Analysis of the association between DNA methylation sites
and cardiovascular disease-related single nucleotide
polymorphisms in the Japanese population
Masahiro Nakatochi *, Sahoko Ichihara †, Ken Yamamoto ‡, Tatsuaki Matsubara §, andMitsuhiro Yokota **Abstract—The development of cardiovascular disease (CVD) depends on environmental and genetic factors.
Although many single nucleotide polymorphisms (SNPs) associated with CVD susceptibility have been
identified to date, the mechanisms through which these polymorphisms contribute to disease development
remain unclear. To investigate the epigenetic basis of myocardial infarction (MI), a type of CVD, we analyzed
the associations between DNA methylation (DNAm) status at methylation sites and CVD-associated SNPs inelderly Japanese individuals. A total of 192 patients with MI and 192 controls were recruited from hospitalattendees and the general population, respectively. Genome-wide DNAm profiles in whole blood were obtainedby analysis with an Infinium HumanMethylation450 BeadChip. The CVD-associated SNPs were genotyped
using an SNP array and were imputed using 1000 genomes phase 3 as a reference. Association analysis
identified 431 genome-wide or suggestive significant combinations of DNAm sites and SNPs. A total of 225
gene symbols were located near the DNAm sites. These genes were significantly enriched in the 6p21 region.
Our results suggested that SNPs located at chromosome 6p21 may influence the development of CVD byaffecting DNAm in blood cells.

Keyword: Single nucleotide polymorphisms, DNA methylation, cardiovascular disease, myocardial
infarction
1 Introduction
Cardiovascular disease (CVD) is the leading cause of mortality, morbidity, and hospitalization worldwide.
The prevalence of CVD is increasing more rapidly in Asia than in Western countries. Improving ourunderstanding of the pathogenesis of CVD may help mitigate further increases in the incidence of CVD.
Genetic factors have been found to contribute to the development of CVD. Genome-wide association
studies (GWAS) for CVD have revealed many associated susceptibility genes and single nucleotide
polymorphisms (SNPs). Although many SNPs associated with CVD susceptibility have been identified todate, the mechanisms through which these polymorphisms contribute to disease development remain
unclear. Recent progress in epigenetic epidemiology has facilitated investigations of the relationships
among genomic coding, modifiable exposure, and manifestations of disease phenotypes. DNA
methylation (DNAm), a major type of epigenetic modification, may be an important mechanism
underlying these relationships.
In this study, we measured the genome-wide DNAm status for DNA samples prepared from whole
blood of patients with myocardial infarction (MI), a type of CVD, attending hospitals in Japan and ofelderly Japanese participants of the Kita-Nagoya Genomic Epidemiology (KING) study. These samples
were also genotyped using an SNP array. We thereby assessed the relationships between DNAm sites
identified in our study and SNPs previously found to be associated with CVD in GWAS.

* Statistical Analysis Section, Center for Advanced Medicine and Clinical Research, Nagoya University Hospital,
65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan, E-mail: mnakatochi@med.nagoya-u.ac.jp, Tel:
+81-52-744-1957† Graduate School of Regional Innovation Studies, Mie University, Tsu 514-8507, Japan‡ Department of Medical Biochemistry, School of Medicine, Kurume University, Kurume 830-0011, Japan§ Department of Internal Medicine, School of Dentistry, Aichi Gakuin University, Nagoya 464-8561, Japan** Department of Genome Science, School of Dentistry, Aichi Gakuin University, Nagoya 464-8561, Japan

2 Methods
2.1 Study participantsThis was an exploratory study examining the association of DNAm status at various sites withCVD-associated SNPs. A total of 192 male cases with MI and 192 male controls without MI wereenrolled. All participants were at least 55 years old, and the two groups were matched by age (within 5years). Genome-wide DNAm profiles for DNA isolated from whole blood were obtained by analysis withan Infinium HumanMethylation450 BeadChip. The details of DNAm profile analysis were described
previously (Nakatochi et al., 2015 and Lehn et al., 2015).
2.2 Genotyping and imputing SNPs
All samples were also genotyped using an Illumina HumanOmniExpress-12 BeadChip. Quality control ofsamples and SNPs was performed. After the quality control process, we imputed genotypes using
SHAPEIT2 and minimac3 with data from the 1000 genomes project phase 3 as a reference panel. SNPs
with poor imputation quality (Rsq < 0.3) and low minor allele frequency (MAF < 0.01) were excluded. Of
SNPs that passed the postimputation process, 146 CVD-associated SNPs were registered in the GWAS
catalog database (https://www.ebi.ac.uk/gwas/). Furthermore, we used PLINK software to exclude SNPs
with a pairwise genotypic r2 of greater than 0.2 on the data of East Asian samples from the 1000 genomes
project 3. A total of 105 CVD-associated SNPs remained for further analysis.
2.3 Statistical analysis
To explore DNAm sites associated with 105 CVD-associated SNPs, association analysis was performed
between each DNAm site and each SNP in the case and control groups. A general linear model wasapplied for each combination of DNAm site and SNP; the dependent variable was DNAm status at eachsite, and independent variables included the genotype of each SNP and covariates. The covariates
included age, smoking status (not a current smoker = 0, current smoker = 1), the first 30 principal
component scores calculated from Infinium 450K assay control probes, the first five principal componentscores calculated from the residuals after adjustment for technical and biological factors, and the cell typecomposition of samples. We coded genotypes as 0, 1, or 2 on the basis of the number of minor alleles.
Next, the results from case and control groups were then combined into a fixed effects meta-analysis
with inverse variance weighting.
Finally, we choose DNAm sites suggestive significantly associated with CVD-related SNPs (p < 1 ×10-6), based on the results of the meta-analysis. We inspected gene symbols located near the significantly
associated DNAm sites. To identify significantly enriched pathways, the Database for Annotation,
Visualization and Integrated Discovery (DAVID, https://david-d.ncifcrf.gov/) was used.
For the association analysis between DNAm sites and SNPs, the genome wide significance level andsuggestive significance level were set to 1 × 10−8 and 1 × 10−6, respectively. For the DAVID analysis, afalse discovery rate (FDR) of less than 0.05 was considered significant. All statistical analyses wereperformed with the R project (version 3.3.0, www.r-project.org).
3 Results
The baseline characteristics of the study participants are shown in Table 1. The frequency of current
smokers was significantly higher in the case group than in the control group.
We performed genome-wide DNAm profiling for whole-blood DNA from 192 cases and 192 controls.
After initial processing, 188 cases and 190 controls, including 348,595 DNAm sites and 105
CVD-associated SNPs, remained for subsequent analysis. We initially performed an association analysis
between DNAm status at each site and each CVD-associated SNP. As a result, 431 combinations ofDNAm sites and SNPs showed suggestive significance. Of these combinations, 187 combinations showed
genome-wide significance. The combination of DNAm site and SNP located near the ABO gene showed
the most significant association (β = 1.330 ± 0.032, p < 1 × 10−99 in meta-analysis).
We identified 225 gene symbols located near the DNAm sites, which included the 431 combinations.
The top 10 gene ontology (GO) terms based on DAVID analysis are shown in Table 2. All of these GOterms, including GO:0071556, GO:0046978, and GO:0032395, were related to the 6p21 region.

4 Discussion and summary
In this study, we performed an association analysis of DNAm sites with CVD-related SNPs and found
431 significant combinations of DNAm sites and SNPs. The most significant combination was theDNAm site and SNP located near the ABO gene. This relationship with the ABO gene was previously
reported by Grunberg et al (2013). The ABO gene encodes proteins related to the first discovered bloodgroup system, ABO; thus, the result observed in our blood samples was consistent with their reports.
Furthermore, genes for which the DNAm sites had significant associations with CVD-related SNPs weresignificantly enriched in GO terms related to the 6p21 region. We have previously reported that theHLA-DQB1 SNP located on chromosome 6p21 is associated with MI (Takeuchi et al., 2012). The 6p21
region is known as the most polymorphic region in the human genome. Our results suggested that theseSNPs located on chromosome 6p21 may influence the development of CVD by affecting DNAm in blood
cells.

Table 1 Characteristics of the study participants
CharacteristicControls(n = 192)

Cases(n = 192)

pMale, n (%)

192 (100%)

192 (100%)

1.000

Age (years)

65.8 ± 6.0

65.9 ± 6.40.915

Current smoker, n (%)

43 (22.4%)

83 (43.2%)

2.01 × 10–5

Continuous data are means ± SDs. Differences in characteristics between cases and controls wereevaluated by Student’s t tests or Fisher’s exact tests.

Table 2 Top five GO terms based on DAVID analysis
Numberof genes

FDR

77.06 × 10–5

56.59 × 10–4

TAP1 binding

4

1.13 × 10–3

GO:0042605

Peptide antigen binding

6

1.28 × 10–3

GO:0042613

MHC class II protein complex

51.94 × 10–3RankGO ID

GO term1

GO:0071556

2

GO:0032395

Integral component of lumenal side ofthe endoplasmic reticulum membrane
MHC class II receptor activity

3

GO:0046978

45

Acknowledgments
This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports,Science, and Technology of Japan, including those for the Funding Program for Next Generation
World-Leading Researchers (NEXT Program) to S.I. (LS056), for Scientific Research (B) to M.Y.
(24390169 and 16H05250) and to T.M. (25293144), for Scientific Research (C) to K.Y. (15K08290), andfor Young Scientists (B) to M.N. (15 K19242).
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