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Genetics of cardiovascular disease: Importance of sex and ethnicity

Open AccessPublished:March 17, 2015DOI:https://doi.org/10.1016/j.atherosclerosis.2015.03.021

      Highlights

      • Incidence and prevalence of and morbidity and mortality from cardiovascular diseases differ between men and women.
      • Cardiovascular diseases are complex traits.
      • Genetic analysis of cardiovascular disease traits often fail to include the sex chromosomes.
      • Hormonal status and ethnicity are confounding variables in sex-based genetic analyses.
      • Understanding the genetics of cardiovascular diseases requires considering interactions among genetic sex, genetic race and hormonal status.

      Abstract

      Sex differences in incidence and prevalence of and morbidity and mortality from cardiovascular disease are well documented. However, many studies examining the genetic basis for cardiovascular disease fail to consider sex as a variable in the study design, in part, because there is an inherent difficulty in studying the contribution of the sex chromosomes in women due to X chromosome inactivation. This paper will provide general background on the X and Y chromosomes (including gene content, the pseudoautosomal regions, and X chromosome inactivation), discuss how sex chromosomes have been ignored in Genome-wide Association Studies (GWAS) of cardiovascular diseases, and discuss genetics influencing development of cardiovascular risk factors and atherosclerosis with particular attention to carotid intima-medial thickness, and coronary arterial calcification based on sex-specific studies. In addition, a brief discussion of how ethnicity and hormonal status act as confounding variables in sex-based analysis will be considered along with methods for statistical analysis to account for sex in cardiovascular disease.

      Keywords

      1. Introduction

      Sex differences in incidence, prevalence, morbidity and mortality from cardiovascular diseases are well documented and represent important health disparities [
      • Go A.S.
      • Mozaffarian D.
      • Roger V.L.
      • Benjamin E.J.
      • Berry J.D.
      • Blaha M.J.
      • et al.
      Heart disease and stroke statistics–2014 update: a report from the American Heart Association.
      ,

      World Health Organization – The top 10 causes of death, Fact Sheet N310 (2011).

      ,
      • Deo R.
      • Albert C.M.
      Epidemiology and genetics of sudden cardiac death.
      ]. These sex differences can be classified as those that are sex-specific such as erectile dysfunction in men and hypertensive pregnancy disorders in women, or conditions common to both sexes but which show sex differences in presentation and outcomes such as hypertension, atherosclerosis, angina, and stroke.
      Sex differences in cardiovascular diseases result from a complex interaction among genetic, hormonal and environmental factors that provide a profile of individual risk and phenotypic presentation of disease. Therefore, there is an increased interest in identifying the genetic components of disease to optimize treatments and outcomes. However, investigations into the genetics of cardiovascular diseases in men and women are hampered by the failure to include the sex chromosomes in genome-wide association studies (GWAS), to account for sex as a variable in targeted genetic analyses, and to examine hormone-gene interactions. In this paper, we will provide general background on the X and Y chromosomes, discuss how they have been ignored in GWAS of cardiovascular disease, and discuss genetic factors contributing to sex differences in conventional cardiovascular risk factors and atherosclerosis with examples of sex-specific studies of carotid intima-medial thickness, and coronary arterial calcification. Confounding factors of ethnicity and hormonal status also will be considered along with some proposed methods for statistical analysis for future studies.

      2. Background: the genetics of sex

      In humans, sex is determined via the X and Y chromosomes. Each somatic cell of the human body contains 23 pairs of chromosomes, 22 of which are the same in both sexes (autosomes); however females have two copies of the X chromosome, whereas males have one copy of the X and one copy of the Y [
      • Li X.
      Sex chromosomes and sex chromosome abnormalities.
      ]. Therefore, differences between the sexes result from the particular combination of sex chromosomes an individual possesses, as well as, differing levels of the sex hormones. Often early studies of sex differences compared persons with XX and XY genotypes to those with sex chromosome monosomy (e.g. XO karyotype in Turner syndrome) or trisomy (e.g. XXY karyotype in Klinefelter syndrome) to distinguish whether observed sex differences were due to sex chromosome dosage effects independent of hormonal influences [
      • Abramowitz L.K.
      • Olivier-Van Stichelen S.
      • Hanover J.A.
      Chromosome imbalance as a driver of sex disparity in disease.
      ].
      As genotyping technologies have rapidly improved, studies of the genetic basis of sex differences are no longer limited to investigating global differences in sex chromosome dosage effects, but can directly examine the individual genes and genetic variants on the X and Y chromosomes. In humans, the larger X chromosome contains 813 protein coding genes, whereas the much smaller Y chromosome contains 143 protein-coding genes, although many of the Y genes are multi-copy genes or present in both males and females (Ensemble Genome Browser, http://www.ensembl.org); only 46 genes are on the male-specific region of the Y (MSY). The sex chromosomes evolved from ancestral autosomes [
      • Ross M.T.
      • Grafham D.V.
      • Coffey A.J.
      • Scherer S.
      • McLay K.
      • Muzny D.
      • et al.
      The DNA sequence of the human X chromosome.
      ,
      • Lahn B.T.
      • Page D.C.
      Four evolutionary strata on the human X chromosome.
      ], and over the course of evolution, the X has retained many of the ancestral genes (98% retained), but the Y lost many (3% retained) [
      • Bellott D.W.
      • Hughes J.F.
      • Skaletsky H.
      • Brown L.G.
      • Pyntikova T.
      • Cho T.J.
      • et al.
      Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators.
      ]. Only 14 ancestral X–Y gene pairs are still present in humans, and many of these have important regulatory functions in regards to transcription, translation, and protein stability [
      • Bellott D.W.
      • Hughes J.F.
      • Skaletsky H.
      • Brown L.G.
      • Pyntikova T.
      • Cho T.J.
      • et al.
      Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators.
      ,
      • Lahn B.T.
      • Page D.C.
      Functional coherence of the human Y chromosome.
      ]. Some of the genes in the ancestral gene pairs retain the same function across the X and Y, but others have diverged. Additionally, both the X and Y have acquired new genes through evolution, with an emphasis on reproductive function [
      • Mueller J.L.
      • Skaletsky H.
      • Brown L.G.
      • Zaghlul S.
      • Rock S.
      • Graves T.
      • et al.
      Independent specialization of the human and mouse X chromosomes for the male germ line.
      ,
      • Skaletsky H.
      • Kuroda-Kawaguchi T.
      • Minx P.J.
      • Cordum H.S.
      • Hillier L.
      • Brown L.G.
      • et al.
      The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes.
      ]. The Y chromosome contains the SRY gene, or sex-determining region Y, which encodes the testes determining factor [
      • Sinclair A.H.
      • Berta P.
      • Palmer M.S.
      • Hawkins J.R.
      • Griffiths B.L.
      • Smith M.J.
      • et al.
      A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif.
      ]. Many other genes on the Y chromosome are related to male sexual development and reproduction, including multicopy gene families expressed exclusively in the testes [
      • Bellott D.W.
      • Hughes J.F.
      • Skaletsky H.
      • Brown L.G.
      • Pyntikova T.
      • Cho T.J.
      • et al.
      Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators.
      ,
      • Skaletsky H.
      • Kuroda-Kawaguchi T.
      • Minx P.J.
      • Cordum H.S.
      • Hillier L.
      • Brown L.G.
      • et al.
      The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes.
      ]. The X chromosome also includes reproductive-related genes, such as the androgen receptor [
      • Khil P.P.
      • Smirnova N.A.
      • Romanienko P.J.
      • Camerini-Otero R.D.
      The mouse X chromosome is enriched for sex-biased genes not subject to selection by meiotic sex chromosome inactivation.
      ], and genes important for brain development, blood clotting, and visual pigmentation [
      • Graves J.A.
      Review: sex chromosome evolution and the expression of sex-specific genes in the placenta.
      ].
      The X and Y chromosomes have developed unique properties as compared to the autosomes, as illustrated schematically in Fig. 1 (this schematic diagram does not reflect the large difference in genetic content between the X and Y chromosomes, such as the large block of non-coding heterochromatin on the Y located in the MSY region designated in the figure at * [
      • Skaletsky H.
      • Kuroda-Kawaguchi T.
      • Minx P.J.
      • Cordum H.S.
      • Hillier L.
      • Brown L.G.
      • et al.
      The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes.
      ]). Unlike autosomal pairs, the X and Y do not generally undergo recombination due to evolutionary divergence. However, there are two regions on each end of the X and Y chromosomes that are homologous, called the pseudoautosomal regions (PAR), which behave like the autosomes and do recombine [
      • Ross M.T.
      • Grafham D.V.
      • Coffey A.J.
      • Scherer S.
      • McLay K.
      • Muzny D.
      • et al.
      The DNA sequence of the human X chromosome.
      ] (Fig. 1A). For most genes in the PAR, both copies are expressed across both sex chromosomes (XX for females or XY for males). However, for other regions, that is, the genes on the non-pseudo-autosomal regions, the aneuploidy causes unequal patterns of expression across the sexes, which requires some form of dosage compensation across males and females [
      • Disteche C.M.
      Dosage compensation of the sex chromosomes.
      ]. One method is upregulation of X-linked genes [
      • Deng X.X.
      • Hiatt J.B.
      • Nguyen D.K.
      • Ercan S.
      • Sturgill D.
      • Hillier L.W.
      • et al.
      Evidence for compensatory upregulation of expressed X-linked genes in mammals, Caenorhabditis elegans and Drosophila melanogaster.
      ]. In humans, one of the female copies of the X chromosome is silenced during embryogenesis to achieve dosage compensation during a process called X chromosome inactivation [
      • Ross M.T.
      • Grafham D.V.
      • Coffey A.J.
      • Scherer S.
      • McLay K.
      • Muzny D.
      • et al.
      The DNA sequence of the human X chromosome.
      ] (Fig. 1B). The X-linked gene XIST is expressed from the inactive chromosome, triggering DNA methylation that is responsible for the chromosome-wide silencing [
      • Brown C.J.
      • Ballabio A.
      • Rupert J.L.
      • Lafreniere R.G.
      • Grompe M.
      • Tonlorenzi R.
      • et al.
      A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome.
      ]. The inactive chromosome remains silent in somatic cells, but is reactivated during meiosis. This inactivation is tissue and cell specific, such that the maternally inherited allele may be expressed in some cells, while the paternally inherited allele may be expressed in others [
      • Wu H.
      • Luo J.
      • Yu H.
      • Rattner A.
      • Mo A.
      • Wang Y.
      • et al.
      Cellular resolution maps of X chromosome inactivation: implications for neural development, function, and disease.
      ]. This process occurs randomly so that usually each chromosome is inactivated in 50% of cells, although preferentially (skewed) inactivation of one chromosome can occur either globally or in specific tissues, as demonstrated in cardiac muscle of mice [
      • Wu H.
      • Luo J.
      • Yu H.
      • Rattner A.
      • Mo A.
      • Wang Y.
      • et al.
      Cellular resolution maps of X chromosome inactivation: implications for neural development, function, and disease.
      ]. Skewed X-inactivation has been observed in samples from human arteries, suggesting that the cells in the plaque arose from a single or similar group of cells [
      • Chung I.M.
      • Schwartz S.M.
      • Murry C.E.
      Clonal architecture of normal and atherosclerotic aorta: implications for atherogenesis and vascular development.
      ]. To further complicate matters, approximately 15% of X-linked genes escape inactivation in humans [
      • Berletch J.B.
      • Yang F.
      • Xu J.
      • Carrel L.
      • Disteche C.M.
      Genes that escape from X inactivation.
      ,
      • Carrel L.
      • Willard H.F.
      X-inactivation profile reveals extensive variability in X-linked gene expression in females.
      ]. For these ‘escapee genes,’ both copies are active in females (whereas males may have a single active copy if there is no homologous gene on the Y). Mechanisms of dosage compensation, whether concerning X or Y specific genes undergoing upregulation or X inactivation, or shared homologous X–Y gene pairs or pseudoautosomal genes, may be related to differential gene expression across the sexes and are important considerations for sex specific disease risk.
      Figure thumbnail gr1
      Fig. 1(A) Schematic of the X and Y chromosomes in males and females showing regions of shared or specific expression. This schematic is not drawn to scale, and does not include the large heterochromatin block within the male-specific region of the Y on the q arm (denoted by *) which is not expressed. (B) Schematic of X chromosome inactivation in multiple cells of the female body.
      In contrast to the PAR on the X and Y chromosomes that is present in both sexes, the non-PAR region of the Y chromosome (MSY) encompasses the vast majority of the Y chromosome [
      • Skaletsky H.
      • Kuroda-Kawaguchi T.
      • Minx P.J.
      • Cordum H.S.
      • Hillier L.
      • Brown L.G.
      • et al.
      The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes.
      ]. This region does not undergo recombination, and is inherited from father to son as a single haplotype, reflecting the paternal lineage only. Many of these genes are widely-expressed (at the RNA level), exhibiting global regulatory functions in transcription, translation, and other biological processes (non-reproductive), and hence may be important to men's health beyond sex determination and sexual reproduction [
      • Bellott D.W.
      • Hughes J.F.
      • Skaletsky H.
      • Brown L.G.
      • Pyntikova T.
      • Cho T.J.
      • et al.
      Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators.
      ].
      Both X and Y chromosomes contribute to disease. Many rare Mendelian genetic disorders are linked to the sex chromosomes. According to the Online Mendelian Inheritance in Man (OMIM) catalog of genetic disorders in humans, approximately 7% of cataloged phenotypes are X-linked [
      • Wise A.L.
      • Gyi L.
      • Manolio T.A.
      eXclusion: toward integrating the X chromosome in genome-wide association analyses.
      ]. XY males are at increased risk compared to XX women for X-linked disorders, such as learning disabilities and mental retardation caused by X-linked genes important in brain development and function [
      • Graves J.A.
      Review: sex chromosome evolution and the expression of sex-specific genes in the placenta.
      ]. Hearing impairment (DFNY1, MIM 400043) reported in a Chinese family was the only documented Mendelian disorder showing Y-linkage in humans [
      • Wang Q.J.
      • Lu C.Y.
      • Li N.
      • Rao S.Q.
      • Shi Y.B.
      • Han D.Y.
      • et al.
      Y-linked inheritance of non-syndromic hearing impairment in a large Chinese family.
      ]. However, subsequent analysis found that the condition was related to an insertion of chromosome 1 into the Y gene rather than a mutation of a Y-chromosomal gene [
      • Wang Q.
      • Xue Y.
      • Zhang Y.
      • Long Q.
      • Asan
      • Yang F.
      • et al.
      Genetic basis of Y-linked hearing impairment.
      ].
      Much work to link the sex chromosomes to disease has focused on such rare disorders caused by a single gene, and less is known about the influence on complex traits with multiple genetic and etiological components. In particular, variants on the X chromosome are understudied for complex traits, with significantly fewer genome-wide associations than the autosomes [
      • Wise A.L.
      • Gyi L.
      • Manolio T.A.
      eXclusion: toward integrating the X chromosome in genome-wide association analyses.
      ] (Fig. 2). Of the over 2800 genome-wide significant associations reported for over 300 traits in the NHGRI GWAS Catalog [
      • Hindorff L.A.
      • Sethupathy P.
      • Junkins H.A.
      • Ramos E.M.
      • Mehta J.P.
      • Collins F.S.
      • et al.
      Potential etiologic and functional implications of genome-wide association loci for human diseases and traits.
      ], only 15 published associations reside on the X despite representing approximately 5% of the human genome, including 153 million nucleotide base pairs and 1669 genes [
      • Kent W.J.
      • Sugnet C.W.
      • Furey T.S.
      • Roskin K.M.
      • Pringle T.H.
      • Zahler A.M.
      • et al.
      The human genome browser at UCSC.
      ]. This representation is in stark contrast to the similarly sized chromosome 7, which holds 120 published associations [
      • Wise A.L.
      • Gyi L.
      • Manolio T.A.
      eXclusion: toward integrating the X chromosome in genome-wide association analyses.
      ]. There are a number of reasons for this discrepancy, including reduced power due to the use of sex-specific analyses or low data quality, reflecting poor genotyping accuracy on current genome-wide arrays and quality control issues. However, an important reason is also that although the X chromosome is routinely included in standard genotyping arrays, it is simply excluded from analysis. Indeed, of the first 53 studies that have released publically available GWAS data, only 31 include the X chromosome [
      • Wise A.L.
      • Gyi L.
      • Manolio T.A.
      eXclusion: toward integrating the X chromosome in genome-wide association analyses.
      ]. Since the number of copies of X chromosome variants is confounded with sex, special analysis methods and computational tools are required (discussed below); therefore the sex chromosomes have historically been excluded to simplify analyses, setting a critical precedent that needs revision.
      Figure thumbnail gr2
      Fig. 2Plot of the number of published GWAS associations studies reported in the NHGRI catalog by number of genes. Each dot represents a pair of autosomal chromosomes. The ‘X’ and ‘Y’ chromosomes are plotted in red.
      Important issues regarding the genetics of sex differences are not limited to large scale GWAS studies in humans but also are critical concerning preclinical work involving model organisms. In particular, basic science research utilizes experiments in model organisms as critical tools to investigate human disease. However, the genetics and biology of the sex chromosomes differs from species to species regarding genes that are conserved and mechanisms of dosage compensation [
      • Bellott D.W.
      • Hughes J.F.
      • Skaletsky H.
      • Brown L.G.
      • Pyntikova T.
      • Cho T.J.
      • et al.
      Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators.
      ,
      • Disteche C.M.
      Dosage compensation of the sex chromosomes.
      ]. Therefore the choice of the model organism may be critical to the study of sex differences. The mouse has become a favorite model organism for preclinical disease research because many genes are shared with humans, although the two species also differ in important areas, highlighted by the work of the Mouse ENCODE Consortium [
      • Yue F.
      • Cheng Y.
      • Breschi A.
      • Vierstra J.
      • Wu W.
      • Ryba T.
      • et al.
      A comparative encyclopedia of DNA elements in the mouse genome.
      ]. Mice have fewer conserved X–Y gene pairs [
      • Bellott D.W.
      • Hughes J.F.
      • Skaletsky H.
      • Brown L.G.
      • Pyntikova T.
      • Cho T.J.
      • et al.
      Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators.
      ] and fewer genes that escape X inactivation as compared to humans [
      • Carrel L.
      • Willard H.F.
      X-inactivation profile reveals extensive variability in X-linked gene expression in females.
      ,
      • Yang P.C.
      • Clancy C.E.
      Effects of sex hormones on cardiac repolarization.
      ], which may suggest that they may NOT be an ideal model organism to study the genetics of sex differences for all conditions in humans.

      3. Genetics of cardiovascular disease

      3.1 General considerations

      Cardiovascular diseases are complex traits with both sex-specific (i.e. cardiovascular diseases associated with pregnancy, erectile dysfunction) and sex-different etiology, pathophysiology, symptom presentation, severity and progression [
      • Deo R.
      • Albert C.M.
      Epidemiology and genetics of sudden cardiac death.
      ,
      • Olivotto I.
      • Maron M.S.
      • Adabag A.S.
      • Casey S.A.
      • Vargiu D.
      • Link M.S.
      • et al.
      Gender-related differences in the clinical presentation and outcome of hypertrophic cardiomyopathy.
      ,
      • Meyer S.
      • van der Meer P.
      • van Tintelen J.P.
      • van den Berg M.P.
      Sex differences in cardiomyopathies.
      ]. The concept that there are sex by trait interactions is not new [
      • Towne B.
      • Siervogel R.M.
      • Blangero J.
      Effects of genotype-by-sex interaction on quantitative trait linkage analysis.
      ] and several methods are used to examine genetic components of complex traits including GWAS, linkage analysis, association studies, and candidate–gene association studies. However, not all studies of populations of men and women account for sex in the analysis and if sex is considered, it is treated only as a confounding variable [
      • Williams S.R.
      • Yang Q.
      • Chen F.
      • Liu X.
      • Keene K.L.
      • Jacques P.
      • et al.
      Genome-wide meta-analysis of homocysteine and methionine metabolism identifies five one carbon metabolism loci and a novel association of ALDH1L1 with ischemic stroke.
      ,
      • Wang D.
      • Yang H.
      • Quinones M.J.
      • Bulnes-Enriquez I.
      • Jimenez X.
      • De La Rosa R.
      • et al.
      A genome-wide scan for carotid artery intima-media thickness: the Mexican-American Coronary Artery Disease family study.
      ,
      • O'Donnell C.J.
      • Cupples L.A.
      • D'Agostino R.B.
      • Fox C.S.
      • Hoffmann U.
      • Hwang S.J.
      • et al.
      Genome-wide association study for subclinical atherosclerosis in major arterial territories in the NHLBI's Framingham Heart Study.
      ,
      • Lange L.A.
      • Lange E.M.
      • Bielak L.F.
      • Langefeld C.D.
      • Kardia S.L.
      • Royston P.
      • et al.
      Autosomal genome-wide scan for coronary artery calcification loci in sibships at high risk for hypertension.
      ]. However, when sex and sex chromosomes are considered, there is the potential to gain new insight into etiology of disease, differences in mechanisms of progression and new targets for preventive and therapeutic interventions [
      • Stamova B.
      • Tian Y.
      • Jickling G.
      • Bushnell C.
      • Zhan X.
      • Liu D.
      • et al.
      The X-chromosome has a different pattern of gene expression in women compared with men with ischemic stroke.
      ]. Failing to do so, hampers medical progress toward optimizing individual outcomes.

      3.2 Cardiovascular risk factors

      Major risk factors for cardiovascular disease include smoking, high blood pressure, high glucose, central adiposity and high blood lipids. While some psychosocial, economic and environmental conditions as well as life-style choices (i.e. access to health care, stress, smoking, diet, and activity) have physiological consequences, it is important to determine which biological factors (regulation of blood pressure, glucose, and lipid metabolism) reflect inherited components in order to optimize preventive and therapeutic strategies.

      3.2.1 Hypertension

      Men develop hypertension at younger ages than women, thus carrying an increased life-long burden of disease including the risk of stroke [
      • Chen Y.C.
      • Guo X.
      • Raffel L.J.
      • Xiang A.H.
      • Fang B.
      • Hsueh W.A.
      • et al.
      Carotid intima-media thickness (cIMT) cosegregates with blood pressure and renal function in hypertensive Hispanic families.
      ,
      • Jiang Y.
      • Kohara K.
      • Hiwada K.
      Association between risk factors for atherosclerosis and mechanical forces in carotid artery.
      ,
      • Xiang A.H.
      • Azen S.P.
      • Buchanan T.A.
      • Raffel L.J.
      • Tan S.
      • Cheng L.S.
      • et al.
      Heritability of subclinical atherosclerosis in Latino families ascertained through a hypertensive parent.
      ,
      • Fox C.S.
      • Polak J.F.
      • Chazaro I.
      • Cupples A.
      • Wolf P.A.
      • D'Agostino R.A.
      • et al.
      Genetic and environmental contributions to atherosclerosis phenotypes in men and women: heritability of carotid intima-media thickness in the Framingham Heart Study.
      ]. Recall that sexual dimorphism is impacted by the sex chromosomal complement independent of hormonal influences. The Sry locus of the Y chromosome, in addition to being required for the development of the testes, was found in experimental animals to regulate tyrosine hydroxylase, a critical enzyme in the synthesis of norepinephrine [
      • Turner M.E.
      • Farkas J.
      • Dunmire J.
      • Ely D.
      • Milsted A.
      Which sry locus is the hypertensive Y chromosome locus?.
      ]. Greater activity of tyrosine hydroxylase in men would pre-disposes them to hypertension differently than women [
      • Ehret G.B.
      • Munroe P.B.
      • Rice K.M.
      • Bochud M.
      • Johnson A.D.
      • Chasman D.I.
      • et al.
      Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.
      ] and indeed, several large genetic studies have identified loci on the Y chromosome associated with hypertension in men (for review see [
      • Charchar F.J.
      • Tomaszewski M.
      • Strahorn P.
      • Champagne B.
      • Dominiczak A.F.
      Y is there a risk to being male?.
      ]).
      In addition to synthesis of adrenergic transmitter, genetic variants in alpha and beta adrenergic receptors affect vascular tone. In women with non-obstructive coronary disease, genetic variants in adrenergic receptors associate with resting hemodynamics and increased risk for stroke, myocardial infarction and heart failure [
      • Humma L.M.
      • Puckett B.J.
      • Richardson H.E.
      • Terra S.G.
      • Andrisin T.E.
      • Lejeune B.L.
      • et al.
      Effects of beta1-adrenoceptor genetic polymorphisms on resting hemodynamics in patients undergoing diagnostic testing for ischemia.
      ,
      • Pacanowski M.A.
      • Zineh I.
      • Li H.
      • Johnson B.D.
      • Cooper-DeHoff R.M.
      • Bittner V.
      • et al.
      Adrenergic gene polymorphisms and cardiovascular risk in the NHLBI-sponsored Women's Ischemia Syndrome Evaluation.
      ,
      • Pacanowski M.A.
      • Gong Y.
      • Cooper-Dehoff R.M.
      • Schork N.J.
      • Shriver M.D.
      • Langaee T.Y.
      • et al.
      Beta-adrenergic receptor gene polymorphisms and beta-blocker treatment outcomes in hypertension.
      ]. Polymorphisms in the genes encoding enzymes and receptors for the renin-angiotensin system would influence development of hypertension in both women and men but these polymorphisms in women may predispose them to pregnancy related hypertension [
      • Rahimi Z.
      • Aghaei A.
      • Vaisi-Raygani A.
      AT2R -1332 G: A polymorphism and its interaction with AT1R 1166 A: C, ACE I/D and MMP-9 -1562 C: T polymorphisms: risk factors for susceptibility to preeclampsia.
      ]. Genetic polymorphisms in these receptors and enzyme systems also would affect a woman's response to pharmacological agents that are used in the treatment of hypertension, i.e. beta and alpha adrenergic antagonists, angiotensin receptor antagonists, and angiotensin converting enzyme (ACE) inhibitors. There are no sex-specific guidelines for treatment of hypertension [
      • Bairey Merz C.N.
      • Mark S.
      • Boyan B.D.
      • Jacobs A.K.
      • Shah P.K.
      • Shaw L.J.
      • et al.
      Proceedings from the scientific symposium: sex differences in cardiovascular disease and implications for therapies.
      ,
      • Mosca L.
      • Benjamin E.J.
      • Berra K.
      • Bezanson J.L.
      • Dolor R.J.
      • Lloyd-Jones D.M.
      • et al.
      Effectiveness-based guidelines for the prevention of cardiovascular disease in women–2011 update: a guideline from the american heart association.
      ].

      3.2.2 Inflammation

      The Y chromosome also includes genes involved with macrophage activation affecting innate immunity [
      • Bloomer L.D.
      • Nelson C.P.
      • Eales J.
      • Denniff M.
      • Christofidou P.
      • Debiec R.
      • et al.
      Male-specific region of the Y chromosome and cardiovascular risk: phylogenetic analysis and gene expression studies.
      ,
      • Charchar F.J.
      • Bloomer L.D.S.
      • Barnes T.A.
      • Cowley M.J.
      • Nelson C.P.
      • Wang Y.
      • et al.
      New insights into inheritance of coronary artery disease in men – the role of the Y sex chromosome.
      ]. Systemic inflammation characterized by activation of macrophages and increases in circulating levels of various cytokines contributes to development of atherosclerosis [
      • Libby P.
      • Ridker P.M.
      • Hansson G.K.
      Inflammation in atherosclerosis: from pathophysiology to practice.
      ]. Thus, in males, genetic risk for development of other cardiovascular disease apart from hypertension may be imparted simply by inheriting the Y chromosome.
      Genes on the X chromosome also affect phenotypic expression of inflammatory risk factors, including genes associated with apoptosis, lipid oxidation, and generation of oxygen-derived free radicals produced by the mitochondria. In an analysis of RNA levels of 683 genes on the X chromosome obtained from individuals at specified time intervals following a stroke, X related genes showed greater and differential upregulation in females compared to males. In females, up-regulated genes were associated with post-translational modification of proteins, small molecule biochemistry and cell–cell signaling; in men, up-regulated genes were associated with cellular migration, cell trafficking and cell death [
      • Stamova B.
      • Tian Y.
      • Jickling G.
      • Bushnell C.
      • Zhan X.
      • Liu D.
      • et al.
      The X-chromosome has a different pattern of gene expression in women compared with men with ischemic stroke.
      ]. This study provides evidence that cellular pathways involved with manifestation of a particular cardiovascular outcome, in this case stroke, differs between men and women.

      3.2.3 Lipids

      Much research and clinical preventive strategies have focused on evaluating and managing serum lipids. Indeed, decreases in high density lipoprotein (HDL) cholesterol and increases in low density lipoprotein (LDL) cholesterol and total cholesterol are considered hallmarks of cardiovascular risk. Sex steroids regulate serum lipids and in women, genetic variants in genes for either estrogen receptor α or β associated with serum lipids, but the associations varied by ethnic groups [
      • Sowers M.R.
      • Symons J.P.
      • Jannausch M.L.
      • Chu J.
      • Kardia S.R.
      Sex steroid hormone polymorphisms, high-density lipoprotein cholesterol, and apolipoprotein A-1 from the Study of Women's Health across the Nation (SWAN).
      ]. The extent to which lipid metabolism shows a sex-difference independent of sex steroids is unclear including mechanisms of lipid deposition in vascular lesions. Male pattern of lipid deposition results in obstructive lesions within the coronary arteries while in women the pattern is diffuse and manifests as non-obstructive ischemic disease [
      • Olson M.B.
      • Kelsey S.F.
      • Matthews K.
      • Shaw L.J.
      • Bharaf B.L.
      • Pohost G.M.
      • et al.
      Symptoms, myocardial ischaemia and quality of life in women: results from the NHLBI-sponsored WISE Study.
      ].
      In a genome wide association study, genes of the metabolic pathways for HDL, LDL and triglycerides associated with obstructive coronary artery disease [
      • Willer C.J.
      • Sanna S.
      • Jackson A.U.
      • Scuteri A.
      • Bonnycastle L.L.
      • Clarke R.
      • et al.
      Newly identified loci that influence lipid concentrations and risk of coronary artery disease.
      ] and with serum lipids in healthy volunteers [
      • Carlquist J.F.
      • McKinney J.T.
      • Horne B.D.
      • Camp N.J.
      • Cannon-Albright L.
      • Muhlestein J.B.
      • et al.
      Common variants in 6 lipid-related genes discovered by high-resolution DNA melting analysis and their association with plasma lipids.
      ]. While the details of these reports are beyond the scope of the present review, several key points serve as an example relevant to studying the genetics of sex differences. In these studies, there was large variability in the number of SNPs among the genes studied and some of the variants were specific to either Caucasian or minority ethnic groups. In these studies, sex was not considered in the analysis. In a genome-wide analysis of loci affecting lipid metabolism conducted in samples from approximately 20,000 persons from European countries, three loci showed sex-specific effects. The strongest effects were in the gene for 3-hyroxy-3-methylglutaryl-Coenzyme A reductase (HMGCoA) and in the gene that encodes chondroitin sulfate proteoglycan (NCAN). The former enzyme is the rate-limiting step in synthesis of cholesterol (greater effect in females, P = 0.001) and a target for statin therapy; the latter enzyme is involved in cell adhesion and migration (greater effect in males P = 0.002). Both these loci associated with total cholesterol. The gene encoding lipoprotein lipase (LPL) showed greater association with HDL in males compared to females (P = 0.006) [
      • Aulchenko Y.S.
      • Ripatti S.
      • Lindqvist I.
      • Boomsma D.
      • Heid I.M.
      • Pramstaller P.P.
      • et al.
      Loci influencing lipid levels and coronary heart disease risk in 16 European population cohorts.
      ]. The age distribution among participants in that study was broad (18–104 years). It is unclear at this time whether these associations would remain if hormonal status was considered in females as estrogen modulates lipid metabolism.
      Two meta-analyses of genome-wide association studies evaluating loci for lipid metabolism with coronary artery disease, sex-differences, perhaps not surprisingly, were confirmed in genes regulating total cholesterol, LDL, HDL and triglycerides [
      • Asselbergs F.W.
      • Guo Y.
      • van Iperen E.P.
      • Sivapalaratnam S.
      • Tragante V.
      • Lanktree M.B.
      • et al.
      Large-scale gene-centric meta-analysis across 32 studies identifies multiple lipid loci.
      ,
      • Teslovich T.M.
      • Musunuru K.
      • Smith A.V.
      • Edmondson A.C.
      • Stylianou I.M.
      • Koseki M.
      • et al.
      Biological, clinical and population relevance of 95 loci for blood lipids.
      ]. Both of these studies identified stronger associations of SNPS to serum triglycerides in women compared to men. Because associations in one analysis were based on those that also associated with coronary artery disease, both women and men in those studies would most likely have phenotypic obstructive disease [
      • Teslovich T.M.
      • Musunuru K.
      • Smith A.V.
      • Edmondson A.C.
      • Stylianou I.M.
      • Koseki M.
      • et al.
      Biological, clinical and population relevance of 95 loci for blood lipids.
      ].
      Statin therapy, which inhibits HMGCoA, has become a main approach to primary prevention of coronary artery disease and stroke. The safety and efficacy of this treatment in a meta-analysis of 27 clinical trials found the treatment of similar effectiveness in women and men who had equivalent cardiovascular risk [
      Efficacy and safety of LDL-lowering therapy among men and women: meta-analysis of individual data from 174 000 participants in 27 randomised trials.
      ]. The number of women included in this analysis was less than 30%. As stated above, genetic polymorphisms in the gene for HMGCoA had greater association lipid levels in women compared to men. Genetic polymorphisms in that gene associate with development of type 2 diabetes [
      • Swerdlow D.I.
      • Preiss D.
      • Kuchenbaecker K.B.
      • Holmes M.V.
      • Engmann J.E.
      • Shah T.
      • et al.
      HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight: evidence from genetic analysis and randomised trials.
      ] that was shown to be increased with statin use in the Women's Health Initiative [
      • Goodarzi M.O.
      • Li X.
      • Krauss R.M.
      • Otter J.I.
      • Chien Y.I.
      Relationship of sex to diabetes risk in statin trials.
      ]. Although there are no sex-specific guidelines for use of statins in primary prevention strategies for cardiovascular disease, the appropriateness related to “equivalent risk” is recommended [
      • Stone N.J.
      • Robinson J.G.
      • Lichtenstein A.H.
      • Goff Jr., D.C.
      • Lloyd-Jones D.M.
      • Smith Jr., S.C.
      • et al.
      Treatment of blood cholesterol to reduce atherosclerotic cardiovascular disease risk in adults: synopsis of the 2013 American College of Cardiology/American Heart Association cholesterol guideline.
      ,
      • Mosca L.
      Sex, statins, and statistics.
      ].

      3.2.4 Metabolic syndrome

      The term “metabolic syndrome” is used to define a constellation of at least three of five risk factors for cardiovascular disease: waist circumference (>88 cm for female and 102 cm for male), high-density lipoprotein cholesterol (HDL-C) <50 mg/dL, triglyceride level >150 mg/dL, fasting blood glucose >100 mg/dL, and systemic blood pressure (systolic blood pressure ≥130 or diastolic blood pressure ≥85 mmHg) [
      • Alexander C.M.
      • Landsman P.B.
      • Teutsch S.M.
      • Haffner S.M.
      NCEP-defined metabolic syndrome, diabetes, and prevalence of coronary heart disease among NHANES III participants age 50 years and older.
      ]. Although two individuals with “metabolic syndrome” may exhibit a different combination of three of the five characteristics [
      • Jayachandran M.
      • Litwiller R.D.
      • Lahr B.D.
      • Bailey K.R.
      • Owen W.G.
      • Mulvagh S.L.
      • et al.
      Alterations in platelet function and cell-derived microvesicles in recently menopausal women: relationship to metabolic syndrome and atherogenic risk.
      ], the collection poses greater risk for cardiovascular disease than any characteristic separately. Not surprisingly there are sex differences in incidence and prevalence of metabolic syndrome (independent of the target values to define the syndrome) [
      • Regitz-Zagrosek V.
      • Lehmkuhl E.
      • Weickert M.O.
      Gender differences in the metabolic syndrome and their role for cardiovascular disease.
      ] due to sex differences in incidence of hypertension and hyperlipidemia. Abdominal obesity shows sexual dimorphism distinct from body mass index. In women, abdominal obesity (as measured by waist circumference) is gaining acceptance as a measure to be considered in assessing cardiovascular risk, but this measure may be influenced by race/ethnicity [
      • Heid I.M.
      • Jackson A.U.
      • Randall J.C.
      • Winkler T.W.
      • Qi L.
      • Steinthorsdottir V.
      • et al.
      Meta-analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution.
      ,
      • Li H.Y.
      • Wu R.L.
      • Lin M.
      • Terra S.G.
      • Pepine C.J.
      • McGorray S.P.
      • et al.
      Epistatic control of human obesity as revealed by linkage disequilibrium mapping: a report from the NHLBI-sponsored WISE study.
      ]. Data from a genome-wide association study of individuals of European and European-American descent to evaluate phenotypic traits of height, weight, body mass index, waist circumference, hip circumference and wait-to-hip-ratio confirms these assertions as only sex-differences in loci for waist phenotypes were found [
      • Randall J.C.
      • Winkler T.W.
      • Kutalik Z.
      • Berndt S.I.
      • Jackson A.U.
      • Monda K.L.
      • et al.
      Sex-stratified genome-wide association studies including 270,000 individuals show sexual dimorphism in genetic loci for anthropometric traits.
      ]. Of 7 loci associated with waist phenotype, all were found in women and not men. Of particular interest is the variant in the gene for PPARG encoding peroxisome proliferator-activator receptor γ (PPARγ), a type II nuclear hormone receptor regulating adipocyte-differentiation, susceptibility for obesity and insulin resistance. PPARγ binds to a site in HSD17B4, which encodes the enzyme hydroxysteroid (17-beta) dehydrogenase involved in the conversion of estrogen to estrone and beta-oxidation of fatty acids [
      • Randall J.C.
      • Winkler T.W.
      • Kutalik Z.
      • Berndt S.I.
      • Jackson A.U.
      • Monda K.L.
      • et al.
      Sex-stratified genome-wide association studies including 270,000 individuals show sexual dimorphism in genetic loci for anthropometric traits.
      ]. As statins also target PPARγ, and as there are documented interactions with estrogen and endocrine disruptors in regulation of PPARγ [
      • Ohlstein J.F.
      • Strong A.L.
      • McLachlan J.A.
      • Gimble J.M.
      • Burow M.E.
      • Bunnell B.A.
      Bisphenol A enhances adipogenic differentiation of human adipose stromal/stem cells.
      ,
      • Ma H.
      • Sprecher H.W.
      • Kolattukudy P.E.
      Estrogen-induced production of a peroxisome proliferator-activated receptor (PPAR) ligand in a PPARgamma-expressing tissue.
      ,
      • Lehrke M.
      • Lazar M.A.
      The many faces of PPARgamma.
      ], sex differences observed with statins and type 2 diabetes may be related to genetic variants in PPARγ [
      • Yano M.
      • Matsumura T.
      • Senokuchi T.
      • Ishii N.
      • Murata Y.
      • Taketa K.
      • et al.
      Statins activate peroxisome proliferator-activated receptor gamma through extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase-dependent cyclooxygenase-2 expression in macrophages.
      ]. Collectively, these data identify the interaction between sex and the genetic components of a phenotype associated with sex differences in adiposity, and demonstrate the utility of pathway analysis to assess sex differences in genetic components of cardiovascular risk susceptibility.
      Central adiposity is functionally linked to elevated glucose, insulin resistance and type 2 diabetes. Diabetes poses about a 3 to 6 times greater risk for myocardial infarction in women compared to men across several ethnic groups [
      • Regitz-Zagrosek V.
      • Lehmkuhl E.
      • Weickert M.O.
      Gender differences in the metabolic syndrome and their role for cardiovascular disease.
      ]. Numerous studies have identified candidate genes involved in development of type 2 diabetes although discussion is beyond the scope of this review. However, one study utilizing a meta-analysis of genetic variants on the Metabochip identified two sex-differentiated variants in combined case–control populations of European and Pakistani origin: CCND2, which encodes cyclin proteins involved with cell division, was most significant in males and GIPR, which encodes G-protein coupled receptor for gastric inhibitory polypeptide that when activated stimulates release of insulin in response to elevated glucose, was most significant in females [
      • Morris A.P.
      • Voight B.F.
      • Teslovich T.M.
      • Ferreira T.
      • Segre A.V.
      • Steinthorsdottir V.
      • et al.
      Large-scale association analysis provides insights into the genetic architecture and pathophysiology of type 2 diabetes.
      ]. However, in the subsequent pathway analyses, sex was not considered and the constellation of pathways may interact in different ways in females compared to males, given the other interactions among lipid metabolism, glucose, metabolism and adiposity [
      • Morris A.P.
      • Voight B.F.
      • Teslovich T.M.
      • Ferreira T.
      • Segre A.V.
      • Steinthorsdottir V.
      • et al.
      Large-scale association analysis provides insights into the genetic architecture and pathophysiology of type 2 diabetes.
      ].
      Using a metabolomics approach to investigate genetic regulatory networks associated with complex disease traits, 102 of 131 serum metabolites showed sex differences in expression from more than 3300 population based samples (Cooperative Health Study in the region of Augsburg, KORA). A sex-specific GWAS identified a locus of the carbamoyl-phosphate synthase 1 gene (CPS1) for glycine, a key component in amino acid synthesis [
      • Mittelstrass K.
      • Ried J.S.
      • Yu Z.
      • Krumsiek J.
      • Gieger C.
      • Prehn C.
      • et al.
      Discovery of sexual dimorphisms in metabolic and genetic biomarkers.
      ]. A sex-based analysis revealed sex specific SNPs that have a greater effect on glycine in females compared to males. CPS1 encodes a mitochondrial enzyme involved in hepatic nitrogen urea metabolism and synthesis of arginine, a precursor to formation of nitric oxide, an endothelium-derived relaxing factor the absence of which is considered to contribute to endothelial dysfunction and progression of vascular disease. These studies underscore the ability to analyze existing genetic data sets by sex and their potential to provide a better understanding of mechanisms of cardiovascular disease that may extend to understanding sex-specific risks such as gestational diabetes and insulin resistance associated with polycystic ovarian syndrome (PCOS) in women [
      • Chang A.Y.
      • Wild R.A.
      Characterizing cardiovascular risk in women with polycystic ovary syndrome: more than the sum of its parts?.
      ].

      3.3 Cardiovascular anatomical pathologies

      As non-invasive imaging techniques are used to evaluate the presence and progression of asymptomatic vascular lesion characteristic of cardiovascular disease, genetic components of these complex traits warrant mention.

      3.3.1 Carotid intima-medial thickness (CIMT)

      CIMT is a quantitative trait showing some degree of heritability in different ethnic populations [
      • Juo S.H.
      Genetics of carotid atherosclerosis.
      ]. Using candidate gene analysis, variants associated with CIMT cluster into several gene classes: hemostasis, extracellular matrix remodeling, endothelial function, the renin angiotensin system, inflammation and antioxidation [
      • Wang L.
      • Beecham A.
      • Zhuo D.
      • Dong C.
      • Blanton S.H.
      • Rundek T.
      • et al.
      Fine mapping study reveals novel candidate genes for carotid intima-media thickness in dominican republican families.
      ]. In recently menopausal women, a candidate gene analysis of genes related to the anticoagulant, procoagulant, fibrinolytic, and innate immunity pathways, only variants in genes of the innate immunity pathway associated with CIMT [
      • Miller V.M.
      • Petterson T.M.
      • Jeavons E.N.
      • Lnu A.S.
      • Rider D.N.
      • Heit J.A.
      • et al.
      Genetic polymorphisms associated carotid artery intima-media thickness and coronary artery calcification in women of the Kronos Early Estrogen Prevention Study.
      ]. These women had endogenous estradiol levels <40 pg/ml, and it is unknown if these associations may change with use of menopausal hormone treatments.
      Genetic contributions to carotid thickening differ along segments of artery, which may reflect differences in exposure to physical forces at the carotid bifurcation [
      • Juo S.H.
      Genetics of carotid atherosclerosis.
      ]. Vascular stiffness in women but not men was nominally associated with variants in endothelial nitric oxide synthase (NOS3) gene [
      • Mitchell G.F.
      • Guo C.Y.
      • Kathiresan S.
      • Vasan R.S.
      • Larson M.G.
      • Vita J.A.
      • et al.
      Vascular stiffness and genetic variation at the endothelial nitric oxide synthase locus: the Framingham Heart study.
      ]. NOS3 is modulated by estrogen, thus, phenotypic expression of NOS3 variants should vary by hormonal status. With an average age of study participants of 62 years, most of the women would have been postmenopausal and estrogen deficient (see section below).
      A meta-analysis of GWAS studies from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium consisting of nine-community-based studies (age 44–76 years) identified four genetic variants associated with CIMT: a transcriptional factor (rs11781551), APOC1 (rs445925) associated with dyslipidemia, a telomerase inhibitor involved in chromosomal segregation in mitosis (rs6601530) and a regulator of uric acid (rs4712972). Contrary to these results, another GWAS study of the Framingham community failed to identify genes associated with increases in CIMT, in spite of large sample sizes and equal representation of men and women [
      • O'Donnell C.J.
      • Cupples L.A.
      • D'Agostino R.B.
      • Fox C.S.
      • Hoffmann U.
      • Hwang S.J.
      • et al.
      Genome-wide association study for subclinical atherosclerosis in major arterial territories in the NHLBI's Framingham Heart Study.
      ]. Neither of these studies stratified analysis by sex. However, overall heritability for intima-medial thickness of the internal carotid artery was higher for men than women, whereas heritability for intima-medial thickness in the common carotid artery was greater for women than men [
      • Fox C.S.
      • Polak J.F.
      • Chazaro I.
      • Cupples A.
      • Wolf P.A.
      • D'Agostino R.A.
      • et al.
      Genetic and environmental contributions to atherosclerosis phenotypes in men and women: heritability of carotid intima-media thickness in the Framingham Heart Study.
      ]. Analyses that do adjust for but do not stratify by both age and sex preclude identification of sex-specific genetic variance contributing to progression of CIMT. For example, apolipoprotein E4 (APOE4) associated with CIMT in several studies [
      • Juo S.H.
      Genetics of carotid atherosclerosis.
      ] but this gene may have sex-specific considerations, since it is a more critical risk factor for Alzheimer's disease in women compared to men [
      • Payami H.
      • Zareparsi S.
      • Montee K.R.
      • Sexton G.J.
      • Kaye J.A.
      • Bird T.D.
      • et al.
      Gender difference in apolipoprotein E-associated risk for familial Alzheimer disease: a possible clue to the higher incidence of Alzheimer disease in women.
      ].
      Several studies suggest sex-specific genetic factors contribute to CIMT. CIMT is an independent predictor of stroke [
      • Chambless L.E.
      • Folsom A.R.
      • Clegg L.X.
      • Sharrett A.R.
      • Shahar E.
      • Nieto F.J.
      • et al.
      Carotid wall thickness is predictive of incident clinical stroke: the Atherosclerosis Risk in Communities (ARIC) study.
      ] and incidence, prevalence and outcomes of stroke show sex disparities. One sex-specific analysis examined the association between CIMT and genetic variants of the phosphodiesterase 4D (PDE4D) gene, a susceptibility gene for stroke where CIMT would provide a phenotype for stroke risk with reduced complexity [
      • Liao Y.C.
      • Lin H.F.
      • Guo Y.C.
      • Yu M.L.
      • Liu C.K.
      • Juo S.H.
      Sex-differential genetic effect of phosphodiesterase 4D (PDE4D) on carotid atherosclerosis.
      ]. One homozygote variant had a significant odds ratio for increased CIMT in the overall study population. However, when these data were analyzed by sex, the odds ratio remained significant for men but not women [
      • Liao Y.C.
      • Lin H.F.
      • Guo Y.C.
      • Yu M.L.
      • Liu C.K.
      • Juo S.H.
      Sex-differential genetic effect of phosphodiesterase 4D (PDE4D) on carotid atherosclerosis.
      ]. PDE4E metabolizes cyclic AMP, a key signaling molecule involved with prostaglandin signaling. As this variant also correlated with stroke in young men, it is possible that the lack of efficacy of aspirin to reduce stroke risk in men may be related to this signaling pathway [
      • Ridker P.M.
      • Cook N.R.
      • Lee I.M.
      • Gordon D.
      • Gaziano J.M.
      • Manson J.E.
      • et al.
      A randomized trial of low-dose aspirin in the primary prevention of cardiovascular disease in women.
      ].
      Other genetic variants showing sex specific associations with subclinical atherosclerosis measured by CIMT include variants in the gene for interleukin-6, an inflammatory cytokine, associated with blood pressure and CIMT in Japanese women but not men [
      • Juo S.H.
      Genetics of carotid atherosclerosis.
      ], for cyclin-dependent kinase inhibitor 2A (CDKN2A), a negative regulator of cell proliferation, which showed association with CIMT in men, while the genetic variation in the gene for oxidized low density lipoprotein receptor 1 (OLR1) showed association with CIMT only in women.

      3.3.2 Coronary calcification

      Prevalence of coronary calcification is greater in men than women and varies by ethnic group [
      • Bild D.E.
      • Detrano R.
      • Peterson D.
      • Guerci A.
      • Liu K.
      • Shahar E.
      • et al.
      Ethnic differences in coronary calcification: the Multi-Ethnic Study of Atherosclerosis (MESA).
      ]. Cardiovascular disease risk factors such as hypertension, hypercholesterolemia and type 2 diabetes can exacerbate accumulation of calcium in the coronary arteries. For example, in individuals at high risk for hypertension, coronary calcification is associated with genes regulating collagen formation, allograft inflammatory factor 1 and bone morphometric protein receptor type 1A [
      • Lange L.A.
      • Lange E.M.
      • Bielak L.F.
      • Langefeld C.D.
      • Kardia S.L.
      • Royston P.
      • et al.
      Autosomal genome-wide scan for coronary artery calcification loci in sibships at high risk for hypertension.
      ]. Other genes implicated in coronary calcification are those for matrix metalloproteinase 9 (MMP9) [
      • Abilleira S.
      • Bevan S.
      • Markus H.S.
      The role of genetic variants of matrix metalloproteinases in coronary and carotid atherosclerosis.
      ] matrix Gla protein (MGP) and osteopontin (OPN) [
      • Taylor B.C.
      • Schreiner P.J.
      • Doherty T.M.
      • Fornage M.
      • Carr J.J.
      • Sidney S.
      Matrix Gla protein and osteopontin genetic associations with coronary artery calcification and bone density: the CARDIA study.
      ]. How these genetic associations might differ with hormonal status (e.g. estrogen) and risk for calcification in women is unknown.
      In diabetics, two SNPs of the gene encoding the inflammatory signaling molecule CD40 were negatively associated with coronary calcification in a candidate gene study in European Americans [
      • Burdon K.P.
      • Langefeld C.D.
      • Beck S.R.
      • Wagenknecht L.E.
      • Carr J.J.
      • Rich S.S.
      • et al.
      Variants of the CD40 gene but not of the CD40L gene are associated with coronary artery calcification in the Diabetes Heart Study (DHS).
      ]. However, a GWAS of African Americans did not uncover any genetic variants significantly associated with coronary calcification [
      • Wojczynski M.K.
      • Li M.
      • Bielak L.F.
      • Kerr K.F.
      • Reiner A.P.
      • Wong N.D.
      • et al.
      Genetics of coronary artery calcification among African Americans, a meta-analysis.
      ]. This negative finding may reflect failure to examine sex-specific effects or consider that multiple genetic pathways converge to promote calcification. Clinically relevant calcification also can exist in persons without significant conventional cardiovascular risk factors. For example, in women being screened for the Kronos Early Estrogen Prevention Study (KEEPS), about 14% were excluded because of the presence of clinically relevant coronary calcification (Agatston score >50) [
      • Miller V.M.
      • Black D.M.
      • Brinton E.A.
      • Budoff M.J.
      • Cedars M.I.
      • Hodis H.N.
      • et al.
      Using basic science to design a clinical trial: baseline characteristics of women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS).
      ]. Women included in KEEPS had Agatston score <50 with most having no calcium in their coronary arteries. However, in those women who had Agatston score >0 but <50, a candidate gene study associated variants in genes related to innate immunity, elastin remodeling and post-translational modification of proteins with coronary calcification [
      • Miller V.M.
      • Petterson T.M.
      • Jeavons E.N.
      • Lnu A.S.
      • Rider D.N.
      • Heit J.A.
      • et al.
      Genetic polymorphisms associated carotid artery intima-media thickness and coronary artery calcification in women of the Kronos Early Estrogen Prevention Study.
      ]. Because these women were estrogen deplete, it is unknown if these genetic variants would associate with calcification following the use of menopausal hormone therapy, as use of conjugated equine estrogen slowed coronary calcification in women of the Women's Health Initiative [
      • Manson J.
      • Allison M.
      • Rossouw J.E.
      • Carr J.
      • Langer R.
      • Hsia J.
      • et al.
      Estrogen therapy and coronary-artery calcification.
      ].

      4. Confounding variables: ethnicity and hormonal status

      4.1 Ethnicity

      Although the genetics of ethnicity are not considered in depth in this review, some mention is warranted given that development of cardiovascular disease is influenced by ethnicity/race [
      • Wang D.
      • Yang H.
      • Quinones M.J.
      • Bulnes-Enriquez I.
      • Jimenez X.
      • De La Rosa R.
      • et al.
      A genome-wide scan for carotid artery intima-media thickness: the Mexican-American Coronary Artery Disease family study.
      ,
      • Xiang A.H.
      • Azen S.P.
      • Buchanan T.A.
      • Raffel L.J.
      • Tan S.
      • Cheng L.S.
      • et al.
      Heritability of subclinical atherosclerosis in Latino families ascertained through a hypertensive parent.
      ,
      • Wang L.
      • Beecham A.
      • Zhuo D.
      • Dong C.
      • Blanton S.H.
      • Rundek T.
      • et al.
      Fine mapping study reveals novel candidate genes for carotid intima-media thickness in dominican republican families.
      ,
      • Liao Y.C.
      • Lin H.F.
      • Rundek T.
      • Cheng R.
      • Guo Y.C.
      • Sacco R.L.
      • et al.
      Segment-specific genetic effects on carotid intima-media thickness: the Northern Manhattan study.
      ,
      • Goodarzi M.O.
      • Taylor K.D.
      • Guo X.
      • Quinones M.J.
      • Cui J.
      • Li Y.
      • et al.
      Association of the diabetes gene calpain-10 with subclinical atherosclerosis: the Mexican-American Coronary Artery Disease Study.
      ]. As indicated in the preceding sections of this review, some studies account for race and ethnicity with specific designations, e.g. Caucasian of European ancestry, Mexican-American, Han Chinese, Japanese, Dominican Republic, etc. However, studies of populations which may consist of mixed racial origin, fail to adjust for population stratification in genetic analyses. For example, studies of individuals identifying as “Hispanic” are by self-report, unrelated individuals from Northern Manhattan [
      • Liao Y.C.
      • Lin H.F.
      • Rundek T.
      • Cheng R.
      • Guo Y.C.
      • Sacco R.L.
      • et al.
      Segment-specific genetic effects on carotid intima-media thickness: the Northern Manhattan study.
      ] or families of specific background (i.e. the Dominican Republic or Mexican-American) [
      • Xiang A.H.
      • Azen S.P.
      • Buchanan T.A.
      • Raffel L.J.
      • Tan S.
      • Cheng L.S.
      • et al.
      Heritability of subclinical atherosclerosis in Latino families ascertained through a hypertensive parent.
      ,
      • Wang L.
      • Beecham A.
      • Zhuo D.
      • Dong C.
      • Blanton S.H.
      • Rundek T.
      • et al.
      Fine mapping study reveals novel candidate genes for carotid intima-media thickness in dominican republican families.
      ,
      • Goodarzi M.O.
      • Taylor K.D.
      • Guo X.
      • Quinones M.J.
      • Cui J.
      • Li Y.
      • et al.
      Association of the diabetes gene calpain-10 with subclinical atherosclerosis: the Mexican-American Coronary Artery Disease Study.
      ]. The type of analyses varied among studies but none studied the effect of ancestry. Self-reported ethnicity is not necessarily the same as the genotypic ethnicity [
      • Miller V.M.
      • Petterson T.M.
      • Jeavons E.N.
      • Lnu A.S.
      • Rider D.N.
      • Heit J.A.
      • et al.
      Genetic polymorphisms associated carotid artery intima-media thickness and coronary artery calcification in women of the Kronos Early Estrogen Prevention Study.
      ]. For instance, “Hispanic” designation from the USA West Coast is most likely to be a mixture of Asian, Native-American and European ancestry, whereas “Hispanic” from the USA East Coast is most likely to be a mixture of Native American, European and African ancestry (Fig. 3). Therefore, studies that evaluate ethnicity/race as a contributor to cardiovascular risk require attention to the geographical origins of ancestry using genetic data [
      • Guthery S.L.
      • Salisbury B.A.
      • Pungliya M.S.
      • Stephens J.C.
      • Bamshad M.
      The structure of common genetic variation in United States populations.
      ]. A validation study of susceptibility loci for coronary artery disease is a case in point [
      • Lu X.
      • Wang L.
      • Chen S.
      • He L.
      • Yang X.
      • Shi Y.
      • et al.
      Genome-wide association study in Han Chinese identifies four new susceptibility loci for coronary artery disease.
      ]. Although 4 loci associating with coronary artery disease were common between the Han Chinese population and European populations, 4 were unique to the Han Chinese (case control study and the number of males or females was not included in the report) [
      • Lu X.
      • Wang L.
      • Chen S.
      • He L.
      • Yang X.
      • Shi Y.
      • et al.
      Genome-wide association study in Han Chinese identifies four new susceptibility loci for coronary artery disease.
      ]. Due to globalization, persons with multiracial and multiethnic backgrounds are increasingly common place; therefore, consideration of ancestral components of disease may become more relevant to understanding inherited cardiovascular risk. In the era of pharmacogenomics, biological factors will drive response to treatment while self-report of ethnicity may be driven by socio-political factors independent of biological factors.
      Figure thumbnail gr3
      Fig. 3Ancestry informative markers (492) were used to stratify participants of the Kronos Early Estrogen Prevention Study (KEEPS) by ethnicity using the program STRUCTURE
      [
      • Miller V.M.
      • Petterson T.M.
      • Jeavons E.N.
      • Lnu A.S.
      • Rider D.N.
      • Heit J.A.
      • et al.
      Genetic polymorphisms associated carotid artery intima-media thickness and coronary artery calcification in women of the Kronos Early Estrogen Prevention Study.
      ]
      . The STRUCTURE program uses ancestry informative markers that are evenly distributed across the genome to identify the ancestry of each subject by using the three HapMap 2 populations (CEU: Utah residents from the CEPH; YRI: Yoruba from Ibudan, Nigeria; and CHB/JPT: Han Chinese from Beijing, China, and Japanese from Tokyo, Japan) or the 1000 Genomes populations
      [
      • Pritchard J.K.
      • Stephens M.
      • Donnelly P.
      Inference of population structure using multilocus genotype data.
      ]
      . For KEEPS, CHB/JPT represents 89 HapMap Asian samples, URI represents 60 HapMap African samples and CEU represents 60 HapMap Caucasian samples. Each circle represents an individual; the colors represent self-identified ethnicity. E and W represent site center as from either the east coast or west coast of the United States, respectively. Women who self-report as USA Hispanic from the West Coast are a mixture of Native Americans and European Ancestry, while those from the East Coast were a mixture of African and European Ancestry.

      4.2 Hormonal status

      Nuclear receptors for the sex steroids affect gene transcription. Therefore, if a gene carrying a particular SNP has a sex steroid receptor in the promoter region, the relative contribution of that SNP to expression of the gene product will be affected by 1) hormonal status; 2) genetic variants in receptors for sex steroids; 3) genetic variants in enzymes required for metabolism (synthesis or degradation) of sex steroids.
      The first studies suggesting that hormonal genetic variant accelerate development of atherosclerosis was in a man with a deletion of estrogen receptor alpha [
      • Sudhir K.
      • Chou T.M.
      • Chatterjee K.
      • Smith E.P.
      • Williams T.C.
      • Kane J.P.
      • et al.
      Premature coronary artery disease associated with a disruptive mutation in the estrogen receptor gene in a man.
      ,
      • Sudhir K.
      • Chou T.M.
      • Messina L.M.
      • Hutchison S.J.
      • Korach K.S.
      • Chatterjee K.
      • et al.
      Endothelial dysfunction in a man with disruptive mutation in oestrogen-receptor gene.
      ]. As estrogen modulates endothelial nitric oxide synthase, this pathway is considered an important mechanism by which estrogenic hormones slow progression of atherosclerosis. Variants in the gene for estrogen receptor alpha also associated with levels of high density lipoprotein cholesterol and some inflammatory cytokines in women using estrogen for secondary prevention of cardiovascular disease [
      • Herrington D.M.
      • Howard T.D.
      • Hawkins G.A.
      • Reboussin D.M.
      • Xu J.
      • Zheng S.L.
      • et al.
      Estrogen-receptor polymorphisms and effects of estrogen replacement on high-density lipoprotein cholesterol in women with coronary disease.
      ,
      • Herrington D.M.
      • Howard T.D.
      • Brosnihan K.B.
      • McDonnell D.P.
      • Li X.
      • Hawkins G.A.
      • et al.
      Common estrogen receptor polymorphism augments effects of hormone replacement therapy on E-selectin but not C-reactive protein.
      ]. However, neither variants of estrogen receptors alpha nor beta significantly associate with cardiovascular events in women of the Women's Health Initiative where the use of estrogenic treatments was used to evaluate primary prevention of cardiovascular disease [
      • Rossouw J.
      • Bray P.
      • Liu J.
      • Kooperberg C.
      • Hsia J.
      • Lewis C.
      • et al.
      Estrogen receptor polymorphisms and the vascular effects of hormone therapy.
      ]. There were no consistent relationships between genetic variants in either estrogen receptor alpha or beta and HDL levels in perimenopausal women of various ethnicities [
      • Sowers M.R.
      • Symons J.P.
      • Jannausch M.L.
      • Chu J.
      • Kardia S.R.
      Sex steroid hormone polymorphisms, high-density lipoprotein cholesterol, and apolipoprotein A-1 from the Study of Women's Health across the Nation (SWAN).
      ]. Collectively, these data suggest that perhaps variants in estrogen receptor genes impact cardiovascular disease to a greater extent in men compared to women.
      The gene for the androgen receptor, which resides on the X chromosome, contains a functional polymorphism consisting of highly variable numbers of CAG repeats. Given the mosaic pattern of X inactivation in women, variants in this gene will have a greater impact in males than in females. There is significant heterogeneity in the CAG repeats among ethnic groups [
      • Ackerman C.M.
      • Lowe L.P.
      • Lee H.
      • Hayes M.G.
      • Dyer A.R.
      • Metzger B.E.
      • et al.
      Ethnic variation in allele distribution of the androgen receptor (AR) (CAG)n repeat.
      ] which may explain, in part, the inability to detect an association between these repeats and heart disease in Caucasian men [
      • Page S.T.
      • Kupelian V.
      • Bremner W.J.
      • McKinlay J.B.
      The androgen receptor gene CAG repeat polymorphism does not predict increased risk of heart disease: longitudinal results from the Massachusetts Male Ageing Study.
      ].
      These CAG repeats in hypothalamic/pituitary regions of the brain have a small effect on the feedback control for testosterone production in men such that the higher number of repeats, the higher the serum free testosterone levels [
      • Pausova Z.
      • Abrahamowicz M.
      • Mahboubi A.
      • Syme C.
      • Leonard G.T.
      • Perron M.
      • et al.
      Functional variation in the androgen-receptor gene is associated with visceral adiposity and blood pressure in male adolescents.
      ]. These repeats are associated with male infertility. In addition, they may impart some cardiovascular risk as low numbers of repeats associated with abdominal adiposity and higher sympathetic modulation of vascular tone in boys than in girls. Data are conflicting regarding the association of CAG repeats with serum levels of high density lipoprotein cholesterol (HDL) and development of type 2 diabetes in men [
      • Zitzmann M.
      • Nieschlag E.
      The CAG repeat polymorphism within the androgen receptor gene and maleness.
      ]. In women, CAG repeats associate with polycystic ovarian syndrome, characterized by male-pattern adiposity and propensity for development of type 2 diabetes [
      • Kim J.J.
      • Choung S.H.
      • Choi Y.M.
      • Yoon S.H.
      • Kim S.H.
      • Moon S.Y.
      Androgen receptor gene CAG repeat polymorphism in women with polycystic ovary syndrome.
      ,
      • Shah N.A.
      • Antoine H.J.
      • Pall M.
      • Taylor K.D.
      • Azziz R.
      • Goodarzi M.O.
      Association of androgen receptor CAG repeat polymorphism and polycystic ovary syndrome.
      ].
      In addition to hormonal receptors, there are genetic variants in enzymes involved with synthesis, conversion and degradation of the sex steroids. However, little attention has focused on these variants with hormonal modulation of cardiovascular disease processes in men or women. For example, SULT1A1, an ubiquitous enzyme that is the predominant sulfotransferase present in the liver, is highly polymorphic including copy number and multiple single nucleotide polymorphisms that affect enzyme activity [
      • Hebbring S.J.
      • Adjei A.A.
      • Baer J.L.
      • Jenkins G.D.
      • Zhang J.
      • Cunningham J.M.
      • et al.
      Human SULT1A1 gene: copy number differences and functional implications.
      ,
      • Raftogianis R.B.
      • Wood T.C.
      • Weinshilboum R.M.
      Human phenol sulfotransferases SULT1A2 and SULT1A1: genetic polymorphisms, allozyme properties, and human liver genotype-phenotype correlations.
      ]. Thus, a gene “dose” effect of SNP by copy number could affect response to various hormonal therapies impacting vascular function including endothelium-derived nitric oxide. In addition, biological effects of SNPS on this enzyme seem to vary by ethnicity as an SNP in the promoter region decreased SULTA1A1 activity in African Americans but increased activity in Caucasians [
      • Ning B.
      • Nowell S.
      • Sweeney C.
      • Ambrosone C.B.
      • Williams S.
      • Miao X.
      • et al.
      Common genetic polymorphisms in the 5′-flanking region of the SULT1A1 gene: haplotypes and their association with platelet enzymatic activity.
      ]. Variation in genes for other enzymes in the estrogen metabolic pathway and their association with menopausal vasomotor symptoms also show differences among ethnic groups [
      • Sowers M.R.
      • Wilson A.L.
      • Karvonen-Gutierrez C.A.
      • Kardia S.R.
      Sex steroid hormone pathway genes and health-related measures in women of 4 races/ethnicities: the Study of Women's Health across the Nation (SWAN).
      ,
      • Crandall C.J.
      • Crawford S.L.
      • Gold E.B.
      Vasomotor symptom prevalence is associated with polymorphisms in sex steroid-metabolizing enzymes and receptors.
      ,
      • Woods N.F.
      • Mitchell E.S.
      • Tao Y.
      • Viernes H.M.
      • Stapleton P.L.
      • Farin F.M.
      Polymorphisms in the estrogen synthesis and metabolism pathways and symptoms during the menopausal transition: observations from the Seattle Midlife Women's Health Study.
      ]. However, studies linking menopausal symptoms and genotypes to cardiovascular disease are lacking.
      Hormonal status affects phenotypic consequences of SNP expression. For example, in unpublished data from our group (Moyer, et al.), the significance of an association of the G allele in SNP of interleukin regulatory factor 4 with change in carotid intima-medial thickness in women of the KEEPS trial varied depending on the treatment assignment (Fig. 4).
      Figure thumbnail gr4
      Fig. 4Association of allele variants for the SNP rs7768807 of the gene for interleukin regulatory factor 4 involved in leukocyte function with changes in carotid intima-medial thickness varied by the type of hormone treatment in women compliant to treatment with either placebo (PL), transdermal 17β estradiol (E2) or oral conjugated equine estrogen (oCEE) in the Kronos Early Estrogen Prevention Study (KEEPS). Minor allele frequency for rs7768807 is 27%.
      These observations point to several important considerations in evaluating genetic contributions to cardiovascular disease. First, attention should be given to ethnicity verified by genetic data rather than self-identification. Second, since hormonal status varies across the life span in both men and women, hormonal status, age and sex should be considered when evaluating specific genetic contributions to cardiovascular risk. Finally, studies to examine genetic basis of hormonal influences on cardiovascular function should continue given the on-going use of hormonal products by women and increased use of exogenous testosterone by men.

      5. Statistical considerations for future studies

      As genetic studies go forward, there are several important considerations that need to be incorporated into analyses. For example, population geneticists study the distribution and changes of allele frequency in a given population due to natural selection, genetic drift, mutation and gene flow by taking into account recombination, population subdivision and population structure. Therefore, in genetic studies of admixed populations, it is relevant to assess if the self-reported ancestry is the same as genotypic ancestry. As discussed above, this can be accomplished by calculating the proportion of ancestry derived from each geographical region using ancestry informative markers (see Fig. 3).
      It is also important to consider the statistical analysis method in genetic association studies because of possible confounding of the number of X chromosomal variants by sex. Univariate linear or logistic regression or standard chi-square tests are typically used for the autosomes and these tests can still be applied for the pseudoautosomal regions of the X and Y chromosomes. However, different analysis methods are necessary when analyzing X chromosome variants outside of this region. For the regions undergoing X-inactivation, the statistical analysis should account for this process.
      However, some methods to account for X-inactivation have not yet been adopted by the research community because they are not readily available in statistical software packages. Those currently available include PLINK for genome-wide analysis [
      • Purcell S.
      • Neale B.
      • Todd-Brown K.
      • Thomas L.
      • Ferreira M.A.
      • Bender D.
      • et al.
      PLINK: a tool set for whole-genome association and population-based linkage analyses.
      ] and R/Bioconductor package ‘snpMatrix’ (http://www.bioconductor.org) [
      • Clayton D.
      Testing for association on the X chromosome.
      ] which both allow for statistical tests of X chromosome variants, and IMPUTE2 [
      • Howie B.
      • Fuchsberger C.
      • Stephens M.
      • Marchini J.
      • Abecasis G.R.
      Fast and accurate genotype imputation in genome-wide association studies through pre-phasing.
      ], which allows for genome-wide imputation of untyped/missing variants on the X chromosome.
      Most methods to analyze X chromosome data have been developed and evaluated univariately, rather than considering multivariate models with multiple genetic factors. As complex trait etiology likely involves a large number of genetic and environmental components, this is a critical research area. Furthermore, studies of the genetics of sex differences should not be limited to variants on the sex chromosomes. Autosomal genetic effects can also be modified by sex, and gene–sex interactions effects may underlie observed sex differences in disease traits. Interactions between autosomal variants and sex may be surrogates for gene–gene interactions between variants on the autosomes and the X or Y chromosomes, as well as gene by hormone interaction effects [
      • Bloomer L.D.
      • Nelson C.P.
      • Denniff M.
      • Christofidou P.
      • Debiec R.
      • Thompson J.
      • et al.
      Coronary artery disease predisposing haplogroup I of the Y chromosome, aggression and sex steroids–genetic association analysis.
      ]. In fact, many autosomal sequence variants that are associated with gene expression (eQTLs) display sex differences, and such sex-biased effects on expression are observed for important regulatory genes [
      • Dimas A.S.
      • Nica A.C.
      • Montgomery S.B.
      • Stranger B.E.
      • Raj T.
      • Buil A.
      • et al.
      Sex-biased genetic effects on gene regulation in humans.
      ]; thus, studies of gene–sex interactions may be critical tools to explore the genetics of sex differences. Recently many statistical methods have been developed to explore gene–environment interactions [
      • Winham S.J.
      • Biernacka J.M.
      Gene-environment interactions in genome-wide association studies: current approaches and new directions.
      ,
      • Thomas D.
      Gene-environment-wide association studies: emerging approaches.
      ]. Such methods could be applied to study sex-specific genetic effects, and represent an important opportunity for future research especially related to cardiovascular disease.

      6. Summary/conclusions

      Cardiovascular diseases represent complex genetic traits with phenotypes being influenced by genetic, hormonal, environmental, and cultural variables. Although many studies have attempted to gain insight into etiology of various cardiovascular diseases and their risk factors through genetic analyses, most have ignored the basic genetics contributed by the sex chromosomes and have failed to conduct sex-stratified analyses, despite emerging tools to study the genetics of sex differences for complex traits. Furthermore, the confounding effects of ancestry and hormonal status across the life-span also need to be considered. Given that sex differences in incidence, prevalence, morbidity and mortality from cardiovascular diseases are documented around the world, sex must be considered in future investigations of genetic components of the disease if preventive, diagnostic and treatment strategies are to be individualized to optimize outcomes.

      Conflicts of interest

      There are no conflicts of interest to declare.

      Acknowledgments

      Grants from the NIA AG 44170 , ORWH HD65987 and the Mayo Foundation .

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      Linked Article

      • Importance of sex and gender in atherosclerosis and cardiovascular disease
        AtherosclerosisVol. 241Issue 1
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          In this special issue of the journal, there are papers on bone health and coronary artery calcification, age and sex differences in the effect of parental stroke on the progression of carotid intima-media thickness, macrophage subsets in the adipose tissue by sex and by reproductive age of women, uric acid levels and metabolic syndrome, sex differences in cardiovascular risk factors and disease prevention, severity of stable coronary artery disease and its biomarkers, cardiovascular disease and autoimmune diseases genetics of cardiovascular disease, outcome after CABG; association of serum phosphorus with subclinical atherosclerosis in chronic kidney disease and relationship of uric acid levels to coronary disease.
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      • Sex matters to the heart: A special issue dedicated to the impact of sex related differences of cardiovascular diseases
        AtherosclerosisVol. 241Issue 1
        • Preview
          Ever since the early 1980s most cardiovascular research has focused on men [1]. This phenomenon has led to the under appreciation of sex-differences in cardiovascular disease (CVD) from an etiological, prognostic, diagnostic and therapeutic perspective. Several initiatives to promote women's health, such as the Women's Health Initiative [2] have been initiated and have changed the practice of cardiovascular disease prevention in women over the past decade. This ultimately led to the first guidelines for cardiovascular disease prevention in women by the American Heart Association in 1999 [3].
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