Cardiovascular Disease Genomic Research

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Clin Chem. Author manuscript; available in PMC 2013 May 10.
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Clin Chem. 2012 January ; 58(1): 113–126. doi:10.1373/clinchem.2011.170423.
Next Steps in Cardiovascular Disease Genomic Research –

Sequencing, Epigenetics, and Transcriptomics
Renate B. Schnabel, MD, MSc1, Andrea Baccarelli, MD, PhD2, Honghuang Lin, PhD3,4,
Patrick T. Ellinor, MD, PhD5,6, and Emelia J. Benjamin, MD, ScM3,7
1Department of General and Interventional Cardiology, University Heart Center Hamburg,
Hamburg, Germany
2Laboratory of Environmental Epigenetics, Exposure Epidemiology and Risk Program, Harvard
School of Public Health, Boston, Massachusetts
3National
Heart Lung and Blood Institute’s and Boston University’s Framingham Heart Study,
Framingham, Massachusetts
4Department of Medicine, School of Medicine, Boston University, Boston, Massachusetts
5Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts

6Cardiac Arrhythmia Service, Massachusetts General Hospital, Boston, Massachusetts
7Cardiologyand Preventive Medicine Sections, Department of Medicine, School of Medicine,
Boston University, Boston, Massachusetts
Abstract
BACKGROUND—Genomic research in cardiovascular disease (CVD) has progressed rapidly
over the last five years. However, in most cases these ground-breaking observations have not yet
been accompanied by clinically applicable tools for risk prediction, diagnosis, or therapeutic
interventions.
CONTENT—We reviewed the English literature for novel methods and promising genomic
targets that will permit large-scale screening and follow-up of recent genomic findings for CVD.
We anticipate that advances in three key areas will be critical for the success of these projects.
First, exome-centered and whole genome next generation sequencing will identify rare and novel
genetic variants associated with CVD and its risk factors. Improvements in methods will also
greatly advance the field of epigenetics and gene expression in humans. Second, research
increasingly acknowledges that static DNA sequence variation explains only a fraction of the
inherited phenotype. Therefore we expect that multifold epigenetic and gene expression signatures
will be related to CVD in experimental and clinical settings. Leveraging existing large-scale
consortia and clinical biobanks combined with electronic health records holds promise to integrate
epidemiological and clinical genomics data. Finally, a systems biology approach will be needed to
integrate the accumulated multidimensional data.
SUMMARY—Novel methods in sequencing, epigenetics and transcriptomics, and unprecedented
large-scale cooperative efforts promise to generate insights into complex CVD. The rapid
accumulation and integration of knowledge will shed light onto the considerable proportion of the
missing heritability of CVD.
Correspondence to: Renate B. Schnabel.

Schnabel et al. Page 2
Keywords
genomics; sequencing; epigenetics; transcriptomics
Cardiovascular disease (CVD) morbidity and mortality pose a significant public health
burden worldwide (1). Understanding the multifactorial, complex underpinnings of CVD
holds promise to have global impact on health promotion. CVD is a heritable condition(2),
with heritability estimates of up to 60 percent for coronary heart disease(3). The completion
of the Human Genome Project(4) has raised high expectations for substantial insights into
the polygenetic architecture of complex human diseases such as CVD. However, despite the
rapid identification of a few genetic variations for CVD and its risk factors, the number of
findings that have been translated into clinical practice has remained disappointingly low.
For this review, we searched the current literature for novel methods and scientific
approaches that may follow-up the recent genomic findings for CVD variants and ultimately
enable large-scale screening in clinical samples and the general population.
Common complex disease

Unlike Mendelian disorders, there not exist a single genetic variant that is responsible for
CVD (Table 1). Rather, CVD phenotypes and other complex diseases result from the sum of
multiple polymorphisms, each with relatively small effects on gene expression and disease.
Genome-wide association studies (GWAS) have been conducted to dissect the contribution
of common polymorphisms and have lead to the identification of more than 20 new loci
related to myocardial infarction and other CVD phenotypes(5). Figure 1 summarizes the
results of GWAS findings for CVD and its risk factors.
Whereas GWAS has had great success in identifying genetic loci for CVD, many challenges
remain. First, these studies typically use only common SNPs which by definition have an
allele frequency of over 1%. Thus, rarer SNPs with stronger effects may not be identified by
GWAS. Second, as for all association analyses, a clear CVD phenotype is required. For
instance, distinct phenotypes of coronary artery disease with different heritability patterns
exist(6) that have often been largely ignored in genomic analyses to date. Clinically
important variation in manifestation, therapeutic approach and prognosis exist for
phenotypes such as left main disease, diffuse versus focal coronary artery disease, ectatic
lesions, and degree of coronary calcification. The harmonization of clinically relevant and
rigorously defined phenotypes across cohorts is essential to better delineate genetic
associations. For continuous and well-standardized phenotypes such as blood lipids highly
significant statistical associations have been shown, but they generally have very modest
effect sizes(7).
The exact pathophysiological mechanisms behind the vast majority of common genetic
polymorphisms from GWAS screening are largely unknown. In many cases the search for
the functional variants has been rendered more difficult by the unexpected observation that
many genome-wide hits are located in non-coding genomic regions and gene deserts as
shown for the 9p21 locus consistently related to CVD(8;9) and the 4q25 locus for atrial
fibrillation(10). Finally, the identified genetic variants often explain less than 10 percent of
trait or diseasevariability (11). Even the combination of information on an increasing
number of common genetic variants promises low discriminatory ability due to small
absolute variation in risk between carriers and non-carriers of the alleles (12). Hundreds of
frequent genetic variants explain a small proportion of inter-individual common phenotype
variability (13;14), and the hypothesis has been raised that some of the missing heritability

could be explained by rare/low frequency variants, structural alterations, and epigenetics

(15;16).
Despite these challenges, there are a few examples of genetic loci in which the functional
importance of a SNP has been elucidated. In a recent elegant investigation of a low-density
lipoprotein cholesterol levels GWAS result, a common noncoding polymorphism at the
chromosome 1p13 locus was reported to create a transcription factor binding site and alter
the expression of the SORT1 gene. In turn, SORT1 affects very low-density lipoprotein
secretion in the liver, and potentially may represent a causal pathway from blood lipids to
myocardial infarction. Such experiments show the value of whole-genome association study
efforts when complemented with follow up functional investigations (17).
Given the challenges posed by the recent GWAS findings and the gaps that remain in
translating these findings into clinical practice, in the remainder of the article we will focus
on three emerging areas that will be critical to advancing our understanding of CVD
phenotypes. Specifically, we will review the application of next generation sequencing,
epigenetics, and transcriptomics to CVD phenotypes. We will discuss the importance of
utilizing systems biology to integrate data across genomic platforms, and we will consider
the potential relevance of genomic findings to the clinical setting.
Next Generation Sequencing

Traditional Sanger sequencing has high-fidelity, but is slow and quite expensive relative to
next generation methods. Recent advances in sequence-based technology permit massive
parallel sequencing (Table 2). Real-time sequencing replaces natural nucleotides or
reversible terminators by detection of continuously added fluorescence labeled nucleotides
to the growing DNA strand enhancing the speed and output length of nucleotides(18). The
establishment of sequencing libraries and post-sequencing bioinformatics algorithms
facilitate the generation, reconstruction, and analysis of sequence reads. Optimizing
sequencing accuracy and redundant sequencing reduce sequencing errors. Bioinformatics
tools are continuously being refined to store and process the massive amount of sequence
data.
Several challenges remain with next generation sequencing. Platforms differ by template
preparation, sequencing chemistry, imaging, read length and quantity per run. Quality
measures are provided by the respective manufacturers, but a uniform quality assessment
protocol has not been implemented. Statistical analyses need to account for type I error in
the resulting huge data sets and need to develop methods to dissect phenotype relevant
variants from commonly shared alleles. Clustering of the total data into conceptually related
parts facilitates information digestion(19). Finally, the identification of disease specific
genetic variants from bystanders remains a challenge.
Despite these limitations, next generation sequencing has already shown great success and
the methods for sequencing have evolved to the point that within the last year sequencing of
an entire genome has become considerably less expensive and straightforward. Next
generation sequencing has been applied to follow up GWAS loci for CVD phenotypes, to
identify rare forms of CVD traits by exome sequencing, and to identify structural variation
in the genome.
Evaluation of GWAS loci for CVD phenotypes—The design of GWAS largely has
been restricted to genotyping common alleles. However, the accumulating evidence of
GWAS suggests that frequent polymorphisms with small odds ratios are unlikely to explain
all familial clustering of CVD. Hence, the common disease – common variant rule has to be
reconsidered. Instead, many heterogeneous rare or low frequency variants in the population

are hypothesized to explain larger proportions of heritability than previously assumed. Re-
sequencing or deep sequencing of the candidate genomic region provides a focused
approach to assess common as well as less frequent genetic variation at the site of interest as
a follow-up of GWAS. Sequencing in larger clinical or even population-based samples

requires different prerequisites in methodology compared to Mendelian cardiovascular
disorders (Table 1).
Exome sequencing in rare and common forms of CVD—Time and effort can be
reduced by focusing on transcribed gene regions in transcriptome or exome sequencing, but
leaves out non-coding regions, which may also be of scientific interest, as shown in many
GWAS findings. Exome sequencing also will provide a means to shed light on alternative
splicing variants in relation to CVD on the way from transcription to translation. In addition,
the completion of the 1000 Genomes Project (http://www.1000genomes.org) will give a
comprehensive genotype map in HapMap populations that will provide novel variants and
may answer many questions without the need of re-sequencing.
Whereas to date, sequencing has mainly been limited to unraveling single or oligogenic
CVD causes, the determination of complete DNA sequences of human individuals will bring
next generation sequencing technology to the population level. A publicly funded large-
scale effort on the application of next generation sequencing to study the protein coding
regions of the human genome is the Grand Opportunity Exome Sequencing Project. The
study of well-phenotyped cohorts is intended to discover low frequency alleles and novel
polymorphisms with large effect sizes as correlates of complex phenotypes and affiliated
mechanisms contributing to CVD. The sequencing results and discoveries will be made
available to the scientific community in order to improve diagnosis and management of
diseases (https://esp.gs.washington.edu/drupal/).
As more experience is accumulated the understanding of the genetic architecture of CVD

will deepen. For example, to date, it is largely unknown what role modifier sequence
variants play in common CVD. Modifier sequence variants may be important correlates of
phenotypes in single gene disorders as they may determine nuances of phenotype besides the
primary, causal alleles(20). In common diseases we have been lacking the power to
investigate subtle differences that distinguish modifier sequence variants from non-causal
variation that is inherited.
Structural variation—The pathophysiological significance of structural variants to CVD

is incompletely understood and may range from direct disease association, to quantitatively
or qualitatively modifying disease phenotype to no relevant effect. Deletions or insertions
affecting one to more than a million base pairs are frequently observed throughout the
genome(21). Smaller studies have investigated insertions and deletions in relation to CVD
phenotypes such as hypertension and coronary heart disease(22). An insertion/deletion was
found in the angiotensin I converting enzyme and has repeatedly been reported in relation to
CVD and its risk factors(23).
Among structural variation, copy number variation (CNV) has been an emerging focus of
interest. In non-syndromal CVD, an exonic deletion in the BCL2-associated athanogene 3
(BAG3) gene was related to dilated cardiomyopathy in a multigenerational family with
autosomal dominant transmission of the disease(24). At the population level, investigations
of SNPs and CNVs revealed potential associations with aortic root diameter in hypertensive
African Americans(25). In sporadic as well as familial aortic aneurysms and dissections rare
CNVs disrupting smooth muscle adhesion or contraction have been identified in inherited
forms of aneurysms(26).

To date, no robustly replicated CNVs in relation to CVD have been identified that could
have been added to Figure 1. Initial evidence for CNV in early-onset myocardial infarction
did not detect differences for genome-wide common and rare CNVs, the number of genes
intersected by CNVs, or the total extent of CNVs compared to controls(27). More detailed
analyses will be needed to comprehensively assess the implication of CNVs to CVD.
Epigenetics
New sequencing methods also will provide high-resolution maps of epigenetics(28).
Epigenetics investigates mechanisms of gene-expression controls independent to genetic
variations(29). The most investigated epigenetic modifications are DNA methylation,
histone modifications, and nucleosome positioning.
DNA methylation is the covalent methylation of cytosines catalyzed by DNA

methyltransferases located in CG dinucleotides (CpGs). Usually, methylated regions are not
actively transcribed, and are silenced in fully differentiated tissues (30). In contrast, non-
methylated regions mark genes that are either actively expressed or poised to be
expressed(31). DNA methylation has been predominantly investigated in regions enriched
with CpGs named CpG islands. Whereas CpG islands have been classically considered the
main regulatory regions for methylation-related control of gene expression, recent work has
suggested that CpG island “shores”, i.e., regions less dense in CpG content located up to
2Kb away from the islands, may be even more enriched in regulatory sequences(32). DNA-
methylation can be determined using a candidate-gene approach through bisulfite-

pyrosequencing OR MASS-array, or using genome-scale analyses by methylation
microarrays or sequencing-based technologies including reduced representation bisulphite
sequencing, methylC-sequencing, methylated DNA immunoprecipitation sequencing, or
methylated DNA binding domain sequencing(33;34).
The major histones H2A, H2B, H3 and H4 group into globular polymers and form the
nucleosomes around which DNA segments are wrapped(35). Histones are not just structural
units, but undergo covalent posttranscriptional modifications including methylation,
phosphorylation, acetylation, and ubiquitination. Those histone modifications appear to
regulate chromosome condensation, gene expression, splicing, and DNA repair mechanisms.
The third major epigenetic regulator of gene transcription is nucleosome positioning. The
presence and location of nucleosomes directly interfere with the transcription process. DNA
organization in nucleosomes inhibits access of transcription factors and polymerases.
Nucleosome-free gene start sites are needed to allow the assembly of the transcription
complex and activity of RNA polymerases. The initiation and orchestration of epigenetic
mechanisms is little understood. It has been assumed that interaction between epigenetic
factors may be central to direction of DNA methylation, histone modification and chromatin
remodeling(36).
Epigenetics may be a unifying principle in the etiology of complex diseases(37). In contrast

to the static DNA backbone, epigenetic modifications, albeit genetically determined, can
also be modified by environmental influences and are dynamic over time(38). Whereas our
knowledge on epigenetics and expectations are largely derived from cancer research in
which hypomethylation of non-coding DNA and de-novo hypermethylation of tumor
suppressor genes has been regularly reported in malignant cells, experience with CVD
associations is still much less substantiated.
Epigenetic marks are almost entirely established during embryogenesis and early
development and may impact cardiovascular development through stem cell control and
differentiation of endothelial and cardiac and vascular muscle cells. During early

development DNA methylation is mostly dependent on availability of folic acid and other
nutrients in the methyl-donor cycle. Genomic DNA methylation directly correlates with
folate status and prenatal folic acid deficiency has been suggested to represent a link to early
CVD development and the observation that early atherosclerosis may begin in utero(39).
Along these lines, intrauterine exposure to dietary protein restriction can lead to
hypertension and endothelial dysfunction in rat offspring, the exact mechanisms being
unknown(40).
Endothelial shear stress also has been identified as a trigger of early angiogenic processes
and vascular development through by histone deacetylases(41). During early stages of
atherosclerosis in apolipoprotein E null mice, global hypomethylation precedes
histologically detectable vascular lesions(42). Among proteins that specifically recognize
CpG methylated DNA regions is methyl-CpG–binding domain protein 2 which activates
histone deacetylases and suppresses gene transcription. Knockdown of the protein was
shown to exhibit beneficial effects on endothelial nitric oxide synthase, induce angiogenesis,
and to be protective against limb ischemia(43). Observations seemed to be specific to
vascular endothelium and promise success in targeted modulation. In fact, endothelial nitric
oxide synthase shows striking methylation differences in endothelial cells with highest
activity compared to dense methylation in non-endothelial cell types(44).
Hypoxia leads to a global reduction in transcription activity reflected in upregulation of

ubiquitous repressive histone methylation(45). Histone modifications seem to be mandatory

to maintain constitutive endothelial nitric oxide synthase transcriptional activity. Acetylation
of proximal promoter histones under hypoxia results in repression of transcription with
deleterious effects on vascular homeostasis(46). Similarly, histone deacetylase 3 has been
demonstrated to be critical in endothelial survival and atherosclerosis development in
response to disturbed blood flow at vessel bifurcations that are known to be prime locations
of atherosclerosis development(47).
Diabetes mellitus is a major risk factor for CVD with clear environmental and genetic
influences. Epigenetics modifications have been invoked as potentially causal for diabetes in
integrating DNA code and external influences(48). DNA methylation of the insulin
promoter region is inversely related to insulin production. De-methylation in mature human
insulin-producing cells affects insulin secretion, and pathological methylation patterns in
context with environmental influences have been suggested to contribute to development of
diabetes(49). At a population level, a differentially methylated CTCF-binding site at 11p15
was discovered in relation to type 2 diabetes(50).
There is exponentially advancing availability of increasingly robust data sets with the fine
mapping of the human DNA methylome at base resolution(33), genome-wide nucleosome

positioning maps, and the continuing identification of new variants and modifications.
Online epigenetics resources have been assembled recently (for review see(51)).
Whereas we have gained most epigenomic insights from basic science, epidemiologic data
have remained scarce. We are at the beginning of understanding that epigenetic
modifications of the genome may be as important as the DNA information itself in the
stable, vertical transmission of gene expression and heritability of phenotypes.
Transcriptomics
A further layer of complexity is added at the transcriptional level. Gene expression is
determined by genetic and environmental influences. It can be assumed that qualitative, and,
to a larger extent, quantitative differences in gene expression determine an individual’s
phenotype(52). Subtle changes in splice site variations, 3’ untranslated regulatory regions,

non-coding RNAs, and direct interaction of transcription factors may have significant effects
on gene expression patterns and CVD phenotypes that can only be assessed by transcriptome
interrogation. Single time point transcriptomics analysis represents a snapshot of cellular
and tissue architecture with an enormous amount of information to process, though not
reflecting biological variation over time. In particular, the transcriptome in acutely injured
tissues, e.g. during myocardial infarction with widespread necrosis, inflammation and
oxidative stress and invasion of acute-phase response cells, may have only remote
resemblance to diseased or normal tissue under stable conditions. However, acute injury
signatures in myocardial infarction may also provide the means to understand the
consequences of genetics and epigenetics resulting in the actual phenotype.
More recently, transcriptome assessment has led to the identification of small, single-
stranded RNA molecules such as microRNA (miRNA) and short, interfering RNA(53).
Small RNAs are short non-coding RNAs that regulate translation of their target messenger
RNAs (mRNA) through mRNA degradation or suppression of translation. Advantages of
miRNAs are their relative chemical stability, which facilitates the miRNA isolation and
analyses from clinical samples. Some miRNAs are actively secreted in exosomes and can be
measured in blood specimens rendering miRNAs targets for biomarker discovery(54).
Specifically designed short interfering RNAs block miRNA action and may have therapeutic
potential(55).
In contrast to the more stable DNA, the protocol for preanalytic steps, extraction and sample
preparation for RNA are highly demanding. Source RNA quality is crucial to reliably assess
transcription levels(56). In order to analyze the abundance of RNA including non-
polyadenylated and non-coding RNA, total RNA measurement has been implemented(57).
Advances in RNA sequencing are more reliable than former tiling microarrays(58;59).
Conventional microarray systems derived relative image intensities and signal quantification
was limited. Low expression transcripts could hardly be detected against transcriptional
background noise and non-specific probe binding produced false positive results.
Furthermore, probe-hybridization was limited to established genomic sequences. Whole
transcriptome sequencing (RNA-seq) allows the detection of transcripts without exact prior
knowledge of the genomic sequence. The directly cDNA-derived digital signals show good
reproducibility and comparatively few false-positive signals(59), although the challenge of
quantification of the level of expression, which is not an on/off phenomenon remains(60).
Replication is mandatory in large-scale gene expression studies.
Atherosclerotic CVD originates from the arterial endothelium. Distinct gene expression
patterns have been observed for pro-atherosclerotic endothelial phenotypes(61;62), and gene
connectivity network analysis in a systems biology approach showed transcript profiles and
susceptibility to oxidative stress in coronary arteries(63). Mouse aortic gene expression

patterns at different stages of atherosclerotic disease revealed striking similarities to human
atherectomy samples(64).
In humans, expression signatures have been related to prognosis in CVD such as first
manifestation of heart failure(65). In large human populations the assessment of the
transcriptome in CVD is mostly restricted to non-cardiac tissues and cells unless patients
undergo risk prone myocardial biopsy. Peripheral blood mononuclear cells and platelets are
obvious candidates due to their central role in the atherosclerotic disease process and
abundant availability. In fact, transcript levels have been related to atherosclerosis burden of
the coronary and carotid arteries(66;67).
Promising is the simultaneous determination of genome-wide gene expression and genetic

variability that will unravel direct links between transcriptional regulation and CVD(68;69).

First results in peripheral blood thrombocyte transcriptome assessment at the epidemiologic

level revealed the heritability of transcripts(70). Inflammatory expression signatures from
the nuclear factor-κB pathway were related to body mass index. Genome-wide mononcyte
expression interrogation of over 1600 expression traits were related to at least one of the
classical cardiovascular risk factors(71). Recently, a novel susceptibility locus for coronary
artery disease (LIPA, lipase A) was identified in the general population by combining
genome-wide genetic and gene expression data(72).
An emerging field of transcriptomics is miRNA expression profiling. Of the 940 known

mature human miRNAs, eighteen have consistently been shown to dominate cardiac miRNA
expression(73). They can be detected during physiological cardiac development and in
pathological states of CVD(74;75). Endothelial alterations in atherosclerosis are reflected by
miRNA expression(76). Cardiac damage leads to detectable release of cardiomyocyte-
specific miRNAs into the circulation, whether actively or through cell degradation remains
to be investigated(77). Distinct miRNA patterns differentiate between entities of heart
failure(75), and the dynamic in miRNA expression indicates therapeutic success in cardiac
recompensation(78).
Differential splicing has been related to disease phenotypes such as heart failure. Expression
patterns of the same exon in a specific gene may vary largely by alternative RNA
generation, although the total amount of messenger RNA for the respective gene may
remain constant(79). To better understand the regulation of gene expression, nuclear

transcription and RNA stability controlled by miRNA need to be assessed at the same time.
New sequencing methods will render differential splicing accessible to large-scale
investigation.
First experiences in the clinical setting revealed downregulation of miR-126 and miR-17
cluster usually detected in the endothelium and miR-155 related to inflammation, whereas
cardiac muscle–enriched miRNAs were elevated in the blood of patients with coronary
artery disease(80). Candidate miRNAs from experimental data showed associations with
acute myocardial infarction in human plasma(81). miRNA derived from peripheral blood
mononuclear cells showed similarities and differential expression signatures in ischemic
versus non-ischemic cardiomyopathy patients and largely differed from control
individuals(82). Not only the presence and quantity of miRNA may be relevant to CVD, but
initial data suggest that genetic variation in binding sites may be related to CVD, too(83).
Intriguingly, miRNA can be easily and effectively antagonized. Antagomirs targeted at

miR-21 reduced remodeling as a response to pressure overload(84), and miR-23a knock-
down in mice prevented cardiac hypertrophy(54). Effects of miRNA depletion provided
different results after knock-down or biochemical blockade, and systemic application of

miRNA mimics may act through effects on remote tissues indicating complex interactions
that need to be elucidated. Furthermore, non-coding RNAs are critically involved in
epigenetic modifications such as methylation regulation and histone variant replacement in
coordinating nucleosome positioning(85). Transcriptomics thus remains a complex field
with intensive research work ahead to understand multi-layer interrelationships.
Outlook Systems Biology

The paradigm of an almost exclusively unidirectional process from DNA to RNA to protein
has been challenged. The task for the future remains to integrate knowledge of genomic and
nongenomic variation, and accelerate understanding of the multidimensional nature of the
genome and transcriptome. Interactions between proteins, regulatory RNA and DNA
provide a more comprehensive picture of genetic regulation, transcription and translation
into proteins. Further complexity is added at the translational step and posttranslational

processing of immature proteins through cleavage, folding, and chemical alterations as well
as environmental interactions that finally determine phenotype plasticity of cardiovascular
health and disease (Figure 2). We are only beginning to unravel the pathophysiology behind
the chromosome 9 finding, one of the most intensively examined genomic loci in relation to
coronary heart disease discovered by GWAS. Researchers have applied genetic, epigenetic,
and transcriptomic assessment without a conclusive answer to date(86–88).
Systems biology tries to integrate interactions between DNA and regulatory elements at the
RNA and protein level. We have overcome the notion that complex CVD has a specific
molecular basis and we will be able to reduce pathophysiology to singular or few molecular
causes. Although most widely applied for ease of use we know that linear models of
associations are unlikely to capture the complexity of genome-phenotype determinants. In
the past, investigations focused on a single or few intermediate phenotypes, with modest
success. In modern systems biology the concept favors modular networks and subnetworks
of disease that, ideally, account for time-variation in the nodes and links(89). A simplified
gene network enriched for GWAS coronary heart disease loci is depicted in Figure 3.
Systems approaches need to appropriately integrate all known (patho-)physiological
components to create a dense network including genetics, epigenetics, posttranslational
modifications of the proteome and metabolome as well as environmental exposures.
Intermediate phenotypes such as inflammatory activity or endothelial dysfunction will

overlap between several types of CVD, but other constituents may be unique alone or in
their specific interplay with neighboring nodes. Previously unknown relations will emerge
and provide causal links whose preventive or therapeutic modulation may alter disease
occurrence and phenotype. A holistic view as suggested by systems biology, integrating all
available knowledge in context with CVD will guide the next steps in cardiovascular
genomics and enhance our understanding of disease susceptibility, treatment and monitoring
and influence preventive actions.
Prospect of clinical application

Despite the enormous advances in science, genomic information is not widely applied in
clinical cardiology. Whereas, for example, in oncology gene expression profiling is actively
used in clinical practice to determine cancer entity and guide therapy(90), large-scale
transcriptome assessment in CVD has not entered clinical routine owing to limited
experience and controversial findings with restricted clinical relevance.
In contrast to the fairly stable primary DNA sequence scaffold, gene-gene, RNA-DNA,
protein-DNA interactions and chemical modifications are significant determinants of
transcription and gene expression(91), and we are only beginning to understand the complex
picture that may shed light on the dark matter of heritability. The wealth of GWAS findings
has generated hypotheses in CVD pathophysiology for the clinician. To fully exploit the
power of GWAS, functional work-up is needed over the next years to establish causative
links. Advances in laboratory medicine with the refinement of genotyping methods through
next generation sequencing, the assessment of epigenetics and the transcriptome as well as
the identification of novel genomic markers such as miRNAs will rapidly advance genomic
cardiovascular medicine and change the definition of complex CVD.
To accelerate clinically relevant discoveries the National Institutes of Health has supported
several large database studies involving de-identified electronic health record phenotypes
and genomic investigations. Current research efforts are being directed towards issues such
as data safety, database architecture, and widely accessible exploration algorithms.
Informatics knowledge is essential to compile and manage project-related clinical data for
research. The National Institutes of Health supports the i2b2 (Informatics for Integrating

Biology and the Bedside) initiative that has been aiming to provide researchers with the
informatics tools to accelerate clinical translation of genomic findings. Similarly, the
National Institutes of Health sponsors the eMERGE Network to accelerate research
integrating DNA biorepositories with electronic medical record (EMR) systems(92).
Pharmacogenomics has advanced the field of drug response assessment, e.g. first
experiences with guidance of vitamin K antagonist therapy with the help of CYP2C9 or
VKORC1 polymorphisms(93) and cytochrome p-450 polymorphisms in relation to
clopidogrel response have entered US Food and Drug Administration recommendations(94).
Disease prevention lags behind. Gene chips and modern sequencing allow large-scale
interrogation of the genome at the population level and will generate novel hypotheses for
disease causation. Furthermore, with the continuing drop in costs of whole genome
sequencing the practicing physician may soon be faced with the need to comment on a
patient’s over four million sequence variants regarding disease risk before clinical signs
occur, a task that no certified genetic counselor could fulfill at present. With the advent of
GWAS ethical and practical concerns of reporting genetic research results have become
apparent. Initial efforts at defining rules of reporting large-scale association results and to
assess the level of evidence have recently been suggested that also apply for next-generation
large-scale genomics(95;96). Reports have suggested that consumer side genome-wide
genetic profiling in employees of health and technology companies did not change anxiety
symptoms, dietary fat intake, or exercise behavior, i.e. lifestyle factors over a six month
period(97). However, the risk association of genetic variation and the dissection between
objective markers of risk versus risk factors which reside in the causal pathway of disease
will need careful assessment before entering clinical decision making(98).
To be of clinical utility the determination of genetic polymorphisms needs to fulfill the basic
requirements of a biomarker. As risk predictors they need to account for sufficient absolute
or population attributable risk. So far, even the combination of multiple newly identified and
replicated loci as shown for blood cholesterol or C-reactive protein concentrations as
intermediate phenotypes does not add appreciably optimized risk prediction of
CVD(99;100). Concrete diagnostic or interventional steps should result from the
determination of genetic variation to serve as pre-clinical markers or indicate pathogenic
mechanisms. Whereas modifiability of the DNA code is impractical for genetic variation,
epigenetic and transcriptional patterns will be accessible for targeted
modifications(43;54;84).
Standardization of measurement methods and determination across populations is desirable

and robustly feasible for DNA, however, at present less reliable for gene expression. Full
DNA sequencing of the human genome was performed at an error rate less than 1 in 10,000
bases in the Human Genome Projects (http://www.genome.gov/11006943). The biochemical

characteristics of miRNA including analyte stability and presence of circulating extracellular
molecules render small RNAs interesting targets if relevant disease relations can be
established. In addition, gene expression, measured either at the mRNA or protein level, is
highly variable over time, as it can show dramatic changes on the scale of hours to minutes.
Clinical trials will need to prove utility and provide evidence for cost-benefit assessment
before general recommendations regarding the use of genomics information for common
diseases can be expected.
To date, unbiased genome-wide approaches have unraveled novel biologic pathways in

CVD. Multi-stage, multi-disciplinary (basic scientist, biostatistician, physician) efforts are
now necessary to understand the functional annotation of risk alleles and explain residual
disease susceptibility (Table 3). With the growing number of databases and the
establishment of large-scale consortial cooperation unprecedented opportunities for effective

genomic research at the population level have arisen. With accumulating evidence and
simplification and standardization of methods, genomic findings will find their translation in
clinical practice in the near future.
Acknowledgments
We hope for the understanding of all authors whose important work in the field could not be cited due to word
count limitations. Renate B. Schnabel is supported by Deutsche Forschungsgemeinschaft (German Research
Foundation) Emmy Noether Program SCHN 1149/3-1. Emelia J. Benjamin is supported by 1R01HL092577;
1RC1HL101056; 1R01HL102214; 1R01AG028321. Patrick T. Ellinor is supported by 1R01HL092577;
5R21DA027021; 5R01HL104156; 1K24HL105780. This work was partially supported by the Evans Center for
Interdisciplinary Biomedical Research ARC on “Atrial Fibrillation at Boston University (http://www.bumc.bu.edu/
evanscenteribr/). Andrea Baccarelli is supported by P30ES000002; R21ES019773; R01ES015172.
Abbreviations
CNV copy number variation

CVD cardiovascular disease
GWAS genome-wide association study
miRNA microRNA
SNP single nucleotide polymorphism
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Figure 1.
Genome-wide association study variants for cardiovascular disease and its risk factors
(http://www.genome.gov/gwastudies/) summarized in a circos plot (http://circos.ca/). The
circle represents the chromosomal and SNP locations. SNPs are indicated by color dots in
relation to their cardiovascular phenotypes. We did not identify replicated genome-wide hits
for copy number variation for any of the cardiovascular disease phenotypes.

Figure 2.
Complex relations between the genome, epigenetic and transcriptional regulations and the
proteome and metabolome that result in cardiovascular disease phenotypes. Environmental
determinants interact at all stages.

Figure 3.
The network was generated using Ingenuity Pathway Analysis (Ingenuity Systems,
www.ingenuity.com). A data set containing 80 genes associated with coronary heart disease
in GWAS was uploaded and overlaid onto the molecular networks developed from
information contained in the Ingenuity Knowledge Base. Networks of Network Eligible

Molecules were then algorithmically generated based on their connectivity. The most
significantly enriched network, as shown, is comprised of 36 genes, of which 20 are
coronary heart disease genes.

Table 1
Genetic epidemiology characteristics of samples for rare and common cardiovascular disease
Rare Mendelian disorders Common genetic variation
Presentation • Usually families • Unrelated individuals, total population

• Small to moderate number of individuals • Large sample sizes
Identification of causal variant
Effect size, absolute risk Large, high Small, low

Population attributable risk Low High
Public health impact Low Possibly high

Table 2
Clinically important characteristics of conventional Sanger sequencing and next generation sequencing
Conventional sequencing Next generation sequencing

Sequencing • Subcloning in vectors, • Direct DNA fragment sequencing
amplification in hosts for
every single DNA fragment • Optional PCR amplification
• Sequencing of 100 fragments • Parallel sequencing of millions of small fragments

in parallel
Yield 100,000 basepairs per sequencing run >100 billion base pairs per sequencing run
Computational requirements Moderate High
Cost per megabase High Low
Accuracy High High
Future directions -- • Direct sequencing of DNA molecules
• Nanotechnology for sequencing
• $1,000 per genome
• Affordable qualitative and quantitative sequencing
of entire transcriptomes without reference database
• Unbiased genome-wide approach to alternative
splice variants, DNA methylation and histone

modifications

Table 3
Resources for the holistic approach of systems biology
Phenotype • Clearly defined, well-characterized

➢ In total
➢ Its components, i.e. biologically meaningful subtypes
• Intermediate phenotypes
• Harmonized across studies
Data sources • High-quality genomic data

• High-quality environmental data
• Online databases for
➢ Genome/epigenome annotations (Ensembl, HEP, NIH Roadmap Epigenomics)
➢ CVD associations (e.g. NHGRI Human Genome Browser)
• Data mining for potential determinants of (patho)biology
➢ Published literature
➢ Experiment
➢ Epidemiology
➢ Clinical biosample repositories linked with electronic health records (e.g. eMERGE,
I2B2)
➢ Clinical trial
• High-throughput profiling
➢ Genome-wide SNP microarray
➢ Next generation sequencing
➢ Expression microarray
➢ Tandem mass spectrometry
Bioinformatics and Statistical • Intensive computational requirement

• Multi-dimensional, dynamic models
• Gene-gene interaction analysis
• Network analysis
• Pathway enrichment analysis
• Pathway simulation
Laboratory resources • Experimental system perturbation

• Core facilities
➢ Access to equipment requiring high capital investments
➢ Frequent platform updates
➢ Large number of users and high volumes of samples processed
• Laboratories
➢ Develop and conduct experimental studies on individual marker
➢ Highly specialized
• Biological repositories
➢ Standardized handling of biological specimens

➢ Management of large collection of samples


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Clin Chem. 2012 January ; 58(1): 113–126. doi:10.1373/clinchem.2011.170423.

Next Steps in Cardiovascular Disease Genomic Research –

5Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts

Correspondence to: Renate B. Schnabel.

Common complex disease

Clin Chem. Author manuscript; available in PMC 2013 May 10.

could be explained by rare/low frequency variants, structural alterations, and epigenetics

Next Generation Sequencing

genetic variants from bystanders remains a challenge.

Clin Chem. Author manuscript; available in PMC 2013 May 10.

a follow-up of GWAS. Sequencing in larger clinical or even population-based samples

As more experience is accumulated the understanding of the genetic architecture of CVD

Structural variation—The pathophysiological significance of structural variants to CVD

Clin Chem. Author manuscript; available in PMC 2013 May 10.

DNA methylation is the covalent methylation of cytosines catalyzed by DNA

methylation can be determined using a candidate-gene approach through bisulfite-

Epigenetics may be a unifying principle in the etiology of complex diseases(37). In contrast

Clin Chem. Author manuscript; available in PMC 2013 May 10.

Hypoxia leads to a global reduction in transcription activity reflected in upregulation of

ubiquitous repressive histone methylation(45). Histone modifications seem to be mandatory

mapping of the human DNA methylome at base resolution(33), genome-wide nucleosome

Clin Chem. Author manuscript; available in PMC 2013 May 10.

susceptibility to oxidative stress in coronary arteries(63). Mouse aortic gene expression

Promising is the simultaneous determination of genome-wide gene expression and genetic

Clin Chem. Author manuscript; available in PMC 2013 May 10.

First results in peripheral blood thrombocyte transcriptome assessment at the epidemiologic

An emerging field of transcriptomics is miRNA expression profiling. Of the 940 known

remain constant(79). To better understand the regulation of gene expression, nuclear

Intriguingly, miRNA can be easily and effectively antagonized. Antagomirs targeted at

different results after knock-down or biochemical blockade, and systemic application of

Outlook Systems Biology

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Intermediate phenotypes such as inflammatory activity or endothelial dysfunction will

Prospect of clinical application

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integrating DNA biorepositories with electronic medical record (EMR) systems(92).

Standardization of measurement methods and determination across populations is desirable

bases in the Human Genome Projects (http://www.genome.gov/11006943). The biochemical

To date, unbiased genome-wide approaches have unraveled novel biologic pathways in

Clin Chem. Author manuscript; available in PMC 2013 May 10.

CNV copy number variation

SNP single nucleotide polymorphism

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Clin Chem. Author manuscript; available in PMC 2013 May 10.

38. Bollati V, Baccarelli A. Environmental epigenetics. Heredity. 2010; 105:105–112. [PubMed:

Clin Chem. Author manuscript; available in PMC 2013 May 10.

50. Kong A, Steinthorsdottir V, Masson G, Thorleifsson G, Sulem P, Besenbacher S, et al. Parental

phenotypes of atherosclerosis. Arterioscler Thromb Vasc Biol. 2004; 24:1922–1927. [PubMed:

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MicroRNA-208b and MicroRNA-499 reflect myocardial damage in cardiovascular disease. Circ

untranslated region: a mechanism for functional single-nucleotide polymorphisms related to

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Clin Chem. Author manuscript; available in PMC 2013 May 10.

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information contained in the Ingenuity Knowledge Base. Networks of Network Eligible

Clin Chem. Author manuscript; available in PMC 2013 May 10.

Rare Mendelian disorders Common genetic variation

Presentation • Usually families • Unrelated individuals, total population