Introduction

High-density lipoprotein cholesterol (HDL-C) levels are associated with decreased risk of cardiovascular disease (CVD).1, 2, 3 Several genome-wide association studies (GWASs) that have examined HDL-C levels reported that the myosin light chain 2 regulatory cardiac slow (MYL2; MIM 160781) gene is a candidate causal gene.4, 5 The MYL2 gene has also been linked with multiple CVDs.6, 7, 8 Continental Asian populations tend to have lower levels of serum cholesterol than those of Europeans.9, 10 Recent GWASs in Asian populations have reported that MYL2 single-nucleotide polymorphisms (SNPs) are strongly associated with HDL-C levels.5 Another recent GWAS on coronary artery disease reported that the rs3782889 SNP in MYL2 is strongly associated with HDL-C level.8 However, they did not analyze the association between rs3782889 and HDL-C in subjects with other risk factors, such as smoking status and body mass index (BMI).

MYL2 regulates myosin ATPase activity in smooth muscle.11, 12 In this study, the association between HDL-C level and the rs3782889 MYL2 SNP was analyzed in a sample of Korean volunteers. The association was also analyzed considering BMI and smoking status.

Materials and methods

Study population

A total of 4294 subjects were the participants who had routine health examinations in the Health Promotion Center in University Hospitals from 1994 to 2012. Among 4294 subjects, 1810 subjects were CVD cases identified by the health insurance reimbursement data from the NHIC. CVD was defined according to the codes of the International classification of Disease (ICD), 10th Revision (I00-I99). In total, 137 subjects who were undergoing lipid-lowering therapy were excluded, and other subjects were excluded owing to missing BMI and HDL-C level data. Thus, the final population included 4025 subjects. Among 4025 subjects, 2403 subjects were the healthy subjects. Other 1622 subjects were CVD patients. The Institutional Review Board of Human Research of Yonsei University approved the protocols for this study, and written informed consent was obtained from all the subjects before enrollment.

Data collection

Each participant was interviewed using a structured questionnaire to collect their personal history of cigarette smoking (never smoked, ex-smoker or current smoker) and demographic characteristics (age, sex and so on). Waist circumference was measured midway between the lower rib and iliac crest. Light clothing was worn to measure weight and height. BMI was calculated as the subject’s weight (kg) divided by the square of the subject’s height (m2).

Serum was separated from peripheral venous blood samples obtained from each participant after a 12-h fast and stored at −70 °C until clinical chemistry assays. Biomarkers of metabolic syndrome, including fasting blood glucose, total cholesterol, triglycerides and HDL-C, were measured with a Hitachi-7600 analyzer (Hitachi, Tokyo, Japan). Quality control was conducted in accordance with the procedures recommended by the Korean Association of Laboratory Quality Control.

Genotyping assays

The rs3782889 MYL2 gene SNP was genotyped via the TaqMan reaction.13 Duplicate genotyping for 1–2.5% of all samples was carried out as a quality control measure. Only those SNPs with a concordance rate >99% in duplicates and a genotype success rate >98% were included in subsequent association analyses.

Statistical analysis

Data are expressed as means±s.d. Most statistical analyses were performed using PLINK and SAS ver. 9.2 (SAS Institute, Cary, NC, USA). Each SNP was tested for possible effects on HDL-C level under an additive model. The multivariate linear regression models used in the study incorporated covariates (age, sex and BMI). Multiple logistic regression analysis was also performed. BMI and waist circumference were divided by quartiles. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to examine the association between the MYL2 SNP and abnormal HDL-C levels (<40 mg dl−1 for men and <50 mg dl−1 for women; NCEP ATP III). All statistical tests were two-sided, and P<0.05 was considered significant.

Results

Mean age was 51.7 for male subjects and 52.3 for female subjects (Table 1). This sample of Korean volunteers had a low mean HDL-C level. Mean HDL-C level was lower in males (48.2 mg dl−1) than that in females (56.7 mg dl−1). About 14.5% of the subjects had abnormal HDL-C levels (<40 mg dl−1). Of the sample data set, 38.2% of men and 3.7% of women were current smokers. And 42.9% of men and 34.7% of women were CVD patients.

Table 1 General characteristics of the study population
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Table 2 shows the P-values from a linear regression model for HDL-C levels in the cohort sample when age, sex and BMI were included as covariates. The rs3782889 SNP in the MYL2 gene was associated with mean HDL-C level (effect per allele, −1.055 mg dl−1, P=0.0005). When analyzed in healthy subjects (N=2403), the association was stronger (effect per allele, −1.472 mg dl−1, P=0.0003). The rs3782889 SNP in the MYL2 gene was also associated with mean GGT level (effect per allele, −5.525 mg dl−1, P<0.0001).

Table 2 Association between the rs3782889 single-nucleotide polymorphism in the MYL2 gene and lipid levels based on a linear regression model
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The association between the MYL2 gene SNP rs3782889 and abnormal HDL-C level was also examined (Table 3). Subjects with the CT/CC genotype had a 1.43-fold (range, 1.19–1.73-fold) higher risk of an abnormal HDL-C level (<40 mg dl−1) than those with the TT genotype. When analyzed by sex, the association between MYL2 and abnormal HDL-C level was stronger in men than that in women.

Table 3 Odds ratios (ORs) of the polymorphic rs3782889 MYL2 genotypes for HDL-cholesterol levelsa in the population (n=4025)
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The analysis by quartiles of BMI in male subjects was presented in Table 4. The association between MYL2 and HDL-C level was much stronger in male subjects with BMI ⩾26.44 kg m−2 (OR=2.68; 95% CI=1.87–3.84, P<0.0001) than that in male subjects with BMI <26.44 kg m−2. In the analysis by quartiles of waist circumference in male subjects, the association between MYL2 and HDL-C level was much stronger in male subjects with WC ⩾92 cm (OR=2.36; 95% CI=1.57–3.56, P<0.0001) than that in male subjects with WC <92 cm. The association between MYL2 and HDL-C level was stronger in current smokers (P=0.0362) and ex-smokers (P=0.0159) than that in nonsmokers (P=0.7304).

Table 4 Odds ratios (OR) of polymorphic rs3782889 MYL2 genotypes for HDL-cholesterol levelsa in Korean men (n=2744)
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Table 5 indicates age-adjusted ORs for abnormal HDL cholesterol levels according to MYL2 (rs3782889) genotype in strata of BMI, waist circumference and smoking status among Korean men. When compared with subjects having the TT genotype and BMI <26.44 kg m−2, ORs (95% CI) were 3.30 (2.41–4.50) in subjects having the CT/CC genotype and BMI ⩾26.44 kg m−2 (P for interaction <0.0001). With regard to waist circumference, ORs (95% CI) were 2.89 (2.02–4.14) for subjects with the CT/CC genotype and WC ⩾92 cm compared with subjects with the TT genotype and WC <92 cm (P for interaction=0.0060).

Table 5 Age-adjusted odds ratios (ORs) for abnormal HDL-cholesterol levelsa according to MYL2 (rs3782889) genotypes in strata of BMI, waist circumference and smoking status in Korean men (n=2744)
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Discussion

In a cohort of 4025 subjects, the rs3782889 SNP in the MYL2 gene was associated with decreased HDL-C level, consistent with previous studies. In a recent GWAS of coronary artery disease, the rs3782889 SNP was strongly associated (P=3.95 × 10−14).8 Another SNP in the 12q24 region, rs12229654 near the MYL2 gene, is associated with HDL-C (P=3.41 × 10−23), hypertension (P=1.79 × 10−8), levels of gamma glutamyl transpeptidase (P=8.76 × 10−58) and plasma glucose levels in KARE study subjects.4, 5, 14 However, a more moderate association with the SNP was observed herein. One possible reason for the difference in the result is that mean HDL-C levels in the present study were much higher than those in the KARE subjects. In recent GWAS, the MYL2 gene was also associated with type 1 diabetes mellitus and clinically defined gout.15, 16

In the present study, we found that MYL2 SNP had a stronger association with HDL-C cholesterol levels in men than that in women, which is similar to several previous studies on the MYL2 gene. A recent GWAS study reported a male-specific association between the rs3782889 SNP in the MYL2 gene and hypertension.4 In another recent study, the association between BMI and MYL2 gene SNP was significantly stronger among men than among women.17

Several studies have reported that HDL-C level is regulated by environmental or lifestyle risk factors, such as obesity and smoking.18, 19 A recent GWAS identified a novel BMI-associated locus near MYL2 (rs12229654) gene.17 In the present study, the rs3782889 SNP in the MYL2 gene was still associated with mean HDL-C level even when adjusted by BMI. We also found that the association between MYL2 (rs3782889) and HDL cholesterol levels can be modified by obesity among Korean men. However, the underlying mechanism has been still unknown. In a study of young type 2 Zucker diabetic fatty rats, some cardiac muscle proteins (Myh6 and Myl2) were downregulated in Zucker diabetic fatty heart compared with control heart.20 In a cohort of MYL2 p.(E22K) founder mutation carriers, the presence of an additional risk factor for hypertrophy such as hypertension and obesity increased the disease penetrance of hypertrophic cardiomyopathy (P=0.0005; combined OR=39.47).21 In the present study, ORs (95% CI) for CVDs were 3.30 (2.41–4.50, P=0.0027) in subjects having the CT/CC genotype and BMI ⩾26.44 kg m−2 compared with subjects having the TT genotype and BMI <26.44 kg m−2 (data not shown). We also examined the association between the MYL2 SNP and HDL-C by smoking status. The association was slightly stronger in current smokers than in nonsmokers. To our knowledge, there are no previous studies that evaluated modification of the association of MYL2 and HDL cholesterol by lifestyle risk factors, such as obesity and smoking, among Korean men.

MYL2 encodes the myosin light chain and is involved in heart morphogenesis. Downregulation of MYL2 may have a role in coronary artery disease.11, 12, 22, 23 MYL2 also mediates the AMP-activated protein kinase pathway, which regulates energy homeostasis in eukaryotes.24 The SNP rs3782889 is located in intron 1 of the MYL2 gene. Differences in the frequency of rs3782889 have been reported in different populations. The C allele frequency is 7.2% in Europeans, and is lower in sub-Saharan Africans (5.3%) and Mexican Americans (1.0%). The frequency in East Asians was 20.9% in Han Chinese in Beijing and 19.2% in Japanese according to HapMap data (NCBI website). In the present study, we found a C allele frequency of 18.5%.

Genetic studies of lipid levels in Asian populations may not necessarily identify the same set of susceptibility genes as those in European-derived populations. However, this Korean cohort showed that the MYL2 gene on chromosome 12 is associated with serum HDL-C levels in Korean men. The association was much stronger in male obese subjects and smokers than that in leaner nonsmoking male subjects.