Detection of Altered Global DNA Methylation in Coronary Artery Disease Patients

Abstract

Epigenetic modifications, especially alteration in DNA methylation, are increasingly being recognized as a key factor in the pathogenesis of complex disorders, including atherosclerosis. However, there are limited data on the epigenetic changes in the coronary artery disease (CAD) patients. In the present study we evaluated the methylation status of genomic DNA from peripheral lymphocytes in a cohort of 287 individuals: 137 angiographically confirmed CAD patients and 150 controls. The differential susceptibility of genomic DNA to methylation-sensitive restriction enzymes was utilized to assess the methylation status of the genome. We observed that the genomic DNA methylation in CAD patients is significantly higher than in controls (p < 0.05). Since elevated homocysteine levels are known to be an independent risk factor for CAD and a key modulator of macromolecular methylation, we investigated the probable correlation between plasma homocysteine levels and global DNA methylation. We observed a significant positive correlation of global DNA methylation with plasma homocysteine levels in CAD patients (p = 0.001). Further, within a higher range of serum homocysteine levels (>/=12-50 muM), global DNA methylation was significantly higher in CAD patients than in controls. The alteration in genomic DNA methylation associated with cardiovascular disease per se appears to be further accentuated by higher homocysteine levels.

Full text

Detection of Altered Global DNA Methylation in Coronary
Artery Disease Patients
Priyanka Sharma,
1,2
Jitender Kumar,
1
Gaurav Garg,
1
Arun Kumar,
1
Ashok Patowary,
1
Ganesan Karthikeyan,
3
Lakshmy Ramakrishnan,
3
Vani Brahmachari,
2
and Shantanu Sengupta
1
Epigenetic modifications, especially alteration in DNA methylation, are increasingly being recognized as a key
factor in the pathogenesis of complex disorders, including atherosclerosis. However, there are limited data on the
epigenetic changes in the coronary artery disease (CAD) patients. In the present study we evaluated the methyl-
ation status of genomic DNA from peripheral lymphocytes in a cohort of 287 individuals: 137 angiographically
confirmed CAD patients and 150 controls. The differential susceptibility of genomic DNA to methylation-sensitive
restriction enzymes was utilized to assess the methylation status of the genome. We observed that the genomic
DNA methylation in CAD patients is significantly higher than in controls ( p< 0.05). Since elevated homocysteine
levels are known to be an independent risk factor for CAD and a key modulator of macromolecular methylation,
we investigated the probable correlation between plasma homocysteine levels and global DNA methylation. We
observed a significant positive correlation of global DNA methylation with plasma homocysteine levels in CAD
patients ( p¼0.001). Further, within a higher range of serum homocysteine levels ($12–50 mM), global DNA
methylation was significantly higher in CAD patients than in controls. The alteration in genomic DNA methyl-
ation associated with cardiovascular disease per se appears to be further accentuated by higher homocysteine
levels.
Introduction
The incidence of coronary artery disease (CAD) is
on the rise worldwide and is expected to become the
main cause of death globally within the next 15 years, owing
to a rapidly increasing prevalence in developing countries
(Dodu, 1988). It is estimated that two-third of the 14 million
cardiovascular fatalities worldwide would occur in the de-
veloping countries. Mortality due to coronary heart diseases
in India increased from 1.17 million to 1.59 million from 1990
to 2000 and is expected to rise to 2.03 million by 2010 (Ah-
mad and Bhopal, 2004) and could reach epidemic propor-
tions by 2030 (Leeder et al., 2004). The early onset of CAD
along with its severity cannot be explained entirely by the
classical risk factors and genetic variations (Wilson et al.,
1998; Winkelmann and Hager, 2000). Further, the epigenetic
code for transcription regulation through DNA methylation
and chromatin modifications can add yet another level of
variability and can escape attention in the typical genetic
association studies (Turner, 2007). There is increasing evi-
dence to show that epigenetic regulation plays an important
role in the course of complex diseases in addition to its role in
X-chromosome inactivation, genomic imprinting, and main-
tenance of cellular transcriptional memory during develop-
ment (Bird, 2002; Feinberg, 2004; Moss and Mallrats, 2007;
Shames et al., 2007).
The modulators of macromolecular methylation could
be important links between the environment and epigenetic
regulation. For instance, an elevated level of homocysteine, a
thiol amino acid, is an independent risk factor for cardiovas-
cular diseases and also a modulator of macromolecular
methylation (Robinson et al., 1995; van Guldener et al., 2005).
Hyperhomocysteinemia has been reported to have a graded
effect on the risk of CAD and the extent and severity of the
disease (Chao et al., 1999). Further, a meta-analysis done by
Wald et al. (2003) along with their data clearly indicated a
significant increase in homocysteine levels with increasing
severity of CAD. It has also been found that in patients with
angiographically confirmed CAD, homocysteine levels are a
significant predictor of mortality, independent of traditional
risk factors (Yoo et al., 1999). The risk of recurrent coronary
events and death in patients with acute myocardial infarction
also was significantly associated with elevated homocysteine
levels, and this was independent of other risk factors.
1
Institute of Genomics and Integrative Biology, Delhi, India.
2
Dr. B.R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India.
3
All India Institute of Medical Sciences, New Delhi, India.
DNA AND CELL BIOLOGY
Volume 27, Number 7, 2008
ªMary Ann Liebert, Inc.
Pp. 357–365
DOI: 10.1089=dna.2007.0694
357

Although hyperhomocysteinemia has been implicated as
an independent risk factor for cardiovascular disease, the
mechanism through which it influences the disease mani-
festation is not fully understood. Several potential mecha-
nisms have been proposed to explain the role of homocysteine
in CAD; prominent among these are oxidative damage to
vascular endothelial cells, thrombotic effects of the coagula-
tion system, impairment of vasodilator properties of endo-
thelium by decreasing the bioavailability of nitric oxide,
endoplasmic reticular stress, and direct modulation through
physical interaction with proteins (Sharma et al., 2006). It
is possible that modulation of homocysteine levels could al-
ter certain fundamental intracellular processes such as the
methylation of macromolecules since homocysteine levels are
believed to dictate the fate of trans-methylation reactions that
are essential for several biological processes (Ulrey et al.,
2005).
In a recent study, using vascular smooth muscle cells in
culture, the authors observed hypomethylation of surrogate
markers Alu and LINE1 elements in spite of elevated activ-
ity of DNMT3a and DNMT3b under a high homocysteine
concentration (Yi-deng et al., 2007a). Similarly in cultured
monocytes, it has been shown that the presence of 100 mM
homocysteine elevates the level of total cholesterol and de-
creases ApoE mRNA through hypermethylation of promoter
sequences (Yi-Deng et al., 2007b).
We have undertaken a comparative analysis of global DNA
methylation at G þC-rich regions targeted by methylation-
sensitive restriction enzyme HpaII and its isoschizomer MspI,
in lymphocytes obtained from peripheral blood of CAD pa-
tients and controls. We found a significant correlation be-
tween global DNA methylation, plasma homocysteine levels,
and CAD. In addition, we observed a considerable effect of
vegetarian diet on plasma homocysteine levels and concur-
rently a correlation with global DNA methylation in CAD
patients. To the best of our knowledge, this is the first study on
the global DNA methylation along with plasma homocysteine
levels and dietary status in CAD patients.
Materials and Methods
Patient population
The study population included 287 subjects mainly from
the northern part of India. Among these, 137 were angio-
graphically confirmed CAD patients and 150 were healthy
controls who tested negative in treadmill test; that is, they
had normal heart rate and blood pressure response and ab-
sence of chest pain during exercise and had no abnormal
ST segment deviation during exercise and recovery. These
samples were randomly selected from a cohort recruited at
the All India Institute of Medical Sciences, New Delhi, India,
as a part of a project to study the genetic polymorphism
and epigenetic alteration associated with CAD. Apart from
clinical history, information on diet was also collected.
People who do not consume any animal products other than
milk and its products were classified as vegetarians (lacto-
vegetarians). The ethics committee of both the All India In-
stitute of Medical Sciences and the Institute of Genomics and
Integrative Biology approved this study. Written informed
consent was obtained from all the participants, and the study
was carried out in accordance with the principles of Helsinki
Declaration.
Blood samples for biochemical markers
Blood samples were collected from volunteers in tubes
containing anticoagulant, immediately chilled on ice, and
plasma was separated from the blood samples within an hour
of collection. Aliquots of plasma were transferred into cryostat
tubes and stored at 808C until further analysis. Genomic
DNA was isolated from blood samples using the modified
salting out method as described earlier and stored at 208C
until further analysis (Kumar et al., 2005). Genomic DNA
concentration was estimated after chelating the DNA with the
fluorescence probe picogreen (Molecular Probes, Eugene, OR).
Plasma levels of homocysteine were determined using high-
performance liquid chromatography (HPLC) equipped with a
fluorescence detector as described earlier (Ji et al., 1995). Vita-
min B12 and folate levels were determined using the Immu-
nolite Vitamin B12 (Diagnostic Product, Los Angeles, CA) and
SimulTrac-SNB Radio assay kits (MP Biomedicals, Orange-
burg, NY), respectively, as per the manufacturer’s protocol.
Assessment of global DNA methylation
The global DNA methylation status in peripheral blood
was determined using the cytosine extension assay as previ-
ously described (Ingrosso et al., 2003). Briefly, 1 mg of genomic
DNA was digested with 20 units of methylation-sensitive
enzymes HpaII and an isoschizomer MspI (New England
Biolabs, Beverly, MA) for 16–18 h in separate tubes. The di-
gested DNA was subjected to single-nucleotide extension re-
action in a 25 mL reaction mixture containing 0.5 mg of DNA, 1
Taq polymerase gold reaction buffer, 1.0 mM MgCl
2
, 0.25
units of heat-activated AmpliTaq Gold DNA polymerase
(Applied Biosystems, Weiterstadt, Germany), and 0.1 mCi of
[
3
H]dCTP (57.4 Ci=mmol; GE Healthcare Biosciences, Piscat-
away, NJ). The reaction mixture was incubated at 568C for 1 h
and chilled on ice. Ten mL aliquot in duplicate from each re-
action was applied on Whatman DE-81 ion exchange filters
with a multiscreen assay system (Millipore, Bedford, MA)
and washed three times with 0.5 M sodium phosphate buf-
fer (pH 7.0) at room temperature. Filters were dried, and the
radioactivity incorporated was counted using scintillation
counter (LS6500; Beckman, San Jose, CA).
Each assay was done in duplicate, and the values were
corrected for nonspecific incorporation by measuring the in-
corporation of [
3
H] dCTP into undigested genomic DNA. The
difference in the mean incorporation of [
3
H]dCTP following
MspI and HpaII digestion at a known concentration of geno-
mic DNA was computed as the global DNA methylation level
expressed as cpm=mg DNA. The deviation between duplicates
was less than 10%. The reproducibility of the cytosine exten-
sion assay was confirmed by repeating the assay in 5% of the
samples selected randomly. Variation between the repeats
was below 10%.
ApoE promoter methylation analysis
The methylation status of ApoE gene was analyzed by direct
sequencing of sodium bisulfite–modified genomic DNA.
Genomic DNA was subjected to bisulfite modification using
the EZ DNA Gold Methylation Kit (Zymo Research, Orange,
CA) following the manufacturer’s protocol. The ApoE forward
primer 50-GGATAATTTTAGGGAGGAGTGTTTTG-30and the
reverse primer 50-CTCCAGAACAATATCATCTCTACTAC-30
358 SHARMA ET AL.

were used to amplify 574 bp representing 25 CpG of ApoE
promoter region. PCR products were purified using QIAquick
Gel Extraction Kit (Qiagen, Chatsworth, CA). These purified
PCR products were cloned using pCR-4-TOPO TA Cloning
vector (Invitrogen). Ligated PCR products were transformed
using TOP10 competent cell (Invitrogen). Plasmids were pre-
pared from selected colonies using Wizard SV 96 Plasmid
DNA Purification System (Promega, Madison, WI) and were
sequenced on ABI 3730 sequencer. For each PCR product, five
clones were sequenced. Bisulfite sequencing results were ana-
lyzed using BiQ Analyzer software tool (Bock et al., 2005).
Statistical analysis
The statistical tests were performed using GraphPad Instat
(version 3.06) software. Mann–Whitney U-test was performed
to compare the biochemical parameters and global DNA
methylation levels between patients and controls. The Spear-
man rank correlation test was used to correlate homocysteine
concentrations with DNA methylation levels in patients and
controls. In all the patients, nonparametric correlation was
employed without Gaussian assumptions as the data were
not normally distributed. Two-tailed p-value <0.05 were con-
onsidered to be significant. We also performed a binary lo-
gistic regression analysis to ascertain the factors associated
with CAD and a multiple regression analysis to check for the
association of global DNA methylation with various param-
eters in patients and controls using SPSS software (version
10.0).
Results
The samples analyzed were randomly selected from a co-
hort recruited on the basis of being angiographically positive
for the patient group and treadmill test negative for controls.
The baseline characteristics of the subjects such as age, dia-
betes, hypertension, cigarette smoking habit, BMI, and diet
were compared between the groups and are shown in Table 1.
The two groups were comparable with reference to all these
parameters ( p>0.05). Further, the number of diabetic and
hypertensive subjects was not significantly different in the
two groups ( p>0.05). To ascertain if global DNA methylation
in CAD patients was different from that in the controls, we
analyzed the global methylation in DNA from peripheral
blood in a cohort of 137 angiographically confirmed CAD
patients and 150 controls. The global DNA methylation level
was significantly higher in CAD patients as shown in Figure 1
(median 11.510
3
cpm=mg DNA) compared to the controls
(median 10.910
3
cpm=mg DNA, p<0.05), irrespective of the
levels of homocysteine, one of the independent risk factors for
CAD, thus suggesting that global methylation could be as-
sociated with CAD per se. We also performed a binary logistic
regression to ascertain the parameters that are associated with
CAD in our study and found that age ( p¼0.003) and global
DNA methylation ( p¼0.02) are significantly associated with
CAD after adjusting for other parameters. But in a back-
ground of CAD, homocysteine levels could have additional
influence as modulators of DNA methylation. Corroborating
this, we observed a significant positive correlation of global
hypermethylation with plasma homocysteine levels in CAD
patients (n¼137, r¼0.2785, p¼0.001, Fig. 2A), but not in
controls (n¼150, r¼0.01662, p>0.05, Fig. 2B). When a subset
of patients and controls with higher than normal levels of
Table 1. Baseline Characteristic of Study Population
Patients (n¼137) Controls (n¼150) p-value
Age (years) 54
a
(30–75) 52
a
(27–75) 0.06
BMI
b
24.69
a
(15.7–36.7) 24.96
a
(16.1–75.6) 0.15
Diabetic [no. (%)] 36 (26.3) 40 (26.7) 0.95
Hypertensive [no. (%)] 73 (53.3) 79 (52.7) 0.95
Vegetarians [no. (%)] 68 (49.6) 64 (42.7) 0.47
Nonvegetarians [no. (%)] 69 (50.4) 86 (57.3) 0.52
Smokers [no. (%)] 39 (28.5) 61 (40.7) 0.13
a
Median values are shown.
b
Body mass index is the weight in kilograms divided by the square of the height in meters.
FIG. 1. Global DNA methylation in CAD patients and
controls. Methylation (cpm10
3
=mg DNA) on the Y-axis
represents the level of methylation estimated in terms of the
incorporation of [
3
H] dCTP. The box represents the inter-
quartile range, which contains the 50% of values. The whis-
kers are lines that extend the box to the highest and lowest
values, excluding outliers. A line across the box indicates the
median value. The global DNA methylation in CAD patients
is significantly higher than healthy controls ( p<0.05) as
shown in the box plot.
GLOBAL DNA METHYLATION AND CORONARY ARTERY DISEASE 359

homocysteine (>12 mM) were compared, we found that the
global DNA methylation levels were significantly higher
(p¼0.0005) in CAD patients (median 12.810
3
cpm=mg
DNA) than in controls (median 10.010
3
cpm=mg DNA,
Fig. 3). This indicates a significant DNA hypermethylation in
CAD patients in the background of higher homocysteine
levels. Our results are in agreement with the recent observa-
tion that global DNA hypermethylation is associated with
inflammation and increased mortality in chronic kidney dis-
ease (CKD) patients especially due to cardiovascular disease
(Stenvinkel et al., 2007).
Aging is known to affect the global DNA methylation in
mammals, and a genome-wide decrease in methylation in
healthy individuals has been observed during aging (Fuke
et al., 2005). We analyzed the correlation of global DNA
methylation with age and observed a negative correlation in
controls as expected, while in CAD patients there was a
trend toward positive correlation, although it was not sta-
tistically significant (data not shown). However, when indi-
viduals in the higher age group (61 to 75 years) were
considered, the global DNA methylation in CAD patients
(median 14.310
3
cpm=mg DNA) was significantly higher
than that in the controls (median 10.110
3
cpm=mg DNA,
p¼0.012, Fig. 4), once again indicating an independent as-
sociation between CAD and DNA hypermethylation.
Homocysteine levels are known to be influenced by diet.
In our study population, almost 46% of the individuals
consumed a vegetarian diet. As expected, the homocysteine
levels were significantly higher in vegetarians (median
15.4 mM) than in the nonvegetarians (13.1 mM, p¼0.0015). We
utilized this parameter for stratification of the cohort and
analyzed the global DNA methylation levels. It was found
that global DNA methylation levels were significantly
higher ( p¼0.0015) in CAD patients following a vegetarian
diet (median 11.710
3
cpm=mg DNA) than in controls (me-
dian 9.210
3
cpm=mg DNA) following a similar diet (Fig. 5A).
However, in the nonvegetarian diet group, global DNA
methylation did not vary significantly between patients and
controls (Fig. 5B). The lower levels of global DNA methyla-
tion in the vegetarian control group are consistent with
higher levels of homocysteine; however, with CAD patients
who are vegetarians, in spite of higher homocysteine levels,
the global DNA methylation is significantly higher. This
again suggests an apparent independent association between
increased global DNA methylation and CAD. We also per-
formed multiple regression analysis in controls and patients
separately to check the association of global DNA methyla-
tion with various parameters, and found that in CAD pa-
tients, global DNA methylation was significantly associated
only with homocysteine levels ( p¼0.003). However, none of
the parameters were associated with global methylation of
DNA in controls.
FIG. 2. Correlation between total homocysteine levels and
global DNA methylation in (A) CAD patients (n¼137,
r¼0.2785, p¼0.001) and (B) controls (n¼150, r¼0.01662,
p>0.05). Y-axis as given in Figure 1.
FIG. 3. A comparison of global DNA methylation levels in
hyperhomocysteinemic cohorts (reference level >12 mM). The
box represents the interquartile range, which contains the 50%
of values. The whiskers are lines that extend the box to the
highest and lowest values, excluding outliers. A line across
the box indicates the median value. The box plot shows that
the global DNA methylation in hyperhomocysteinemic CAD
patients is significantly higher than that in hyperhomocys-
teinemic controls ( p¼0.0005). Y-axis as given in Figure 1.
360 SHARMA ET AL.

Folate and vitamin B12 did not show any significant cor-
relation with global DNA methylation in the study popula-
tion (data not shown).
It has recently been shown that ApoE promoter methylation
varies with homocysteine levels in monocytes in culture (Yi-
Deng et al., 2007b). We therefore analyzed the methylation
status of ApoE promoters using bisulfite sequencing in 15
patients and 15 control samples with varying homocysteine
levels. After bisulfite modification of genomic DNA, PCR
products were amplified and analyzed using bisulfite-specific
primers as mentioned in the Materials and Methods section.
The region included contained 25 CpG sites of which 8 CpG
sites were methylated in most of the patients and controls.
However, we did not find any significant difference in
methylation of this region in patients and controls (Fig. 6).
Even when the patients and controls were classified on the
basis of their homocysteine levels, there was no significant
difference in the methylation pattern (data not shown).
Discussion
The origin of complex diseases is attributed to a combina-
tion of heritable and environmental factors, and epigenetic
modulation could be the basis of the effects of environmental
factors on the disease outcome (van Vliet et al., 2007). DNA
methylation is one of the well-known mediators of epigenetic
effects with several documented instances of impact on dis-
ease phenotype (Tang and Ho, 2007). DNA methylation,
through the localization of gene silencing protein complexes
at the methylated CpG dinucleotides, leads to altered ex-
pression profile (Ehrlich et al., 2001; Wade, 2001). It is known
that cancer cells exhibit a global hypomethylation and CpG
island hypermethylation unlike normal cells (Boltze et al.,
2003; Futscher et al., 2004; Lund and van Lohuizen, 2004;
Yatabe et al., 2004).
The alteration in global DNA methylation along with aging
and dietary habit correlated with pathogenesis is reported in
case of cancer (Liu et al., 2003; Waggoner, 2007). However,
similar investigations in CAD are limited. We undertook the
present study to investigate if epigenetic changes are a part of
the pathogenesis in CAD. We found that the global DNA
FIG. 4. Global DNA methylation in CAD patients and con-
trols based on age groups. Data from CAD and control indi-
viduals in the age group 61 to 75 years is shown. The box
represents the interquartile range, which contains the 50% of
values. The whiskers are lines that extend the box to the
highest and lowest values, excludingoutliers. A line across the
box indicates the median value ( p¼0.0122). Y-axis: as given in
Figure 1.
FIG. 5. Global DNA methylation in CAD patients and
controls based on diet. Data from CAD and healthy con-
trols in (A) vegetarians ( p¼0.0015) and (B) nonvegetarians
(p>0.05). The box represents the interquartile range, which
contains the 50% of values. The whiskers are lines that extend
the box to the highest and lowest values, excluding outliers.
A line across the box indicates the median value. Y-axis: as
given in Figure 1.
GLOBAL DNA METHYLATION AND CORONARY ARTERY DISEASE 361

methylation in peripheral blood is significantly higher in CAD
patients than in controls. Within the cohort of CAD patients,
there is a correlation of plasma homocysteine levels with
higher global methylation. Our results are consistent with a
recent study of chromic kidney patients where an association
of hypermethylation of genomic DNA with inflammation and
increased mortality due to cardiovascular diseases has been
reported (Stenvinkel et al., 2007). The authors analyzed the
global DNA methylation by HpaII=MspI sensitivity assay on
genomic DNA from peripheral blood. We have employed a
similar approach. The CCGG sequences that are the target
sequences in our assays is suitable for genome-wide scan for
methylation in terms of their density and distribution in dif-
ferent chromosomes. HpaII=MspI sensitivity has been utilized
to scan genome-wide promoter methylation by Hatada et al.
(2006).
Aging is known to cause global DNA hypomethylation and
promoter hypermethylation (Fraga and Esteller, 2007). Our
results indicate that there is a trend (albeit not significant) of
global hypomethylation in controls and hypermethylation in
CAD patients with age (data not shown). We also observed
significant hypermethylation in CAD patients in the higher
age group (61 to 75 years) as compared to the controls. Al-
though the exact mechanism of alteration of DNA methyla-
tion is not yet known, it is believed that loss of global DNA
methylation with age may be due to the passive demethyla-
tion of heterochromatic DNA, probably as a result of a pro-
gressive loss of DNMT1 efficacy or defective targeting of the
enzyme by other cofactors (Fraga and Esteller, 2007). Apart
from the general loss of DNA methylation, several regions of
the DNA like the promoter CpG islands of estrogen receptor
(ER), myogenic differentiation antigen 1 (MYOD1), insulin-
like growth factor II (IGF2), and tumor suppressor candidate
33 (N33) become hypermethylated with age. It has been pro-
posed that these methylation changes (global hypomethyla-
tion and promoter hypermethylation) with age are mosaic in
tissues and play a triggering role in age-related diseases like
neoplasia and atherosclerosis (Issa, 2003). We believe that in
CAD patients, age-induced global DNA hypomethylation is
offset by promoter-specific hypermethylation resulting in in-
creased overall DNA methylation in CAD patients, especially
in the higher age group.
It is generally believed that an elevated homocysteine level
would lead to hypomethylation of DNA because increased
homocysteine levels are known to elevate the concentration of
S-adenosyl homocysteine (SAH), which is an inhibitor of
methyltransferases (Yi et al., 2000). In a study focusing on
global DNA methylation status in vascular patients, it was
reported that increased homocysteine levels led to hypo-
methylation of DNA (Castro et al., 2003). The authors reported
that the patients with vascular disease had significantly
higher plasma tHcy and AdoHcy concentrations, and the
DNA hypomethylation status was significantly correlated
with plasma tHcy and AdoHcy but not with plasma Ado-
Met=AdoHcy ratios. This is not consistent with the effect of
SAH as a competitive inhibitor of DNA methyltransferase,
thus indicating that the relationship between homocysteine
levels and DNA methylation in disease background is intri-
cate and not due to inhibitory action of SAH only. In addition,
the sample size in the study was small and also the clinical
attributes pertaining to vascular disease were not restricted to
CAD only (8 patients with stroke, 9 patients with myocardial
infarction, and 15 controls).
In our cohort, the range of plasma homocysteine is similar
between the patients and controls, suggesting that the plasma
level of tHcy alone does not have an effect on methylation.
However, high tHcy is significantly correlated with global
DNA hypermethylation in the background of CAD. We did
not find any significant correlation between the levels of folate
or vitamin B12 and global DNA methylation in our study.
There are reports that support the role of folate in restoring
normal homocysteine levels (Ingrosso et al., 2003), while two
recent reports published from the Norwegian Vitamin trial
(NORVIT) and Heart Outcomes Prevention Evaluation
(HOPE) trials concluded that supplements containing folic
acid, vitamin B12, or vitamin B6 did not significantly reduce
the risk of adverse events in cardiovascular disease patients
although the levels of homocysteine were substantially re-
duced (Bonaa et al., 2006; Lonn et al., 2006), thus questioning
the role of level of vitamin B12=B6 in preventing adverse ef-
fects of homocysteine in CAD. However, in these trials the
epigenetic modulation due to hyperhomocysteinemia has not
been addressed.
The data on DNA methylation from hyperlipidemic ApoE
null mice in aortae as well as peripheral blood cells indicated
both hypo- and hypermethylation of DNA (Lund et al., 2004).
In human macrophage THP-1 cell line, on stimulation of
atherogenic lipid levels, significant DNA hypermethylation
FIG. 6. Methylation pattern of 25 CpG sites in the promoter region of ApoE gene in 15 patients (A) and 15 controls (B). The
filled boxes represent methylated cytosines, while blank boxes represent unmethylated cytosines.
362 SHARMA ET AL.

was observed as compared with untreated cells (Lund et al.,
2004). Based on these results the authors observe that the
initial stages of atherogenesis are associated with alteration in
genomic DNA methylation patterns including hyper- and
hypomethylation, rather than a unidirectional change toward
global hypomethylation (Zaina et al., 2005). The same authors
have reported aberrant methylation in the peripheral blood
also with a higher level of hypomethylation (Lund et al., 2004;
Zaina et al., 2005). Recently, using monocytes in culture, it has
been demonstrated that increase in homocysteine levels leads
to hypermethylation of the ApoE promoter and decrease in its
expression. This was correlated with increase in total choles-
terol, free cholesterol, and cholesteryl ester (Yi-Deng et al.,
2007b). However, we did not find any significant differences
in the methylation pattern in the promoter region of ApoE in
peripheral blood lymphocytes of patients and controls. It
should be noted that methylation differences detected by
Yi-Deng et al. (2007b) in cultured monocytes treated with
homocysteine are significantly different from the samples
used in our study. Additionally, our samples included CAD
patients, which could contribute to further epigenetic alter-
ations.
These results suggest that additional factors, including
nutrition, affect DNA methylation patterns by mechanisms
that are likely to be independent of vitamin B12 or homo-
cysteine levels (Zaina et al., 2005). In this context, it is in-
teresting that we find a significant correlation between
methylation, CAD, and homocysteine levels in vegetarians
when the cohort is stratified with reference to diet. A major
proportion of Indian population follow a strict vegetarian
diet, which results in higher levels of plasma homocysteine as
compared to the global scenario (Refsum et al., 2001; Kumar
et al., 2005). Further, there is no significant difference in global
DNA methylation in vegetarian CAD patients and nonvege-
tarian controls in spite of lower homocysteine levels in the
latter. It remains to be seen if the sequences methylated in
vegetarian CAD patients and the nonvegetarian controls and
patients are the same.
The investigation on the effect of homocysteine on DNA
methylation in relation to cardiovascular diseases in ani-
mal models (Lund et al., 2004; Zaina et al., 2005) and cells
in culture (Yi-deng et al., 2007a, 2007b) and the recent study
on CKD patients (Stenvinkel et al., 2007) support the bidi-
rectional effect of homocysteine leading to both hypo- and
hyper-methylation of DNA and an increase in the activity
of DNMT3a and 3b (Yi-deng et al., 2007a). The promoter of
ApoE gene is hypermethylated under hyperhomocysteine-
mia, hyperlipidemia in spite of the hypomethylation of glo-
bal DNA as measured by MethyLight assay and AluIan
LINE elements as surrogate markers (Yi-Deng et al., 2007b).
Therefore, it can be inferred that the effect is gene specific
and could be influenced by additional factors. However, the
significant difference in global DNA methylation we ob-
served is consistent with the increased activity of DNMT3a
and 3b under hyperhomocysteimic conditions in vascular
cells in culture (Yi-Deng et al., 2007b).
Others and we have shown that elevated levels of homo-
cysteine induce endoplasmic reticulum stress in models such
as yeast and human cell lines (Werstuck et al., 2001; Kumar
et al., 2006). Further, homocysteine-induced ER stress acti-
vates sterol regulatory binding proteins (SREBP), which is
associated with the increased expression of genes involved in
cholesterol transport and metabolism. This has been sug-
gested as a possible mechanism for homocysteine-induced
development and progression of hepatic steatosis and ath-
erosclerotic lesions. Hepatic steatosis is also a hallmark of
chronic alcoholism, and Bonsch et al. (2004) have shown that
in patients with chronic alcoholism, homocysteine levels are
positively correlated with global DNA methylation and hy-
permethylation of homocysteine-induced endoplasmic retic-
ulum protein (HERP) promoter region. This resulted in the
decreased expression of HERP gene, which is an endoplasmic
reticulum stress response gene (Bleich et al., 2006). Recently,
Azfer et al. (2006) have shown the activation of ER stress re-
sponse in the development of ischemic heart disease in mice.
These studies suggest that in CAD patients there are complex
interactions, where elevated levels of homocysteine lead to
the counter intuitive consequence, namely hypermethylation.
The genes coding for ER and HERP may be examples of genes
contributing to the pool of global DNA hypermethylation.
Further studies are required to elucidate the functional con-
sequences of these interactions in CAD.
In summary, our results strongly suggest that an alteration
in DNA methylation profile could be associated with CAD
condition itself. A comparative analysis of the methylation
profile at individual genes in CAD patients is underway to
understand the effect of environment through modulation of
homocysteine levels superimposed on the disease pheno-
type.
Acknowledgments
We are grateful to Dr. Rita Castro, Dr. Dwaipayan Bhar-
adwaj, Dr. Sridhar Sivasubbu, and Dr. Beena Pillai for pro-
viding critical inputs and Dr. Saurabh Ghosh (Indian
Statistical Institute, Kolkata) for inputs in statistical analysis.
We also acknowledge the help of Dr. Elayanambi Sundar-
amoorthy in preparation of the manuscript and Lijo John
for help in data analysis. P.S. and J.K. thank the University
Grants Commission for financial support through a research
fellowship. A.K. is thankful to the Council of Scientific and
Industrial Research for research fellowship. This study was
supported by funds provided by the Department of Bio-
technology, Govt. of India (S.S., G.K., and V.B.), under pro-
ject BT=PR4525=Med=14=533=2003.
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Address reprint requests to:
Shantanu Sengupta, Ph.D.
Institute of Genomics and Integrative Biology
Mall Road
Delhi 110007
India
E-mail: shantanus@igib.res.in
Vani Brahmachari, Ph.D.
Dr. B.R. Ambedkar Centre for Biomedical Research
University of Delhi
Delhi 110007
India
E-mail: vbrahmachari@acbr.du.ac.in
Received for publication October 22, 2007; received in re-
vised form March 17, 2008; accepted March 25, 2008.
GLOBAL DNA METHYLATION AND CORONARY ARTERY DISEASE 365

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