenic B phenotype of low-density lipoprotein-cholesterol particles, and paternal history of myocardial infarction.5 Our recent phylogenetic analysis showed that one of the most common European lineages of the Y chromosome (haplogroup I) was associated with increased risk of coronary artery disease (CAD) among British men.6 Indeed, the analysis of ≈3000 men from 2 British cohorts revealed that compared with other lineages of the Y chromosome, haplogroup I increased risk of CAD by ≈50%.6 This makes haplogroup I of the Y chromosome one of the strongest common genetic risk factors of CAD known to date.6 The biological mechanisms underlying the association between CAD and human Y chromosome are not fully understood. Our previous transcriptome-wide analysis of human macrophages revealed that compared with other Y chromosome lineages, haplogroup I was associated with suppression of adaptive immunity and increased response to inflammation.6 Specifically, 19 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways connected by genes of immunity and inflammation showed differential regulation in macrophages between men with haplogroup I and carriers of the other MSY lineages.6 However, it is not clear which genes of the MSY may drive the association between haplogroup I Received on: October 27, 2012; final version accepted on: April 11, 2013. From the Department of Cardiovascular Sciences (L.D.S.B., C.P.N., J.E., M.D., P.C., R.D., J.M., A.H.G., N.J.S., F.J.C., M.T.) and Department of Health Sciences (J.T.), University of Leicester, Leicester, United Kingdom; Leicester National Institute for Health Research Biomedical Research Unit in Cardio vascular Disease, Glenfield Hospital, Leicester, United Kingdom (C.P.N., A.H.G., J.T., N.J.S., M.T.); Department of Internal Medicine, Diabetology, and Nephrology, Medical University of Silesia, Zabrze, Poland (E.Z.-S.); and School of Science and Engineering, University of Ballarat, Ballarat, Australia (F.J.C.). The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.301608/-/DC1. Correspondence to Maciej Tomaszewski, MD, FAHA, FRCP, Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield Hospital, Leicester LE3 9QP, United Kingdom. E-mail [email protected] © 2013 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org 1722 DOI: 10.1161/ATVBAHA.113.301608 Bloomer et al Y Chromosome and Cardiovascular Risk 1723 Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 and predisposition to CAD. Of the 27 distinct protein-coding genes of the MSY, 15 single-copy genes with homologs on the X chromosome (so-called X-degenerate genes) show mostly ubiquitous tissue expression (including cells and organs of cardiovascular system) and thus are the strongest biological candidates for further functional studies on MSY and cardiovascular disorders. So far little is known about their function and specific roles in male health and disease. It is also not clear whether the altered immune response may entirely explain the increased susceptibility to CAD in men with haplogroup I. The effect of the MSY on CAD risk could be mediated, at least to some extent, by traditional cardiovascular and metabolic risk factors, such as adiposity, blood pressure, lipids, glucose, or insulin resistance. Taken together these risk factors explain <50% individual variation in susceptibility to CAD. Although in our previous study the association between haplogroup I and CAD in the West of Scotland Coronary Prevention Study was independent of some sociodemographic and conventional cardiovascular risk factors, this analysis was limited only to 1 cohort of middle-age Scottish men, many of whom were on pharmacological treatment with a potential to affect measurements of several key phenotypes.6 Thus, it is difficult to extrapolate these data into apparently healthy general population of European men. Here, we examined whether CAD-related haplogroup I of the Y chromosome is associated with either previously investigated or new traditional cardiovascular risk factors in young men of white European ethnicity recruited from the general population. This was followed by a systematic comparative expression analysis of 15 ubiquitous X-degenerate genes of the MSY in macrophages from carriers of haplogroup I and men from other MSY lineages. Materials and Methods Materials and Methods are available in the online-only Supplement. In brief, 1940 biologically unrelated, apparently healthy men from 3 populations of white European ethnicity (Young Men Cardiovascular Association Study 1-YMCA1,5 Young Men Cardiovascular Association Study 2-YMCA27 and Genetic Regulation of Arterial Pressure in Humans in the Community-GRAPHIC8) were included in the genetic association analysis. Macrophage RNA from 48 white British men with a history of premature myocardial infarction (recruited into Cardiogenics Transcriptome Project)6 was used in MSY genes expression studies. Reconstruction of the MSY phylogenetic tree was based on information provided by 11 bi-allelic polymorphisms (M9, M35, M45, M89, M170, M173, M201, M207, M269, M304, and SRY10831) genotyped in all cohorts, as reported before (Table I in the online-only Data Supplement).6 This set of polymorphic variants allows classification of >95% of European Y chromosomes into major haplogroups (Y[xBR], BR[xE1b1b1,F], E1b1b1, F*, G, I, J, K*, P*, R*, R1*, R1a, R1b1b2).9–11 The presence of population stratification/population admixture was examined using information provided by autosomal SNPs from previous array-based experiments (GRAPHIC8, Cardiogenics6) or a panel of 34 ancestrally informative SNPs (YMCA1 and YMCA2).12–15 Gene expression analysis of all ubiquitous single-copy genes of MSY was conducted using quantitative real-time PCR. Results Clinical and Demographic Characteristics A total of 1068 and 509 unrelated men in Young Men Cardiovascular Association Study 1 (YMCA1) and YMCA2, respectively, were informative in the Y chromosome phylogenetic analysis. In the Genetic Regulation of Arterial Pressure in Humans in the Community (GRAPHIC) Study, 363 biologically unrelated men from the offspring generation provided full genetic information for haplogroup resolution. In the Cardiogenics cohort, 48 men had full DNA and mRNA information necessary for further haplogroup I–stratified gene expression analysis. The clinical characteristics of YMCA1, YMCA2, and GRAPHIC sons are shown in Table 1 and are largely consistent with the phenotypic characteristics of young, apparently healthy males from general population. The basic phenotypic information on men from the Cardiogenics Study included in this project is shown in Table II in the online-only Data Supplement. Table 1. Demographic and Clinical Characteristics of Men From YMCA1, YMCA2, and the GRAPHIC Study Phenotype YMCA1 YMCA2 GRAPHIC Sons 1068 509 363 Age, y 19.3 (3.6) 18.9 (3.3) BMI, kg/m2 22.9 (3.0) 22.6 (3.0) 25.1 (4.1) Clinic SBP, mm Hg 118.1 (13.2) 118.8 (13.2) 128.4 (13.0) Clinic DBP, mm Hg n 25.7 (5.0) 74.2 (7.9) 74.1 (7.9) 76.4 (9.6) TC, mmol/L 4.3 (0.9) 3.7 (1.0) 4.6 (0.9) HDL-C, mmol/L 1.2 (0.3) 1.1 (0.3) 1.3 (0.3) Triglycerides, mmol/L 1.1 (1.0–1.1) 0.9 (0.8–0.9) LDL-C, mmol/L 2.6 (0.9) 2.2 (0.8) Glucose, mmol/L Creatinine, µmol/L CRP, mg/L HOMA-IR … 2.5 (0.7) 4.8 (0.7) 4.4 (0.8) 4.9 (0.8) 82.8 (11.1) 74.9 (9.8) 90.8 (9.1) … … 1.6 (1.6–1.7) 1.6 (1.6–1.7) 0.08 (0.07–0.08) … Data are mean and SD or geometric mean and 95% confidence intervals (triglycerides, CRP, and HOMA-IR). BMI indicates body mass index; CRP, C-reactive protein; DBP, diastolic blood pressure; GRAPHIC, Genetic Regulation of Arterial Pressure in Humans in the Community; HDL-C, high-density lipoprotein-cholesterol; HOMAIR, homeostatic model assessment insulin resistance index; LDL-C, low-density lipoprotein-cholesterol; n, No. of subjects; SBP, systolic blood pressure; TC, total cholesterol; and YMCA, Young Men Cardiovascular Association Study. 1724 Arterioscler Thromb Vasc Biol July 2013 Figure 1. Frequency of Y chromosome haplogroups in each cohort. Colored bars and letters represent each haplogroup running from the evolutionarily oldest on the left to the youngest of the right (youngest haplogroup is shown in pink [R1b1b2]); M and SRY markers indicate the terminal marker of each haplogroup; data are percentages of individuals in each haplogroup. GRAPHIC sons indicates Genetic Regulation of Arterial Pressure in Humans in the Community; and YMCA, Young Men Cardiovascular Association Study. Phylogenetic Analysis of the Y Chromosome Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 A total of 1988 individuals from YMCA1, YMCA2, GRAPHIC, and Cardiogenics with sufficient genetic/phenotypic information were classified into 1 of 13 major European MSY haplogroups. Approximately 75% to 93% of the haplotypic variation in these cohorts was attributable to 3 lineages (I, R1a, and R1b1b2; Figure 1). As expected, haplogroup R1a was the most common in Polish populations, whereas R1b1b2 predominated in the British men (Figure 1). Haplogroup I (previously associated with increased risk of CAD) was the second most common haplogroup in each cohort. There was no evidence of population stratification/admixture in any of the cohorts (Figures I–III in the online-only Data Supplement). Association of Haplogroup I and Major Cardiovascular Risk Factors: Individual and Joint Analysis The cohort-specific associations between haplogroup I and conventional risk factors are shown in Tables III–V in the online-only Data Supplement. None of traditional cardiovascular risk factors was associated with haplogroup I in the meta-analysis of 1944 men from 3 populations (Table 2). There was no evidence of heterogeneity in the joint analysis (Table 2). Sensitivity analyses showed that adjustment for blood pressure–lowering effect of antihypertensive treatment did not affect the association findings from analysis of systolic blood pressure and diastolic blood pressure (data not shown). Association Between Haplogroup I and Expression of Single-Copy MSY Genes in Human Macrophages Of 15 MSY genes, 14 were expressed in human macrophages. Two genes (ubiquitously transcribed tetratricopeptide repeat, Y-linked gene [UTY] and protein kinase, Y-linked, pseudogene [PRKY]) showed statistically significant difference in expression between men with haplogroup I and those with other lineages of the Y chromosome after correction for multiple testing (Table VI in the online-only Data Supplement). Specifically, carriers of haplogroup I had ≈0.61- and 0.64-fold Table 2. The Association Between Haplogroup I and Cardiovascular Risk Factors in YMCA1, YMCA2, and GRPAHIC Sons: Fixed-Effect Age-Adjusted Inverse Variance Meta-Analysis Phenotype β-Coefficient (SE) BMI, kg/m −0.07 (0.19) 0.709 0.590 0.14 (0.81) 0.865 0.555 2 Clinic SBP, mm Hg P Value Heterogeneity Clinic DBP, mm Hg −0.32 (0.49) 0.524 0.356 TC, mmol/L −0.01 (0.06) 0.862 0.131 HDL-C, mmol/L 0.00 (0.02) 0.798 0.113 Triglycerides, mmol/L* 0.04 (0.03) 0.161 0.823 LDL-C, mmol/L −0.01 (0.05) 0.775 0.017 Glucose, mmol/L −0.01 (0.05) 0.820 0.445 Creatinine, µmol/L −0.02 (0.62) 0.972 0.937 0.18 (0.17) 0.290 — −0.11 (0.10) 0.263 0.624 CRP, mg/dL** HOMA-IR* Data are expressed as β-coefficients, SE, and levels of statistical significance from meta-analysis; heterogeneity, level of statistical significance from χ2 test for heterogeneity. BMI indicates body mass index; CRP, C-reactive protein; DBP, diastolic blood pressure; GRAPHIC, Genetic Regulation of Arterial Pressure in Humans in the Community; HDL-C, high-density lipoprotein-cholesterol; HOMA-IR, homeostatic model assessment insulin resistance index; LDL-C, low-density lipoprotein-cholesterol; SBP, systolic blood pressure; TC, total cholesterol; and YMCA, Young Men Cardiovascular Association Study. *YMCA1 and YMCA2 only; and **GRPAHIC sons only. Bloomer et al Y Chromosome and Cardiovascular Risk 1725 Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 Figure 2. Macrophage expression of UTY and PRKY mRNA in British men from Cardiogenics cohort. A, Differences in relative (β-actin normalized) expression of PRKY between men with haplogroup I and carriers of other haplogroups. B, Differences in relative (β-actin normalized) expression of UTY between men with haplogroup I and carriers of other haplogroups. C, Linear correlation in macrophage expression of UTY and PRKY. lower age-adjusted levels of UTY and PRKY in macrophages than men with non-I haplogroups (P=0.0001 and P=0.002, respectively; Figure 2). There was a significant linear correlation in mRNA levels of both genes in human macrophages (r=0.486; P=0.0005; Figure 2). Discussion Our data revealed that CAD-predisposing haplogroup I of the Y chromosome6 was not associated with any major conventional cardiovascular risk factors in apparently healthy men from the general population of white European ancestry. The systematic analysis of all ubiquitous single-copy MSY X-degenerate genes revealed that a vast majority of them are indeed expressed in human macrophages. We also uncovered a significant downregulation of 2 MSY genes (UTY and PRKY) in macrophages from men with haplogroup I when compared with carriers of other lineages. Our data from YMCA studies and the GRPAHIC cohort provide important support for previously proposed notion that the association between haplogroup I of the Y chromosome and CAD is independent of traditional cardiovascular risk factors.6 Indeed, using continuous measures of cardiovascular risk (essentially un-confounded by medications), we showed that neither blood pressures nor metabolic phenotypes were associated with haplogroup I of the Y chromosome among apparently healthy men. The absence of association between the Y chromosome and C-reactive protein in this analysis is particularly important given that CAD-related haplogroup I was linked to immunity and inflammation in the recent transcriptome-wide analysis of human macrophages.6 The data collected here show that C-reactive protein, a simple measure of low-grade inflammation16,17 and increasingly recognized surrogate of cardiovascular risk,16 is unlikely to explain the reported associations between haplogroup I and increased susceptibility to CAD6 or possibly higher risk of AIDS progression or failure of retroviral therapy.18 Our data also reveal that other correlates of CAD not included in the previous observation,6 such as creatinine levels (a measure of renal function) and insulin resistance, do not track with haplogroup I of the Y chromosome among young apparently healthy men. Insulin resistance is a recognized contributor to metabolic syndrome, type 2 diabetes mellitus, as well as CAD19 and data from experimental models suggested a linkage between the Y chromosome and measures of insulin sensitivity.20 Estimated glomerular filtration rate, a measure of renal function, is also associated with the risk of CAD and cardiovascular mortality in the general population.21,22 In this study, instead of estimated glomerular filtration rate we used creatinine levels because a significant proportion of YMCA men was aged <18 years, and The Modification of Diet in Renal Disease-estimated glomerular filtration rate equation is simply not validated as a measure of renal function in this age category. The inclusion of these additional cardiovascular risk factors in our analysis extended the range of conventional correlates of CAD that are unlikely to account for its association with the haplogroup I of the Y chromosome. Our gene expression analysis provided the first insights into MSY transcriptome in human macrophages. This cell type is a recognized player in the development of atherosclerosis.23 Perhaps more importantly, macrophages from men with haplogroup I showed suppression of adaptive immunity and upregulation of proinflammatory response pathways when compared with other haplogroups.6 Indeed, we hypothesized that changes in activity/expression of these pathways in human macrophages could possibly mediate the effect of MSY on CAD.6 However, it was not known which MSY genes and transcripts may be the biological drivers of the identified findings. In this context, association between 1726 Arterioscler Thromb Vasc Biol July 2013 Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 CAD-predisposing haplogroup I and 2 mRNAs of MSY (PRKY and UTY) reported here is an important step forward to elucidate the role of the Y chromosome in atherosclerosis. PRKY encodes one of cAMP-dependent serine/threonine protein kinases. These enzymes are key messengers in the cellular responses to cAMP and act through phosphorylation of proteins to activate (or deactivate) receptors in different cells and tissues. PRKY is classified as a transcribed pseudogene; it lost its exon 6 and a part of exon 5 (encoding functionally active domains of the kinase) and is prone to nonsense-mediated decay. The biological role of PRKY is not clear. Its fully functional homolog on the X chromosome (PRKX) is involved in macrophage maturation and kidney development.24,25 The protein product of UTY belongs to one of the histocompatibility antigens recognized by T cells.26 Expressed in a wide variety of tissues,26 UTY elicits T-cell response in allogenic transplantation and is hypothesized to play a major role in graft-versushost disease and male grafts rejections.27 Interestingly, both allograft rejection and graft-versus-host-disease KEGG pathways were associated with haplogroup I in macrophage transcriptome profiling in our previous study.6 The results shown here together with previously published data suggest that of 2 genes, UTY is a biologically more likely mediator of the association between haplogroup I and immune system, and possibly CAD. However, further studies are warranted to confirm the direct association between the UTY (PRKY) and CAD and elucidate the biological mechanisms of these findings. In summary, our results show that haplogroup I of the Y chromosome previously linked to increased risk of CAD does not track with conventional cardiovascular and metabolic risk factors in young men from the general white European population. We also show for the first time that haplogroup I is associated with expression of ≥2 MSY genes, of which 1 is a strong biological candidate linked to immune system. Further studies should focus on functional characterization of biological underpinnings of the association between haplogroup I and UTY/PRKY expression to fully elucidate the mechanism of increased susceptibility to CAD among men with haplogroup I of the Y chromosome. Disclosures This study was funded by the British Heart Foundation project grants (PG/06/097/21331 and PG/12/9/29376) to Maciej Tomaszewski; Departmental PhD scholarship to Lisa D.S. Bloomer and University Hospitals of Leicester National Health Service (NHS) Charitable Funds (M61RT31) to Maciej Tomaszewski and Lisa D.S. Bloomer. Christopher P. Nelson is supported by National Institute for Health Research (NIHR) Leicester Cardiovascular Biomedical Research Unit. James Eales is supported by British Heart Foundation grant (PG/12/9/29376) to Maciej Tomaszewski. Fadi J. Charchar is supported by research grants from the LEW Carty Charitable Fund and National Health and Medical Research Council of Australia. Nilesh J. Samani holds a personal chair supported by the British Heart Foundation and is a UK NIHR senior investigator. The Cardiogenics project was supported by the European Union 6th Framework Program (LSHM-CT-2006–037593). The remaining authors report no conflicts. References 1.Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, et al. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature. 2003;423:825–837. 2. Charchar FJ, Tomaszewski M, Strahorn P, Champagne B, Dominiczak AF. Y is there a risk to being male? Trends Endocrinol Metab. 2003;14: 163–168. 3. Charchar FJ, Tomaszewski M, Padmanabhan S, Lacka B, Upton MN, Inglis GC, Anderson NH, McConnachie A, Zukowska-Szczechowska E, Grzeszczak W, Connell JM, Watt GC, Dominiczak AF. The Y chromosome effect on blood pressure in two European populations. Hypertension. 2002;39(2 Pt 2):353–356. 4. Ellis JA, Stebbing M, Harrap SB. Association of the human Y chromosome with high blood pressure in the general population. Hypertension. 2000;36:731–733. 5.Charchar FJ, Tomaszewski M, Lacka B, Zakrzewski J, ZukowskaSzczechowska E, Grzeszczak W, Dominiczak AF. Association of the human Y chromosome with cholesterol levels in the general population. Arterioscler Thromb Vasc Biol. 2004;24:308–312. 6. Charchar FJ, Bloomer LD, Barnes TA, et al. Inheritance of coronary artery disease in men: an analysis of the role of the Y chromosome. Lancet. 2012;379:915–922. 7.Tomaszewski M, Charchar FJ, Maric C, McClure J, Crawford L, Grzeszczak W, Sattar N, Zukowska-Szczechowska E, Dominiczak AF. Glomerular hyperfiltration: a new marker of metabolic risk. Kidney Int. 2007;71:816–821. 8.Tomaszewski M, Debiec R, Braund PS, et al. Genetic architecture of ambulatory blood pressure in the general population: insights from cardiovascular gene-centric array. Hypertension. 2010;56:1069–1076. 9. King TE, Jobling MA. What’s in a name? Y chromosomes, surnames and the genetic genealogy revolution. Trends Genet. 2009;25:351–360. 10. Y Chromosome Consortium. A nomenclature system for the tree of human Y-chromosomal binary haplogroups. Genome Res. 2002;12:339–348. 11.Karafet TM, Mendez FL, Meilerman MB, Underhill PA, Zegura SL, Hammer MF. New binary polymorphisms reshape and increase resolution of the human Y chromosomal haplogroup tree. Genome Res. 2008;18:830–838. 12. Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000;155:945–959. 13.Falush D, Stephens M, Pritchard JK. Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics. 2003;164:1567–1587. 14.Falush D, Stephens M, Pritchard JK. Inference of population structure using multilocus genotype data: dominant markers and null alleles. Mol Ecol Notes. 2007;7:574–578. 15. Hubisz MJ, Falush D, Stephens M, Pritchard JK. Inferring weak population structure with the assistance of sample group information. Mol Ecol Resour. 2009;9:1322–1332. 16.Ridker PM. High-sensitivity C-reactive protein, inflammation, and cardiovascular risk: from concept to clinical practice to clinical benefit. Am Heart J. 2004;148(1 Suppl):S19–S26. 17.Ridker P, Rifai N, Koenig W, Blumenthal RS. C-reactive protein and cardiovascular risk in the Framingham Study. Arch Intern Med. 2006;166:1327–8; author reply 1328. 18. Sezgin E, Lind JM, Shrestha S, Hendrickson S, Goedert JJ, Donfield S, Kirk GD, Phair JP, Troyer JL, O’Brien SJ, Smith MW. Association of Y chromosome haplogroup I with HIV progression, and HAART outcome. Hum Genet. 2009;125:281–294. 19.Reaven G. Insulin resistance and coronary heart disease in nondiabetic individuals. Arterioscler Thromb Vasc Biol. 2012;32:1754–1759. 20.Strahorn P, Graham D, Charchar FJ, Sattar N, McBride MW, Dominiczak AF. Genetic determinants of metabolic syndrome components in the stroke-prone spontaneously hypertensive rat. J Hypertens. 2005;23:2179–2186. 21.Di Angelantonio E, Chowdhury R, Sarwar N, Aspelund T, Danesh J, Gudnason V. Chronic kidney disease and risk of major cardiovascular disease and non-vascular mortality: prospective population based cohort study. BMJ. 2010;341:c4986. 22.Hallan S, Astor B, Romundstad S, Aasarød K, Kvenild K, Coresh J. Association of kidney function and albuminuria with cardiovascular mortality in older vs younger individuals: The HUNT II Study. Arch Intern Med. 2007;167:2490–2496. 23. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809. 24.Glesne D, Huberman E. Smad6 is a protein kinase X phosphoryla tion substrate and is required for HL-60 cell differentiation. Oncogene. 2006;25:4086–4098. 25. Li X, Li HP, Amsler K, Hyink D, Wilson PD, Burrow CR. PRKX, a phylogenetically and functionally distinct cAMP-dependent protein kinase, Bloomer et al Y Chromosome and Cardiovascular Risk 1727 activates renal epithelial cell migration and morphogenesis. Proc Natl Acad Sci U S A. 2002;99:9260–9265. 26. Warren EH, Gavin MA, Simpson E, Chandler P, Page DC, Disteche C, Stankey KA, Greenberg PD, Riddell SR. The human UTY gene encodes a novel HLA-B8-restricted H-Y antigen. J Immunol. 2000;164:2807–2814. 27.Vogt MH, Goulmy E, Kloosterboer FM, Blokland E, de Paus RA, Willemze R, Falkenburg JH. UTY gene codes for an HLA-B60-restricted human male-specific minor histocompatibility antigen involved in stem cell graft rejection: characterization of the critical polymorphic amino acid residues for T-cell recognition. Blood. 2000;96:3126–3132. Significance Compared with carriers of other haplogroups, men with haplogroup I of the Y chromosome exhibit increased susceptibility to coronary artery disease, possibly because of downregulation of adaptive immunity and upregulation of response to inflammation pathways in macrophages. Traditional cardiovascular risk factors, including body weight, blood pressure, lipids, glucose, C-reactive protein, measures of renal function, and insulin resistance, are unlikely mediators of the association between haplogroup I and coronary artery disease. Fourteen of 15 ubiquitous single-copy genes of the male-specific region of the Y chromosome are expressed in human macrophages. Two of these genes (UTY and PRKY) are downregulated in macrophages from carriers of haplogroup I when compared with men with other haplogroups of the Y chromosome. Future functional studies will clarify contributions of these genes to haplogroup I–driven risk of coronary artery disease. Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 Male-Specific Region of the Y Chromosome and Cardiovascular Risk: Phylogenetic Analysis and Gene Expression Studies Lisa D.S. Bloomer, Christopher P. Nelson, James Eales, Matthew Denniff, Paraskevi Christofidou, Radoslaw Debiec, Jasbir Moore, Cardiogenics Consortium, Ewa Zukowska-Szczechowska, Alison H. Goodall, John Thompson, Nilesh J. Samani, Fadi J. Charchar and Maciej Tomaszewski Arterioscler Thromb Vasc Biol. 2013;33:1722-1727; originally published online May 2, 2013; doi: 10.1161/ATVBAHA.113.301608 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2013 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://atvb.ahajournals.org/content/33/7/1722 Data Supplement (unedited) at: http://atvb.ahajournals.org/content/suppl/2013/05/02/ATVBAHA.113.301608.DC1 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at: http://atvb.ahajournals.org//subscriptions/ Male-specific region of the Y chromosome and cardiovascular risk – phylogenetic analysis and gene expression studies Lisa D. S. Bloomer (1), Christopher P. Nelson (1,2), James Eales (1), Matthew Denniff (1), Paraskevi Christofidou (1), Radoslaw Debiec (1), Jasbir Moore (1), CARDIOGENICS, Ewa ZukowskaSzczechowska (3), Alison H Goodall (1,2), John Thompson (2,4), Nilesh J. Samani (1,2), Fadi J. Charchar (1,5) and Maciej Tomaszewski (1,2) 1. Department of Cardiovascular Sciences, University of Leicester, Leicester, UK 2. Leicester National Institute for Health Research Biomedical Research Unit in Cardiovascular Disease, Glenfield Hospital, Leicester, UK 3. Department of Internal Medicine, Diabetology and Nephrology, Silesian School of Medicine, Zabrze, Poland 4. Department of Health Sciences, University of Leicester, Leicester, UK 5. School of Science and Engineering, University of Ballarat, Ballarat, Australia Corresponding author: Maciej Tomaszewski, MD, FAHA, FRCP Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield Hospital, LE3 9QP, UK, Tel: +44 (0)116 2044752 e-mail: [email protected] 1 Supplementary Table I. SNPs defining the major European Y chromosome haplogroups. Polymorphism symbol db SNP "rs" number M201 rs2032636 M304 rs13447352 M269 rs9786153 M207 rs2032658 M45 rs2032631 SRY10831 rs2534636 M173 rs2032624 M35 N/A M89 rs2032652 M9 rs3900 M170 rs2032597 SRY10831 is a recurrent mutation Alleles G->T A->C C->T A->G G->A A->G A->C G->C C->T C->G A->C 2 Haplogroup G J R1b1b2 R1b1b2 P B-R; R1a R1b1b2 E1b1b1 F-R K-R I Supplementary Table II. Clinical characteristics of men from Cardiogenics with available RNA. Phenotype Values n 48 Age (years) 54.6 (6.0) BMI (kg/m2) 28.7 (4.5) SBP (mmHg) 133.8 (20.1) DBP (mmHg) 82.5 (10.8) Data are means and standard deviations, n – number of subjects, BMI – body mass index, SBP – systolic blood pressure, DBP – diastolic blood pressure 3 Supplementary Table III. Cardiovascular risk factors stratified by Y chromosome haplogroups (I versus others) in Young Men Cardiovascular Association Study 1 (YMCA1). Phenotype I Other P-value n 190 878 BMI (kg/m2) 22.8 (3.1) 22.9 (3.0) 0.607 Clinic SBP (mmHg) 117.9 (12.8) 118.2 (13.3) 0.589 Clinic DBP (mmHg) 73.5 (7.4) 74.3 (8.0) 0.258 TC (mmol/L) 4.1 (0.9) 4.3 (0.9) 0.147 HDL-C (mmol/L) 1.2 (0.4) 1.1 (0.3) 0.213 Triglycerides (mmol/L) 1.1 (1.0 – 1.2) 1.1 (1.0 – 1.1) 0.190 LDL-C (mmol/L) 2.4 (0.9) 2.6 (0.9) 0.034 Glucose (mmol/L) 4.8 (0.8) 4.8 (0.7) 0.669 Creatinine (µmol/L) 82.6 (11.0) 82.9 (11.2) 0.978 HOMA-IR 1.6 (1.5 – 1.8) 1.6 (1.5 – 1.7) 0.548 Data are means and standard deviations or geometric means and 95% confidence intervals (triglycerides and HOMA-IR), n – number of subjects, BMI – body mass index, SBP – systolic blood pressure, DBP – diastolic blood pressure, TC – total cholesterol, HDL-C – high-density lipoprotein cholesterol, LDL-C – low-density lipoprotein cholesterol, HOMA-IR – homeostatic model assessment insulin resistance index, P-value – level of statistical significance after age-adjustment 4 Supplementary Table IV. Cardiovascular risk factors stratified by Y chromosome haplogroups (I versus others) in Young Men Cardiovascular Association Study 2 (YMCA2). Phenotype I Other P-value n 85 424 BMI (kg/m2) 22.7 (2.9) 22.5 (3.1) 0.650 Clinic SBP (mmHg) 120.3 (11.9) 118.6 (13.4) 0.354 Clinic DBP (mmHg) 73.9 (7.2) 74.1 (8.0) 0.786 TC (mmol/L) 3.9 (1.2) 3.7 (1.0) 0.204 HDL-C (mmol/L) 1.0 (0.3) 1.1 (0.3) 0.124 Triglycerides (mmol/L) 0.9 (0.8 – 1.0) 0.9 (0.8 – 0.9) 0.584 LDL-C (mmol/L) 2.4 (0.9) 2.2 (0.8) 0.054 Glucose (mmol/L) 4.4 (0.6) 4.4 (0.8) 0.560 Creatinine (µmol/L) 75.9 (9.7) 74.7 (9.8) 0.874 HOMA-IR 1.6 (1.5 – 1.7) 1.7 (1.6 – 1.7) 0.282 Data are means and standard deviations or geometric means and 95% confidence intervals (triglycerides and HOMA-IR), n – number of subjects, BMI – body mass index, SBP – systolic blood pressure, DBP – diastolic blood pressure, TC – total cholesterol, HDL-C – high-density lipoprotein cholesterol, LDL-C – low-density lipoprotein cholesterol, HOMA-IR – homeostatic model assessment insulin resistance index, P-value – age-adjusted level of statistical significance 5 Supplementary Table V. Cardiovascular risk factors stratified by Y chromosome haplogroups (I versus others) in sons recruited into Genetic Regulation of Arterial Pressure in Humans in the Community (GRAPHIC). Phenotype I Other P-value n 51 312 BMI (kg/m2) 25.5 (4.5) 25.1 (4.0) 0.395 Clinic SBP (mmHg) 128.7 (12.3) 128.4 (13.1) 0.814 Clinic DBP (mmHg) 77.5 (9.9) 76.3 (9.6) 0.292 TC (mmol/L) 4.6 (1.0) 4.6 (0.9) 0.544 HDL-C (mmol/L) 1.3 (0.3) 1.3 (0.3) 0.483 LDL-C (mmol/L) 2.5 (0.8) 2.5 (0.7) 0.884 Glucose (mmol/L) 5.0 (0.9) 4.9 (0.8) 0.285 Creatinine (µmol/L) 90.4 (9.7) 90.8 (9.1) 0.746 C-reactive protein 0.09 (0.06 – 1.12) 0.10 (0.06 – 0.08) 0.291 Data are means and standard deviations or geometric means and 95% confidence intervals, n –number of subjects, BMI – body mass index, SBP – systolic blood pressure, DBP – diastolic blood pressure, TC – total cholesterol, HDL-C – high-density lipoprotein cholesterol, LDL-C – low-density lipoprotein cholesterol, CRP – C-reactive protein, P-value – age-adjusted level of statistical significance 6 Supplementary Table VI. Association between haplogroup I of the Y chromosome and expression of 15 ubiquitous single-copy MSY genes in human macrophages from Cardiogenics. MSY gene Haplogroup I Other haplogroups P-value n 14 34 USP9Y 8.4 (1.0) 8.2 (0.8) 0.464 DDX3Y 6.1 (0.7) 5.8 (0.6) 0.087 NLGN4Y 13.9 (1.7) 13.3 (3.9) 0.637 TBL1Y 3.6 (1.0) 3.3 (0.9) 0.358 SRY NOT EXPRESSED TMSB4Y 10.0 (2.3) 11.1 (0.9) 0.011 EIF1AY 4.2 (0.5) 4.2 (0.5) 0.942 CYorf15A 9.4 (1.0) 9.4 (0.9) 0.693 CYorf15B 8.6 (1.5) 8.3 (1.6) 0.933 RPS4Y1 3.5 (0.5) 3.2 (0.6) 0.202 PRKY 8.2 (0.8) 7.6 (0.6) 0.002 UTY 8.4 (0.6) 7.8 (0.6) 0.0001 RPS4Y2 3.1 (0.4) 3.3 (0.8) 0.391 KDM5D 10.2 (1.9) 9.3 (2.1) 0.212 ZFY 7.0 (0.7) 7.0 (0.6) 0.881 MSY – male specific region of the Y chromosome, data are dCTs (means and standard errors) from real-time quantitative PCR, P-value – level of statistical significance after age-adjustment 7 Supplementary Figure I. Analysis of population admixture in Young Men Cardiovascular Association Study 1 (YMCA1) and 2 (YMCA2). The method predicts the likelihood of ancestry to a predefined set of subpopulations (Asian [blue], African [green] and European [red]). YMCA1 is shown in orange, YMCA2 is shown in purple. 8 Supplementary Figure II. A Analysis of population stratification in the GRAPHIC sons based on autosomal SNPs from previous large scale genetic association analysis. The X and Y axes represent the first two dimensions from multidimensional scaling (MDS) analysis on the matrix of genome-wide identity-by-state pair-wise distances. There is a clear proximity between men from GRPAHIC (red) and HapMap CEU population (yellow). HapMap YRI and JPT+CHB populations are shown in green and blue, respectively. B. individuals with haplogroup I (green) versus carriers of all other haplogroups (red). A B 9 Supplementary Figure III. A. Analysis of population stratification in men with available macrophage RNA from the Cardiogenics based on autosomal SNPs from previous genome-wide association scan. The X and Y axes represent the first two dimensions from multidimensional scaling (MDS) analysis on the matrix of genome-wide identity-by-state pair-wise distances. There is a clear proximity between men from Cardiogenics (red) and HapMap CEU population (yellow). HapMap YRI and JPT+CHB populations are shown in green and blue, respectively. B. individuals with haplogroup I (green) versus carriers of all other haplogroups (red). A B 11 10 Methods Populations Young Men Cardiovascular Association Study 1 and 2 (YMCA1 and YMCA2) The Young Men Cardiovascular Association Study 1 (YMCA1) consists of 1157 biologically unrelated apparently healthy males (mean age: 19.1 years) recruited in Silesia (Southern Poland) (1,2). The Young Men Cardiovascular Association Study 2 (YMCA2) - an extension of YMCA1, recruited an additional sample of unrelated 597 young (mean age: 19.0 years) men in Southern Poland (2). Clinical and biochemical phenotyping protocols of each study were described in detail in previous publications (1,2). In brief, clinical history was collected using anonomysed coded questionnaires. Recorded anthropometric measurement included height and weight as well as three consecutive blood pressure measurements (1,2). Each subject underwent thorough biochemical phenotyping (under fasting conditions) including analysis of fasting lipids (total cholesterol, HDL-C, triglycerides) glucose and creatinine. Circulating levels of insulin were measured by radioimmunoassay on 1470 Wizard automatic gamma counter and were used to calculate homeostatic model assessment insulin resistance index (HOMA-IR) [(insulin glucose)/22.5] (2). LDL-C was calculated using the Friedewald formula. Only 1.6% and 0.3 % men in YMCA1 and YMCA2 were on antihypertensive medication (1,2) and none were prescribed lipid-lowering medications. Blood pressure values from those on antihypertensive treatment were adjusted for blood pressure-lowering effect of therapy using previously reported method (1,2). Genetic Regulation of Arterial Pressure in Humans in the Community (GRAPHIC) The Genetic Regulation of Arterial Pressure in Humans in the Community (GRAPHIC) cohort consists of 2037 healthy individuals from 520 European British families (two parents, two offspring) recruited from the general population of Leicester, UK (3). A total of 1028 men of white European ethnicity were recruited (516 fathers [average age: 53.8 years] and 512 sons [average age 25.0 years]). Of 512 male offspring, 391 biologically unrelated individuals were selected for this analysis – in families with two male offspring only the elder sibling was included in this study. Clinical and biochemical phenotyping protocols of the GRAPHIC were described in detail elsewhere (3). In brief, clinical history was collected through standardised questionnaires alongside anthropometric measurements (height, weight and waist circumference). Each individual had their blood pressure measured using both clinic and 24-hour ambulatory monitoring (3). A blood sample was secured to measure total cholesterol, HDL-cholesterol, glucose, and creatinine. LDL-cholesterol was measured enzymatically. 0.8% of GRAPHIC sons were on antihypertensive medication. Blood pressure values from those on treatment were adjusted to correct for blood pressure-lowering effect of antihypertensive therapy as reported before (3). Cardiogenics Transcriptome Project Of 918 individuals recruited into this international multi-centre collaborative project, 849 and 684 underwent transcriptomic analysis on Illumina Human Ref-8 array, as reported before (4). Of 255 men included in the previous transcriptome-wide analysis, 48 had sufficient amount of RNA for measurement of MSY genes expression in macrophages. Only these individuals (all white British men with a history of premature myocardial infarction) were included in this study. Written, informed consent was obtained from each individual from all cohorts in accordance with the Declaration of Helsinki and all the studies had the approval of relevant institutional ethics committees. DNA and RNA analysis Genotyping and Y chromosome phylogenetic analysis DNA was extracted from peripheral leukocytes using standardised procedures. Genotyping was conducted using the iPLEX genotyping method ([Sequenom (San Diego, USA)] in the GRAPHIC Study or TaqMan [on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Cambridge, UK)] in YMCA1,YMCA2 and Cardiogenics Study. Eleven bi-allelic MSY polymorphisms (M9, M35, M45, M89, M170, M173, M201, M207, M269, M304, and SRY10831) (Supplementary Table I) were genotyped in all cohorts. The genotypes of 1 these polymorphisms were then used to map each Y chromosome into one of 13 major European branches (haplogroups) of the Y chromosome phylogenetic tree, as previously described (4). This set of polymorphic variants allows classification of >95% of European Y chromosomes into major haplogroups [Y(xBR), BR(xE1b1b1,F), E1b1b1, F*, G, I, J, K*, P*, R*, R1*, R1a, R1b1b2] (5). The haplogroups were named in accordance with the Y chromosome Consortium nomenclature (6-7); all males in one haplogroup are monophyletic for investigated SNPs. Analysis of population stratification/admixture The presence of population admixture in YMCA1 and YMCA2 was assessed using the extensively validated Structure program (http://pritch.bsd.uchicago.edu/software.html) (8-11). This method uses information from 34 ancestrally informative SNPs (rs10141763, rs1024116, rs1084344, rs12913832, rs1321333, rs1335873, rs142665, rs149844, rs1573020, rs16891982, rs182549, rs1886510, rs1978806, rs2026721, rs2040411, rs2065160, rs2065982, rs2303798, rs2304925, rs239031, rs2572307, rs2814778, rs3785181, rs4540055, rs5033240, rs5997008, rs722098, rs727811, rs730570, rs773658, rs7897550, rs881929, rs896788, rs917118) and predicts the likelihood of ancestry to a predefined set of subpopulations (Asian, African and European). Reference population genotypes were downloaded from http://spsmart.cesga.es/snpforid.php. The genotyping of the 34 ancestrally informative markers was conducted using KASP genotyping and SNPline instrumentation (KBiosciences). The analysis of population stratification in the GRAPHIC was conducted using SNPs from previously conducted 50 IBC gene-centric array-based experiment (3). In brief, ~1700 ancestrally informative SNPs present on the array were included in the principal component analysis. Non-metric, multidimensional scaling was then applied to visualise the genetic similarity between the major Y chromosome lineages. To ensure all individuals were of European descent, GRPAHIC men were visualised in the context of Hap Map populations of different ethnicities (CEU, JAP+CHB, Yoruba). Exclusion of population stratification in men from the Cardiogenics Study included in this project was conducted using principal component method and non-metric, multi-dimensional scaling, as reported before (4). Monocyte and macrophage isolation and RNA extraction Monocytes were isolated from whole blood using positive selection with CD14 magnetic beads on AutoMACS system (Miltenyi Biotech, Bergisch Gladblach, Germany) (12,13). Macrophages were derived from monocytes after culturing for 7 days in the presence of recombinant human M-CSF, as described previously (12-13). MSY gene expression analysis Only 50% of ubiquitous single-copy MSY genes were present on the microarray platform used in Cardiogenics and survived all quality control filters. Therefore, full expression analysis of all ubiquitous single-copy genes of MSY in this study was conducted using quantitative real-time PCR [on ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Cambridge, UK)] in all Cardiogenics men with available macrophage RNA. Relative expression of each MSY mRNA was calculated after normalisation to a housekeeping control (β-actin). Statistical and bioinformatics analysis Due to skewed distributions, triglycerides, C-reactive protein (CRP) and HOMA-IR were logtransformed prior to statistical analysis. The cohort-specific association analysis was based on comparison of cardiovascular risk factors between men with CAD-predisposing haplogroup I and all carriers of other lineages using multiple linear regression. Inverse-variance fixed-effect meta-analysis was then used to examine the effect of haplogroup I of the Y chromosome on clinical phenotypes across the three cohorts (YMCA1, YMCA2, GRAPHIC). All analyses were conducted after adjustment for age. 2 Our calculations showed that the study had from good to excellent (79-100%) power to detect associations between main cardiovascular risk factors and haplogroup I assuming that its effect on CAD was mediated solely by each of these risk factors (Table below). Using the formula ln(a/b)=ln(a)-ln(b) [whereby a – odds ratio CAD in men with haplogroup I: 1.56 (1.24-1.97) (4), b – the effect size for each individual cardiovascular risk factor from previously published studies (14-20, Table below)] we estimated the magnitude of an effect that the we would expect from haplogroup I to have on each risk factor if it was the major driver of the association between haplogroup I and CAD. The corresponding power estimates were then calculated using sampsi in Stata 12.1. Analysis of association between haplogroup I and expression of MSY genes was conducted using linear regression in men from Cardiogenics. Specifically; age-adjusted residuals of dCt from quantitative real-time PCR were compared between haplogroup I and other lineages. The fold difference in expression between these groups was calculated by previously used formula: fold difference=2−difference in dCt . The threshold of statistical significance after correction for multiple testing was calculated at P≈0.004 given that 14 out of 15 genes were expressed in macrophages (P=0.05/14). Table. Risk factors of coronary artery disease and haplogroup I of the Y chromosome – power calculations. Phenotype Effect size – CAD L95 U95 Reference Effect size – haplo I Power (%) BMI 1.14 1.09 1.2 14 0.31 99.0 (kg/m2) SBP 1.24 1.15 1.35 15 0.23 82.7 (mmHg) DBP 1.13 1.04 1.22 15 0.32 99.3 (mmHg) TC 1.08 1.02 1.15 16 0.37 99.9 (mmol/L) HDL-C 0.78 0.73 0.83 16 0.69 100.0 (mmol/L) LDL-C 1.25 1.18 1.33 17 0.22 79.1 (mmol/L) Triglycerides* 1.19 1.08 1.32 18 0.27 99.9 (mmol/L) Glucose* 1.06 1.00 1.12 19 0.39 99.9 (mmol/L) CRP 1.23 1.07 1.42 20 0.24 85.9 (mg/L) Effect size – CAD – estimated magnitude of effect of each individual phenotype on the risk of coronary artery disease [per 1 SD unit increase/decrease or 1 mmol/L increase (triglycerides and glucose)*], L95 – lower 95% confidence interval of the estimate, U95 – upper 95% confidence interval of the estimate, Reference – a literature source of the estimates, Effect size – haplo I – the expected magnitude of effect of haplogroup I on each individual risk factor, Power – power of the study to detect the expected effect of haplogroup I on each individual cardiovascular risk factor, BMI – body mass index, SBP – systolic blood pressure, DBP – diastolic blood pressure, TC – total cholesterol, HDL-C – high-density lipoprotein cholesterol, LDL-C – low-density lipoprotein cholesterol, CRP – C-reactive protein 3 References 1. 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