Epigenetic studies have identified DNA methylation in coronary artery disease (CAD). How the critical genes interact at the cellular level to cause CAD is still unknown. The discovery of DNA methylation inspired researchers to explore relationships in genomic coding and disease phenotype. In the past two decades, there have been many findings regarding the relationship between DNA methylation and CAD development, and the DNA methylation of critical genes have been found to be significantly changed during CAD, including DNA methylation at homocysteine, Alu and long Interspersed Element 1 (LINE-1) repetitive elements.

1. The Role of DNA Methylation in
Coronary Artery Disease
Bardia Farivar
Department of Medical Biology
Istanbul University
Cerrahpaşa Medical Faculty

2. Abstract:
• Epigenetic studies have identified DNA methylation in coronary
artery disease (CAD). How the critical genes interact at the cellular
level to cause CAD is still unknown. The discovery of DNA
methylation inspired researchers to explore relationships in
genomic coding and disease phenotype. In the past two decades,
there have been many findings regarding the relationship between
DNA methylation and CAD development, and the DNA methylation
of critical genes have been found to be significantly changed during
CAD, including DNA methylation at homocysteine, Alu and long
Interspersed Element 1 (LINE-1) repetitive elements.

3. • Over the past few decades, coronary artery disease (CAD)
has been among the leading cause of morbidity and
mortality worldwide. The number of deaths due to CAD is
56 million globally. CAD is caused by a variety of factors, in
which atherosclerosis is the main pathological basis of
CAD. Epigenetic factors, including DNA methylation,
histone modification, chromatin remodeling and noncoding
RNA regulation, have been reported to cause CAD by
altering the interaction between genes and the
environment.

4. 1. Introduction of DNA methylation
• DNA methylation refers to a covalent methylation
modification on the cytosine base of the 5’ CpG3’
dinucleotide, which is the major DNA modification in
mammals. Largely unmethylated CpG segments are named
CpG islands which always appear at the regions of
promoters. While, methylated CpGs are mainly located at
non-regulatory areas in the genome. CpG islands play an
extremely important role as a key regulatory region of DNA
transcription. Most of the methylated CpGs are established
and maintained by DNA methyltransferases (DNMTs), such
as DNMT3A, DNMT3B and DNMT3L.

5. Mechanism of DNA methylation on genes. In DNA
methylation, a methylation modification appears on
the cytosine base of the 5’ CpG3’ dinucleotide. If the
methylation of the gene is upregulated by DNMTs, the
structure of the DNA linked tightly so that the gene
cannot be transcribed.

6. 2. DNA Methylation in CAD
• In the past two decades, there have been many findings
regarding the relationship between DNA methylation
and CAD development. And the DNA methylation of
critical genes have been found to be significantly
changed during CAD. In cell and animal studies, the
common mode is to focus on a critical gene in CAD and
then examine the methylation of the critical gene or the
critical gene promoter. Generally, hypomethylation of
the gene promoter can upregulate expression of the
gene, while hypermethylation of the gene promoter can
downregulate expression of the gene.

7. • Average gene methylation level was inversely associated
with the expression of the gene. Another mode is to
observe the change of the phenotype with DNA
methylation inhibitor. In the human tissue studies,
genome-wide methylation was often applied between
CAD patients and control with Human Methylation
array or sequencing. Meanwhile, based on some
previous studies, the methylation of critical genes was
also detected in human tissue by pyrosequencing or
methylation specific PCR (MSP).

8. 3.1 The evidence from cell and animal
studies
• DNA methylation inhibits the functions of the critical
genes in CAD. Hypermethylation of the gene promoter
can downregulate expression of the gene. Aldehyde
dehydrogenase 2 (ALDH2) is important to protect
myocardium from ischemia. Hypermethylation of the
ALDH2 promoter inhibited ALDH2 after MI in border
zone tissues, which is related to myocardial ischemia. In
myocardial infarction (MI) rats, ALDH2 was
significantly downregulated. Meanwhile, DNA
methylation of the ALDH2 promoter increased
significantly time-dependently.

9. 3.1.1 Homocysteine
• Homocysteine (Hcy), a thiol amino acid, is also a key
modulator in DNA methylation. Hyperhomocysteinemia
(HHcy) is a candidate risk factor for CAD. Hcy is the
precursor of methionine, which could be converted to
S- adenosylmethionine (SAM). SAM is the main methyl
group donor in DNA methylation. In the process of
transferring the methyl group, SAM is converted to S-
adenosylhomocysteine (SAH). Therefore, Hcy is a bridge
between DNA methylation and CAD.

10. Methionine Cycle
• Hcy is the precursor of methionine, which could be
converted to S- adenosylmethionine (SAM). SAM is the
main methyl group donor in DNA methylation. In the
process of transferring the methyl group, SAM is converted
to S-adenosylhomocysteine (SAH).

11. 3.1.2 Atherosclerosis
• Currently, atherosclerosis is the major basis of CAD, and
DNA methylation play a role of atherosclerosis as DNA
methylation can inhibit the expression of critical genes
or gene promoters related to atherosclerosis. In addition,
recently, investigators have been identifying the
diagnostic value of DNA methylation in atherosclerosis.

12. • Estrogen receptor (ER) modulators benefit
cardiovascular outcomes and lipid improvements. In
1999 it was found that methylation inactivation of the
ER alpha in vascular tissue may play a role in
atherogenesis and the ageing of the vascular system.
Coronary atherosclerotic tissues exhibited higher levels
of methylation in human aorta samples using Human
Methylation 450k array. In swine aorta regions
susceptible to atherosclerosis, methylation of ATF4, the
ER stress gene appears differentially, as well as
microRNA-10a. In addition, DNMTs and methylation of
ER alpha was recruited by insulin so that ER alpha, the
regulator of VSMCs proliferation, decreased to result in
atherosclerosis both in vitro and in vivo.

13. 3.2 The evidence from human tissue studies
• Recently, studies on DNA methylation related to CAD
begin to expand rapidly from cell or animal experiments
to epidemiological research. In CAD or atherosclerosis
patients, genome-wide DNA methylation was examined
to find the critical genes and sequences, such as ABCA1,
DDAH2, LINE-1 and Alu. Different genetic, lifestyle and
environmental factors may affect DNA methylation,
CAD and its risk factors.

14. 3.2.1 CAD
• Genome-wide DNA methylation was often determined in blood
samples of CAD patients and controls with Human
Methylation array. Immune signaling and cellular functions
might be regulated at an epigenetic level in acute coronary
syndrome (ACS) patients. Genome-wide DNA methylation of
whole blood was detected in 102 ACS patients and 101 controls
using Human Methylation 450 array and another replicated
cohort of 100 patients and 102 controls, a significant
enrichment of CpGs was validated again related with smoking
and low-density lipoprotein cholesterol (LDL-C) in T and B
cells.

15. • . The results could be identified twice. In addition, it
indicated DNA methylation could have different
meanings in different cells from the blood of human. In
the same method, another study included a total of 192
subjects with MI and 192 control subjects. Three DNA
methylation sites showed genome-wide significant
associations with MI. Two of these sites still showed
such associations after adjustment for classical risk
factors of MI.

16. • The study provided a potential biomarker for MI. In
addition, among individuals with a history of MI, DNA
methylation appeared deviated. In 729 blood samples
from the northern Sweden population health study,
differential DNA methylation was observed at 211 CpG-
sites in individuals with a history of MI. These sites
represent 196 genes, of which 42 have been described to
be associated with cardiovascular disease. Interestingly,
DNA methylation at specific loci was associated with the
risk of MI in women which is sensitive to prenatal
conditions. No association was observed among men.

17. • Global DNA methylation was positively correlated with
plasma Hcy levels of CAD patients. DNA methylation
was evaluated in a cohort of 137 CAD patients and 150
controls. Significantly higher levels of serum Hcy and
global DNA methylation was observed in CAD
patients[42]. A total of 72 differentially methylated
regions (DMRs) were hypermethylated in CAD patients
with varying Hcy levels

18. • DDAH2 is an enzyme found in all mammalian cells.
DDAH2 degrades methylarginines, specifically
asymmetric dimethylarginine (ADMA).
• Hypermethylation in the DDAH2 promoter was
positively correlated with EPCs dysfunction in CAD
patients. Hcy disrupted EPCs function by inducing the
hypermethylation of the DDAH2 promoter[46]. The
plasma levels of ADMA had been proved to be an
independent cardiovascular risk factor.

19. • Inflammation is the critical factor in CAD and
atherosclerosis. Interleukin-6 (IL-6) is a multifunctional
cytokine in inflammation. Methylation at two CpG sites
in IL-6 promoter was measured by pyrosequencing in
blood leukocyte of a total of 212 cases with CAD and 218
controls. Mean methylation level in IL-6 promoter in
CAD cases was significantly lower, inversely associated
with the risk of CAD.

20. 3.2.2 Atherosclerosis
• With the HumanMethylation array, hypomethylation of
these sites appeared in leukocytes and atherosclerotic
arteries. In addition, several cardiovascular disease risk
factors, as well as medications, might affect methylation
levels of CpG sites. In the same way, a large-scale
analysis of DNA methylation in subjects with
atherosclerosis has indicated that aberrant DNA
methylation in subjects with atherosclerosis affects the
transcription of critical regulatory genes to induce a pro-
atherogenic cellular phenotype.

21. 3.3 The intervention via DNA methylation
• Several experiments have reported that drugs or foods elicit a therapeutic
effect via the methylation of specific genes. For example:
 The intake of aspirin, a conventional drug in the established guidelines, leads to
significantly increased L5 levels in STEMI patients. With low concentration aspirin, L5
impairment of HCAEC function was inhibited through CpG methylation at the FGF2
promoter.
 The Western-type diet could induce transplantable epigenetic changes in bone marrow
cells, alter the haematopoietic system, and increase the susceptibility to atherosclerosis
 Bvitamins intake was negatively associated with DNA-methylation of the candidate
genes. folate and B-vitamins low intake may regulate methylation in critical enzymes of
One-Carbon metabolism and Hcy related to the CAD risk.
• Therefore, DNA methylation could become a therapeutic target for the
prevention and treatment of CAD.

22. 4. Conclusion
 DNA methylation is a key epigenetic process in CAD.
 DNA methylation patterns exhibit different in different
tissues, genes and gene promoters among CAD patients.
 Hcy, a thiol amino acid, plays a critical role in DNA
methylation among CAD patients and is as an independent
risk factor for cardiovascular disease as well as a key
modulator of macromolecular methylation.
 The Alu and LINE-1 element sequences are potential
markers for DNA methylation in CAD patients.
 DNA methylation also influences atherosclerosis in several
aspects and CAD risks.
 DNA methylation of specific genes can play important roles
in the early diagnosis of CAD and atherosclerosis.
 This reveals the potential effect of DNA methylation on
CAD.