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DNA methylation of NOS genes and carotid intima-media thickness in children

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DNA methylation of NOS genes and carotid intima-media thickness in children

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DNA METHYLATION OF NOS GENES AND

CAROTID INTIMA-MEDIA THICKNESS IN CHILDREN

by

Caron Park

________________________________________________________________________

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOSTATISTICS)

May 2012

Copyright 2012 Caron Park
ii

ACKNOWLEDGEMENTS
I would like to thank my advisor Dr. Carrie Breton, for her guidance and support
throughout my research and writing process. I would like to also thank Dr. Jim
Gauderman and Dr. Kim Siegmund for serving as my committee members and providing
valuable suggestions. Finally, I would like to thank my family and friends for all their
love, support, and encouragement.

iii

TABLE OF CONTENTS
Acknowledgements ii
List of Tables iv
Abbreviations v
Abstract vi
Introduction 1
Methods 4
Study Population 4
CIMT Measurements 5
Buccal Cell Sample Collection 6
CpG Methylation Loci Selection 7
DNA Methylation 8
Statistical Analyses 9
Results 12
Figure 1: Distribution of carotid intima-media thickness (μm)
in participants 13
Discussion 21
References 25

iv

LIST OF TABLES
Table 1: Descriptive statistics of the 377 CHS study participants 12
Table 2: Distribution of CIMT (μm) by population study
Characteristics 13
Table 3: Distribution of percent methylation of CpG loci in Nitric
Oxide Synthase and Arginase genes 14
Table 4: Spearman pairwise correlations for NOS1 and NOS3
CpG loci 16
Table 5: Spearman pairwise correlations for NOS2A CpG loci 16
Table 6: Spearman pairwise correlations for ARG CpG loci 16
Table 7: The association between percent methylation of NOS and
ARG genes and CIMT measurement (μm) 17
Table 8: The association between DNA methylation of NOS1 with
CIMT stratified by race and genetic ancestry 19
Table 9: The association between DNA methylation of average CpG
Island of NOS2A and CIMT stratified by BMI status 20

v

ABBREVIATIONS
ARG: Arginase
BMI: Body Mass Index
CCA: Common Carotid Artery
CHS: Children’s Health Study
CIMT: Carotid Intima-media Thickness
CVD: Cardiovascular Disease
DNA: Deoxyribonucleic Acid
FeNO: Fractional Concentration of Nitric Oxide
NO: Nitric Oxide
NOS1/nNOS: Neuronal Nitric Oxide Synthase
NOS2A/iNOS: Inducible Nitric Oxide Synthase
NOS3/eNOS: Endothelial Nitric Oxide Synthase
PCR: Polymerase Chain Reaction
PSQ: Pyrosequencing system
SD: Standard Deviation

vi

ABSTRACT
Carotid intima-media thickness, a biomarker for atherosclerosis, is associated
with cardiovascular disease risk in adults. Atherosclerosis is a lifelong process that
begins in childhood. Nitric Oxide (NO) plays an important role in cardiovascular health
by maintaining and regulating vascular tone and blood flow. It remains unclear as to how
the epigenetic regulation of nitric oxide synthase, the gene responsible for nitric oxide
production, affects carotid intima-media thickness (CIMT) in children. Data from a
subset of 377 participants from the Children’s Health Study on whom buccal cell DNA
and CIMT measurements were collected were analyzed to determine whether DNA
methylation in NOS genes was associated with CIMT. Results showed that an increase
in DNA methylation of NOS1 was significantly associated with an increase in CIMT
measurements in children. Our study suggests that DNA methylation of NOS genes is
associated with CIMT in children, which may help to advance the understanding of
biological processes contributing to atherosclerosis even at an early age.
1

INTRODUCTION
Cardiovascular disease (CVD) is a leading cause of morbidity and mortality in the
general population (1). Atherosclerosis, a progressive disease of the large arteries where
the arterial walls thicken as they accumulate lesions and fatty tissues over time, is a well-
known underlying pathology for a majority of cardiovascular events. Atherosclerosis
begins in childhood with fatty streaks forming in the intima of large arteries in children
(2, 3). Advanced stages of atherosclerosis can result in the lesions growing large enough
to obstruct blood flow and instigate cardiovascular events such as stroke or myocardial
infarction (4). Carotid intima-media thickness has been accepted as a noninvasive
surrogate marker for atherosclerosis and a strong indicator for cardiovascular disease risk
assessment in adults (5). Increased intima-media thickness has been shown to be
predictive of future cardiovascular events. Although no study currently exists that
determines CIMT cut-off points predictive for atherosclerosis in children, a case study
has been reported of significant intima-media thickening of a morbidly obese 2-year old
boy, suggesting that vascular changes can occur early in childhood (6).
Multiple biologic pathways have been implicated in the pathogenesis of
atherosclerosis, one of which is the role for nitric oxide in vascular regulation (7). Nitric
Oxide (NO) is well-known to have a cardio-protective role in regulating vascular tone
and blood pressure, preventing platelet aggregation, and inhibiting smooth muscle cells
from proliferation (8, 9). NO is synthesized from L-arginine by nitric oxide synthase
(NOS) enzymes of which there are three main isoforms including neuronal (nNOS or
NOS1), inducible (iNOS or NOS2A), and endothelial NOS (eNOS or NOS3). All three
2

isoforms are expressed in the cardiovascular system of humans (8). Existing evidence
from in vitro and animal studies has shown that epigenetic changes in iNOS and eNOS
from DNA methylation and histone modification were associated with gene expression
changes, and these epigenetic variations in NOS can in turn affect NO generation and
bioavailability (10, 11).
Previously it was believed that eNOS derived NO was largely responsible for the
regulation of vascular tone. However, recent studies suggest that nNOS may also play a
role. Local nNOS derived NO was found to be the main factor of micro vascular tone
and basal blood flow in the forearm and coronary vascular bed in human studies where S-
methyl-L-thiocitrulline, an nNOS inhibitor, was used (12, 13). The evidence suggests
that NO derived from nNOS may play an essential role in vascular regulation and
cardiovascular health.
Arginase (ARG) competes with NOS for the common substrate of L-arginine,
suggesting that ARG may play a factor in regulating NO synthesis by moderating the
availability of intracellular L-arginine (14, 15). In mammals there are two isoforms of
ARG, one is the cytosolic isoform (ARG1) and the other is the mitochondrial isoform
(ARG2) that are expressed in the endothelial and vascular smooth muscle cells (14).
Changes in NOS and ARG expression instigated by genetic or epigenetic modifications
may have the potential to influence NO synthesis and impact cardiovascular health
outcomes.
Carotid intima-media thickness is predictive of cardiovascular morbidity and
mortality in adults (16). Young children and adolescents with cardiovascular risk factors
3

are even more prone to atherosclerosis later on in life (17). Since atherosclerosis can start
in childhood, it is important to detect the progression early in order to develop
interventions to help reduce or prevent cardiovascular disease risk that can manifest as
serious outcomes in adulthood. Therefore in this study we investigated the potential role
for epigenetic regulation of NO, an important player in CVD development. Specifically,
we evaluated whether DNA methylation in NOS and ARG genes were associated with
level of CIMT in children. Gene loci were chosen based on previous evidence of their
likelihood to influence gene expression or transcription. We also investigated the
association between DNA methylation with ancestry and BMI status. This study was
conducted in population-based cohort of children who had participated in the Southern
California Children’s Health Study (CHS) (18, 19).

4

METHODS
Study Population
In 2002 and 2003, a new cohort was recruited from schools in 13 southern
California communities for the ongoing Children’s Health Study that was started in 1993
by the University of Southern California. Nine of the communities were the same as the
previous original cohorts, but four were new communities that were added in order to
provide a more diverse study population. In this cohort 5,341 kindergarten and first
graders that were between the ages of 5 and 7 were recruited. From this ongoing CHS
cohort, we sampled from 2,498 of the kindergarten and first grade children that had at
least one measurement of fractional concentration of nitric oxide (FeNO) and a buccal
sample collected at the same time. From the 2,498 children there were 6,578 available
buccal cell samples. Buccal cell samples were collected in school years: 2004-2005
(Year 3), 2005-2006 (Year 4), or 2006-2007 (Year 5); and all CIMT measurements were
obtained in the school year of 2007-2008 (Year 6).
For the purpose of this study, samples of subjects were restricted to children
whose ethnicity was either non-Hispanic white or Hispanic white with available genetic
information (n=5,765 samples). Samples were excluded if measurements were taken
outside or there were missing FeNO measurements (n=218 samples), missing air
pollution data or if they lived outside the eight towns under active follow-up (n=1,524
samples), missing asthma status (n=122 samples), samples with little to no DNA
remaining (n=1,893 samples), or if they had low DNA concentration (n=345samples).
From the remaining 1,137 children with 1,663 buccal cell samples, a subset of 940
5

samples was randomly chosen. For children that had multiple buccal cell samples
available, preference was given to the earliest sample collected. From the 940 children,
there were further exclusions if there were missing CIMT measurements (n=561). Since
the sample size was too small to analyze for year 4 of buccal cell collection (n=2), we
only looked at years 3 and 5. There were a total of 377 children that had both genetic
information and CIMT measurement data. This study was approved by the University of
Southern California institutional review board for human studies.

CIMT Measurements
Carotid intima-media thickness measurements were performed at the children’s
school and were assessed by a single physician-imaging specialist from the USC
Atherosclerosis Research Unit (ARU) Core Imaging and Reading Center (CIRC). To
assess CIMT of children at year 6, a B-mode carotid ultrasound was performed and a 3
lead electrocardiograph (ECG) was recorded. A Mindray DC-7 Diagnostic System
attached to a 10-MHz linear array transducer was used to get high-resolution B-mode
ultrasound images of the right and/or left common carotid artery (CCA). To ensure that
CIMT was measured at the same point in the cardiac cycle a single-limb lead
electrocardiogram was simultaneously recorded along with the B-mode image, and the
images contained internal anatomical landmarks for probe angulation reproducibility.
CIMT was measured along the far wall of the distal CCA between the lumen-intima and
media-adventitia along a 1-cm length just 0.25 cm from the carotid artery bulb by
automated computerized edge-detection with an in-house developed software package
6

(Patents 2005, 2006). 70 to 100 individual measurements over the standard 1-cm length
of the carotid artery were averaged to produce the mean CIMT. The left and right CIMT
measurements were then averaged to produce one measurement.

Buccal Cell Sample Collection
Buccal cell specimens were collected using a toothbrush. The children were
provided with two toothbrushes and with the first one they were required to brush their
teeth in order to clean the mouth without the use of toothpaste. The second was used to
brush against the buccal mucosa, the inner lining of the cheeks, with ten strokes on each
side. Afterwards, the children were instructed to swish water in their mouth and to spit
the contents into a tube and to place the second brush in the expelled water. Then
approximately 10 ml of isopropyl alcohol was added using a pipette by the staff. Most of
the specimens were collected at the school in which the children attended under the
supervision of a study staff. The rest of the buccal cell specimens were collected at the
child’s home and sent over through postal mail.
On the day that the sample was received in the laboratory, buccal cell suspensions
were centrifuged at 2,000g and the pellets stored at -80°C until time for DNA extraction.
At the time of extraction, the pellets were re-suspended and left to incubate overnight at
55°C in a 600μl of lysis solution containing 100μg/ml of proteinase K from a
PUREGENE DNA isolation kit (cat #D-5000; GENTRA, Minneapolis, MN). All DNA
extractions were executed according to the manufacturer’s recommendations. Afterwards
7

the DNA samples were re-suspended in a hydration solution (GENTRA) and stored away
at -80°C.

CpG Methylation loci Selection
CpG is the linear paring of cytosine and guanine bases along the chromosome.
CpG loci located in the promoter regions of NOS1, NOS2A, NOS3, ARG1, and ARG2
were examined for this study. For NOS1, three CpG loci in a nuclear hormone receptor
regulatory sequence in exon 2 were chosen on the basis that this nuclear hormone
receptor site contributed to the regulation of NOS1 transcription (20). Within the
NOS2A gene three regions were selected for DNA methylation analysis: the non-CpG
island promoter region, the non-CpG island region between exons 1 and 2, and the CpG
island region. Two loci, position 1 and 2, in the non-CpG island promoter region were
chosen because they showed to be inversely related to NOS2A mRNA expression (10).
Position 3, a locus situated between exon 1 and 2 at a non-CpG region, was chosen to
correspond to a site that was previously examined by Tarantini and colleagues (21).
Positions 4-7, that were located in the CpG island region, were chosen to observe the
transcription factor binding sites conserved in the mammal alignment
(http://genome.ucsc.edu/). For NOS3, two CpG loci located in the positive regulatory
domain in the proximal promoter region were chosen because they contributed to NOS3
transcription (22). According to the assembly of GRCh37/hg19 CpG positions were
located at chr12:117,769,133-117,769,145 for NOS1, chr17:26,127,518-26,127,523 for
non-CpG island in the promoter region of NOS2A, chr17:26,126,265-26,126,267 for
8

non-CpG island between exons 1 and 2 of NOS2A, chr17:26,120,696-26,120,703 for
CpG islands of NOS2A, and chr7:150,690,770-150,690,776 for NOS3.
For ARG1, a CpG locus that was situated in the promoter region but not in a CpG
island was chosen for analysis because it was previously associated with change in
expression (23). Three CpG loci (Position 1-3) located in a CpG island in the promoter
region was selected for ARG2 because this region of promoters consisted of several
transcription factor binding sites, potential binding sites for promoter and enhancer
elements, and because of its possible role in regulation of ARG2 gene expression (24).
CpG positions were located at chr6:131,894,368-131,894,360 for ARG1 and
chr14:68,086,547-68,086,554 for ARG2.

DNA Methylation
Pellets of buccal cell suspensions that were centrifuged and previously frozen
were re-suspended and incubated in lysis solution from a PUREGENE DNA isolation kit
overnight before genomic data was extracted from the buccal cells. Laboratory personnel
performing the DNA methylation analysis were blinded to information regarding the
study subjects. Bisulfite conversion of one microgram of genomic DNA was
implemented by using the EZ-96 DNA Methylation-Gold Kit (Zymo Research, Orange,
CA), according to the recommended protocol of the manufacturer. Final elution was
performed using 40 μl of M-Elusion Buffer (Zymo Research, Orange, CA), and the
bisulfite-converted DNA was stored away until further use at a temperature of -70°C.
Bisulfite-polymerase chain reaction (PCR) pyrosequencing assay was used for
9

methylation analyses using the HotMaster Mix (Eppendorf, Hamburg, Germany) and
PSQ HS 96 Pyrosequencing Sytem (Biotage AB, Uppsala, Sweden). Pyrosequencing
output data was then reported as a percent of DNA methylation at each CpG locus.
Steps for quality control were implemented to confirm the efficiency of the
bisulfite conversion. Duplicate genomic DNA samples were placed on each bisulfite
conversion plate to evaluate the internal plate variation of bisulfite conversion and
pyrosequencing reaction. The results showed conversion efficiency to be greater than
95%. Run-to-run and plate-to-plate variation in the pyrosequencing reaction was
observed by adding universal PCR products amplified from cell line DNA, and quality of
reaction was confirmed for each sample by looking at the pyrogram peak pattern.

Statistical Analyses
Descriptive analyses were performed to examine the distribution of DNA
methylation (ARG and NOS genes), CIMT, and subject characteristics. Linear regression
analyses were then used to examine univariate associations. Spearman correlations were
used to observe pairwise correlations of percent methylation between different CpGs in
the same gene, and for genes that had multiple CpG loci measured we also looked at the
correlation between individual loci. Using CIMT as the dependent outcome for DNA
methylation of ARG and NOS genes, multiple regression analyses were conducted to
assess the hypotheses while adjusting for potential confounders.
Additional analyses were conducted in which methylation variables for ARG and
NOS genes were dichotomized into two categories. For loci that had 20% or more zero
10

values of methylation, the dichotomized variable was categorized as either “un-
methylated” or “methylated”; otherwise they were dichotomized into “low” methylation
or “high” methylation where the cut-off point was at the median. For genes that had
multiple CpG loci measured we also evaluated the average percent methylation which
was dichotomized accordingly.
To investigate the association between percent DNA methylation and CIMT, we
fitted linear regression models adjusted for gender, race, BMI, age at IMT scan, town of
residence, and experimental plate for pyrosequencing reactions. These variables were
chosen because they were known to be predictors of CIMT or were shown to be
confounders that affected the variability observed in the data. For the variable age at
CIMT scan, ages 9 and 10 were combined due to the small sample size in age 9 (n=2).
The residuals were tested to examine if the model assumptions of normality were
met, and scatterplots of residuals against predicted CIMT measurements for each DNA
methylation variables were examined to check for linearity and homoscedasticity. The
residuals and scatterplots revealed that model assumptions were met. Results of the
linear regression models were reported as μm (micrometer) changes in CIMT for a 1%
difference in DNA methylation. For the dichotomized model, results were reported as
micrometer changes in CIMT for DNA that was “methylated” or had “high” methylation.
Statistical interaction terms were added to the linear regression model in order to
assess whether the association between DNA methylation of ARG and NOS genes varied
by ancestry or BMI status. Ancestry was divided into three groups based on self-reported
ethnicity and a genetic ancestry score. This score was computed using the program
11

STUCTURE from a set of ancestral informative markers (AIMS) and was scaled to
represent the proportion of Native American and Caucasian ancestry (25). Children who
reported being “non-Hispanic white” were predominately of Caucasian ancestry.
Children who reported being “Hispanic white” were categorized into “More Caucasian”
if they were genetically more Caucasian than Native American, or they were categorized
as “More Native American” if they were genetically more Native American. BMI status
was dichotomized into one of two categories, “normal” for those with underweight or
healthy weights and “overweight” for those who were overweight or obese. The cutoff
for “normal” (less than 85
th
percentile) and “overweight” (85
th
percentile or greater) were
based on percentiles from the Centers for Disease Control and Prevention BMI percentile
range
(http://www.cdc.gov/healthyweight/assessing/bmi/childrens_bmi/about_childrens_bmi.ht
ml). All tests assumed a two-sided alternative hypothesis with a significant level of 0.05.
The data was analyzed using SAS software version 9.2.
12

RESULTS
The children of this study were on average 11 years old when the carotid intima-
media thicknesses were measured. A little less than half of the children were male and
63% were of Hispanic white ethnicity (Table 1). CIMT measurements were generally
normally distributed with an average of 562.0 μm and standard deviation of 40.6 μm
(Figure 1, Table 2). Although not significant, the means of CIMT measurements were
higher in twelve year olds, males, and non-Hispanic whites (Table 2).

13

Table 3 displays the percent methylation distributions of ARG and NOS loci.
Non-CpG island loci in NOS1, NOS2A, NOS3, and ARG1 were generally heavily
methylated with the exception of NOS2A position 3 that had a maximum methylation of

14

15

16

63.3%. The CpG island loci in NOS2A and ARG2 were mostly un-methylated. The
multiple CpG loci that were measured within each gene were not strongly correlated
(Table 4-6).

After adjusting for the covariates sex, ethnicity, BMI, town, plate, and age at IMT
scan, we found that DNA methylation in NOS1 was significantly associated with carotid
intima-media thickness (Table 7). For every 1% increase in average DNA methylation of
17

NOS1, CIMT measurements increased by 1.2 μm (p = 0.02). Similar results were shown
for analyses that were conducted using a dichotomous variable for categorizing
methylation, further strengthening the analyses. Compared to subjects with “low”
methylation, those with “high” methylation of average NOS1 had CIMT measurement
18

that was 15.7 μm higher (p < 0.01). Individual loci for both continuous and dichotomized
models showed similar but weaker patterns.
We also investigated whether the association between DNA methylation of NOS
and ARG genes and CIMT might vary by ancestry, since self-reported ethnicity may not
be able to accurately categorize subjects into their true ancestry. We found that the
association between methylation in NOS1 and CIMT levels was larger in Hispanic white
children who were “More Native American” than Non-Hispanic whites and Hispanic
whites that were “More Caucasian” (Table 8). Although the p-value for the test of trend
across the 3 genetic ancestry groups was not significant, as the level of Native American
ancestry increased among the 3 groups, we observed that the CIMT levels increased. We
did not see any significant results for the other genes.
Methylation of NOS2A, NOS3, ARG1, and ARG2 genes were not associated with
CIMT. However, when considering BMI as a possible effect modifier of CpG
methylation and CIMT for all CpG targets, we observed that the association between
average DNA methylation in the CpG island of NOS2A and CIMT was stronger in
children who were overweight compared to those who were normal (Table 9). Stratified
analyses showed that there was a protective effect on CIMT ( -3.9 μm, p = 0.02) in
overweight children as the average CpG island methylation of NOS2A increased, but
there was an opposite effect on those with normal weights. The dichotomized model
showed a higher protective effect among obese children (-47.9 μm, p = 0.02). However,
when adjusted for multiple testing using a Bonferroni correction, none of the associations
remained significant.
19

20

21

DISCUSSION
In this study we examined the association between DNA methylation of NOS and
ARG genes with CIMT in children. Our results showed that there was an association
between neuronal nitric oxide synthase and CIMT suggesting that DNA methylation of
NOS1 may play a role in the level of carotid intima-media thickness in early childhood.
Children who had “More Native American” ancestry appeared to demonstrate stronger
associations than those with Caucasian ancestry. We also found that obesity may modify
the association between DNA methylation of NOS2A in the CpG island and CIMT.
However this last result may be spurious due to concerns for multiple testing.
The association between DNA methylation of NOS1 and CIMT is of importance
because an increase in methylation of NOS1 may be reducing its transcriptional activity
resulting in a decrease of NO production and an increase in CIMT levels. NO is
synthesized from L-arginine by NOS enzymes and known to play a cardio-protective role
in vascular regulation (7, 9). Reduced bioavailability of NO is believed to occur early in
the development of atherosclerosis (9). In an animal study, nNOS-knockout mice
exhibited neointimal formation, constrictive vascular remodeling, and narrowing of the
lumen in the ligated carotid artery showing that nNOS takes a part in protecting against
vascular lesion formation (26). Human studies have shown that NO derived from local
nNOS to be a main factor of micro vascular tone and basal blood flow in the forearm and
coronary vascular bed when a nNOS inhibitor, S-methyl-L-thiocitrulline, was used (12,
13). nNOS was also shown to be expressed in early and advanced human atherosclerotic
vessels (27). The results of our study supports the idea demonstrated in animal models
22

that increased DNA methylation of NOS1 reduces transcriptional activity of NOS1,
lowering NO production and decreasing vascular regulation (i.e. vasoconstriction,
increased blood pressure, and platelet aggregation), leading to higher CIMT.
An association between DNA methylation in NOS1 and CIMT was strongest in
children who were predominately Native American. We observed suggestion of a trend
in CIMT levels as Native American ancestry grew stronger; however it was not
statistically significant. Among those who self-reported “Hispanic white” ethnicity,
approximately 32% were found to be more of Caucasian ethnicity than anything else
based on genetic analyses. The inclusion of genetic information to more accurately
identify ancestry helped to identify potential trends in susceptibility that otherwise would
have been missed if self-reported ethnicity was only observed.
We observed interesting results in children who were overweight and obese.
Increased DNA methylation of NOS2A in the CpG islands appeared to be associated with
lower CIMT levels in overweight children. However, the associations did not remain
significant after applying a Bonferroni adjustment suggesting false-positive results.
Many studies have observed that obese children tend to have higher CIMT compared to
healthy non-obese children (6, 28). Risk of atherosclerosis was also shown to be more
prominent as cardiovascular risk factors increased (2, 3). These studies have shown
obese and overweight children to be more susceptible to higher CIMT levels as opposed
to having a protective effect. However, further investigation in a larger sample is
warranted to observe the association between CIMT and NOS in overweight and obese
children.
23

We observed no associations between CIMT and DNA methylation in ARG
genes. Limitation of a small cohort may have prevented us from detecting potential
significant results. Another limitation was that buccal cells were used to obtain DNA
methylation information. A stronger association might have been observed if
methylation data was taken from the appropriate tissue related to the outcome of interest,
or if blood samples that were more closely related to the vasculature were obtained.
However, obtaining such sample would have been difficult and invasive. Therefore,
noninvasive buccal cell samples were used to assess epigenetic changes in cardiovascular
health in children.
Lastly, buccal cell samples and CIMT measurements were not collected on the
same day. Buccal cells were collected in school years 2004-2005 (Year 3) or 2006-2007
(Year 5), and CIMT measurements were collected in 2007-2008 (Year 6). It would have
been ideal to have all the measurements recorded at the same time. However, the
analyses did not show a significant interaction between year of buccal cell collection and
DNA methylation of NOS and ARG genes in our model, suggesting that the effect of
DNA methylation on CIMT did not vary depending on year of collection.
In this study, we provide evidence that differences in percent DNA methylation in
NOS1 are associated with CIMT levels in children. We observed a stronger association
between NOS1 and CIMT levels in children with greater Native American ancestry.
These results indicate that NOS1 may play a role in atherosclerosis by regulating
production of nitric oxide, a key player in vascular health and development of
atherosclerosis, leading the changes in CIMT. More research is warranted in a larger
24

cohort with a more optimal study design to replicate these findings and clarify the
importance of epigenetic regulation of NOS genes for atherosclerosis.
25

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Abstract (if available)

Abstract Carotid intima-media thickness, a biomarker for atherosclerosis, is associated with cardiovascular disease risk in adults. Atherosclerosis is a lifelong process that begins in childhood. Nitric Oxide (NO) plays an important role in cardiovascular health by maintaining and regulating vascular tone and blood flow. It remains unclear as to how the epigenetic regulation of nitric oxide synthase, the gene responsible for nitric oxide production, affects carotid intima-media thickness (CIMT) in children. Data from a subset of 377 participants from the Children’s Health Study on whom buccal cell DNA and CIMT measurements were collected were analyzed to determine whether DNA methylation in NOS genes was associated with CIMT. Results showed that an increase in DNA methylation of NOS1 was significantly associated with an increase in CIMT measurements in children. Our study suggests that DNA methylation of NOS genes is associated with CIMT in children, which may help to advance the understanding of biological processes contributing to atherosclerosis even at an early age.

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Asset Metadata

Creator Park, Caron (author)

Core Title DNA methylation of NOS genes and carotid intima-media thickness in children

School Keck School of Medicine

Degree Master of Science

Degree Program Biostatistics

Degree Conferral Date 2012-05

Publication Date 09/19/2012

Defense Date 02/21/2012

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag CIMT,DNA methylation,nitric oxide synthase,OAI-PMH Harvest

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Breton, Carrie (committee member), Gauderman, William James (committee member), Siegmund, Kimberly D. (committee member)

Creator Email caronpar@usc.edu,caronpark@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC11291671

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