Hee Nam Kim; Sun-Seog Kweon; Jin-Su Choi; Jung-Ae Rhee; Young-Hoon Lee; Hae-Sung
Nam; Seul-Ki Jeong; Kyeong-Soo Park; Seong-
Woo Choi; So-Yeon Ryu; Yong-Woon Yun; Min-Ho Shin

Research Article

J Dis Markers. 2015;2(3): 1028.

HDAC9 Polymorphism is Associated with Carotid Plaque in a Korean Population

Hee Nam Kim¹, Sun-Seog Kweon^2,3, Jin-Su Choi², Jung-Ae Rhee², Young-Hoon Lee⁴, Hae-Sung Nam⁵, Seul-Ki Jeong⁶, Kyeong-Soo Park⁷, Seong- Woo Choi⁸, So-Yeon Ryu⁸, Yong-Woon Yun⁹ and Min-Ho Shin^1,2*

¹Center for Creative Biomedical Scientists, Chonnam National University, Gwangju

²Department of Preventive Medicine, Chonnam National University Medical School, Gwangju, Korea

³Jeonnam Regional Cancer Center, Chonnam National University Hwasun, Hwasun Hospital, Korea

⁴Department of Preventive Medicine & Institute of Wonkwang Medical Science, Wonkwang University College of Medicine, Iksan, Korea

⁵Department of Preventive Medicine, Chungnam National University Medical School, Daejeon, Korea

⁶Department of Neurology & Research Institute of Clinical Medicine, Chobuk National University-Biomedical Institute of Chonbuk National University Hospital, Jeonju, Korea

⁷Department of Preventive Medicine, Seonam University College of Medicine, Namwon, Korea

⁸Department of Preventive Medicine, Chosun University Medical School, Gwangju, Korea

⁹Gwangju-Jeonnam Regional Cardiocerebrovascular Center, Chonnam National University Hospital, Gwangju, Korea

*Corresponding author: Min-Ho Shin, Department of Preventive Medicine, Chonnam National University Medical School, Dong-gu, South Korea

Received: July 02, 2015; Accepted: July 23, 2015; Published: July 27, 2015

Abstract

Background: An association between histone deactylase (HDAC) polymorphism and large vessel ischemic stroke was identified in two genomewide association studies. The aim of this study was to investigate the association between HDAC9rs 2107595 polymorphism and carotid atherosclerosis in Korean adults.

Methods and Findings: The study population consisted of 1954 subjects aged 50 years or more from the Dong-gu Study. Carotid ultrasonography was performed to measure common carotid (CCA) intima-media thickness (IMT) and the presence of carotid plaques. Genotyping was performed by high-resolution melting analysis. After adjustment for age, diabetes, and total cholesterol, carriers of AA and AG had a higher risk ofcarotid plaquethan the GG genotype (odds ratio [OR]AG= 1.38, 95%confidence interval [CI]=1.12-1.70; OR_AA = 1.46:1.05-2.02). After further adjustment forcovariates, ORs of AA and AG for carotid plaque were slightly increased (OR_AG= 1.44: 1.17-1.78; OR_AA = 1.57: 1.12-2.20). However, HDAC9 genotype was not associated with CCA-IMT.

Conclusions: We found that the HDAC9rs 2107595 polymorphism is associated with carotid plaque. These results suggest that the HDAC9 polymorphism may be an independent risk factor for cardiovascular disease.

Keywords: HDAC9; Polymorphism; Carotid artery plaque; Intima-media thickness

Introduction

Stroke is one of the most common causes of morbidity and mortality in the world [1, 2]. In Korea, it is the third common cause of death, after cancer and heart disease [2]. Strokes are classified as is chemic or hemorrhagic, with more than 80% of them being ischemic [3]. Animal models and twin studies have suggested a significant genetic component in ischemic stroke [4]. Many candidate gene studies and several genome-wide association studies (GWAS) have been performed to identify common genetic variants [5-9]. However, these results are inconsistent. Polymorphisms of the β-fibrinogen (FGβ) and phosphodiesterase 4 (PDE4D) genes were associated with the risk of ischemic stroke [7, 8, 10], which is a finding thatwas not confirmed in other studies [9, 11]. Carotid intima-media thickness (IMT) is used to detect atherosclerotic disease and isarisk factor for cardiovascular disease (CVD) and stroke [12-16]. Genetic studies have shown that several genes were associated with carotid IMT [17- 19].

Two genome-wide association study (GWAS) identified an association between two single nucleotide polymorphisms (SNPs) (rs11984041 and rs2107595) in histone deactylase 9 (HDAC9) genes and large vessel ischemic stroke [5, 6]. The underlying mechanism of this association remains unclear. HDAC genes encode proteins that deacetylate nucleosomal histones and alter the chromatin structure to affect the gene transcription factor [20]. HDAC9, one of the class II HDACs, is ubiquitously expressed in the brain, skeletal muscles and cardiac tissue. Additionally, it is expressed in healthy human cerebral and systemic arteries and is upregulated in human atherosclerotic plaques in various arteries [21]. To explore the mechanisms underlying the 7p21.1 association with large artery stroke, a metaanalysis demonstrated thatthese two SNPswere also associated with common carotid artery intima-media thickness (CCA-IMT) and with the presence of carotid plaques, as well [21].

The aim of this study was to evaluate the association between the HDAC9 polymorphism and carotid atherosclerosis in Korean adults. This finding has not been replicated in Asian populations.

Methods

Subjects

The Dong-gu Study is an ongoing prospective study designed to investigate the prevalence, incidence and risk factors for chronic diseases in urban populations. The details ofthe study subjects and measurements have been published previously [22]. In the Dong-gu Study, 9260 subjects aged 50 years and older were enrolled between 2007 and 2010 in the Dong-gu district of Gwangju Metropolitan City in South Korea. In total, 2000 subjects were randomly selected from the cohort of the Dong-gu Study cohort, with stratification by sex. Of the 2000 randomly selected subjects, 46 subjects were excluded because of missing data for carotid ultrasonography, blood lipid tests and life-style. Total 1954participants (978 men and 976 women) were included in the final analyses.

Ethics statement

This study was approved by the Institutional Review Board of Chonnam National University Hospital, and written informed consent was obtained from each subject.

HDAC9 genotyping

Genomic DNA was extracted from peripheral blood with a QIA amp DNA Mini Kit (Qiagen Inc., Chatsworth, CA, USA) according to the manufacturer’s protocol. Because rs11984041 is not polymorphic in the Asian population in the Hap Map project, only rs2107595 genotyping was performed by high-resolution melting analysis as described previously [23]. The primer pair was F: 5’-TTTTGTGTGCTTGTACATTCTTTTT-3’ and R: 5’-ACTCATTGAGAAGGATGAGGAG-3’. The cycling conditions were 5min at 95 °C, followed by 40 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s. In the melting analysis, the temperature was increased from 70 to 81 °C at a rate of 0.1 °C per second.

Carotid ultrasound

Details of the ultrasound methods were published previously [24]. Carotid ultrasonography was performed using ahigh-resolution mode B ultrasound system (SONOACE 9900, Medison, Korea) with an electrical linear array transducer (7.5 MHz). The CCA-IMTwas determined as the average of the maximum IMT values for the left and right CCAs. The presence of carotid plaques was defined as focal structures that encroached into the lumen by at least 100% of the surrounding IMT value.

Other clinical variables

Smoking status was classified as non-smoker or former smoker versus current smoker. The body mass index (BMI) was calculated by dividing the weight (in kilograms) by the height squared (in meters squared). Diabetes was defined as having a fasting glucose level >126 mg/dl (7 mmol/l) or use of hypoglycemic medication. Hypertension was defined by systolic blood pressure ≥ 140 mm Hg or diastolic blood pressure ≥90 mmHg, or using of antihypertensive medication. Blood samples were drawn from an antecubital vein in the morning after a 12-h overnight fast. Serum total cholesterol, high-density lipoprotein (HDL) -cholesterol, triglyceride and fasting blood glucose levels were measured by enzymatic methods using an automatic analyzer (model 7600 chemical analyzer; Hitachi Ltd, Tokyo, Japan).

Statistical analysis

The data are presented as the means ± standard deviation (SD), or as percentages for the categorical variables. Analysis of variance and Pearson’s chi-square test were used to compare the baseline characteristics across the HDAC9 genotypes. Multiple linear regression and logistic regression modelswere used to evaluate the associations of HDAC9 genotype with CCA-IMT and carotid plaque formation. The analyses were adjusted for age, diabetes, and total cholesterol with a P-value of less than 0.2 in association with HADC9 genotype. In addition, we conducted a stratified analysis by age to assess effect modification by age (50-64 years and ≥65 years). Statistical analyses were performed using SPSS version 21.0 (SPSS, Inc., an IBM Company, Chicago, Illinois, USA). The significance level was set at a P value of <0.05.

Results

The HDAC9 genotype frequencies were 47.0% for GG, 42.7% for AG and 10.3% for AA. The overall mean age was 65.2±8.1years. The HDAC9 genotype frequencies were consistent with Hardy-Weinberg equilibrium (p= 0.55). There were no significant differences in the proportion of males, smoking, hypertension, and diabetes, serum HDL-cholesterollevel, and triglyceride levelsexcept for total cholesterol level across the HDAC9 genotypes (Table 1).

Table 1: Characteristics of the study subjects according to HDAC9 genotype.




  
    
    GG
    AG
    AA
    p-value
  
  
    N
    918
    835
    201
     
  
  
    Age (years)
    64.8 ± 8.3
    65.7 ± 7.9
    64.9 ± 8.0
    0.060
  
  
    Men (%)
    442 (48.1)
    432 (51.7)
    104 (51.7)
    0.285
  
  
    Body mass index    (kg/m²)
    24.3 ± 2.8
    24.3 ± 2.9
    24.4 ± 3.0
    0.906
  
  
    Current smoking (%)
    115 (12.5)
    108 (12.9)
    31 (15.4)
    0.541
  
  
    Hypertension (%)
    427 (46.5)
    385 (46.1)
    85 (42.3)
    0.546
  
  
    Diabetes mellitus    (%)
    204 (22.2)
    150(18.0)
    37 (18.4)
    0.070
  
  
    Total cholesterol    (mg/dl)
    201.1 ± 40.9
    197.4 ± 38.3
    194.3 ± 34.8
    0.036
  
  
    HDL cholesterol    (mg/dl)
    51.9 ± 12.2
    51.4 ± 12.2
    50.5 ± 12.2
    0.307
  
  
    Triglyceride    (mg/dl)
    140.1 ± 120.0
    142.6 ± 96.7
    137.8 ± 84.5
    0.808
  
  
    CCA-IMT (mm) 
    0.721 ± 0.155 
    0.727 ± 0.150 
    0.734 ± 0.164 
    0.488 
  
  
    Carotid plaque (%) 
    309 (33.7) 
    353 (42.3) 
    85 (42.3) 
    <0.001 
  
  
    Values are mean ± SD or number    (percentage). 
      HDL, high-density lipoprotein;    CCA-IMT, common    carotid artery intima-media thickness.



Table 1:  Characteristics of the study subjects according to HDAC9 genotype.

HDAC9 polymorphism was not associated with CCA-IMT (Table 2). However, there was a significant association between HDAC9 polymorphism and carotid plaque formation. The carriers of AA and AG had higher risk for carotid plaque formation than the GG genotype (odds ratio [OR]AG= 1.44, 95% confidence interval [CI] = 1.19-1.75; OR_AA = 1.44, 95% CI = 1.06-1.97, respectively). After adjustment forage, diabetes, and total cholesterol, ORs for carotid plaque were slightly increased of AA and AG (OR_AG = 1.43, 95% CI = 1.17-1.75; OR_AA = 1.52, 95% CI = 1.10-2.10, respectively) (Table 3). In a stratified analysis by age, although there was no significant interaction by age (p=0.332), this association was more evident in the younger group (50-64 years) than in the elderly (≥65 years) (Table 4).

Table 2: Association of HDAC9 genotype with intima-media thickness.




  
    Genotype
    Model I
    Model II
  
  
    GG
    0.725 ± 0.005
    0.723 ± 0.005
  
  
    AG
    0.723 ± 0.005
    0.725 ± 0.005
  
  
    AA
    0.736 ± 0.010
    0.737 ± 0.010
  
  
    P for trend 
    0.522
    0.301
  
  
     
      Data are presented as mean ± standard    error. 
      Model I,    adjusted age and    sex. 
      Model II, further adjusted for BMI,    smoking, diabetes, hypertension, total cholesterol, HDL cholesterol, and    log-transformed triglycerides.



Table 2:  Association of HDAC9 genotype with intima-media thickness.

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Citation:Kim HN, Kweon S-S, Choi J-S, Rhee J-A, Lee Y-H, Nam H-S, et al. HDAC9 Polymorphism is Associated with Carotid Plaque in a Korean Population. J Dis Markers. 2015;2(3): 1028. ISSN : 2380-0682

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Genotype	Model I	Model II
GG	1 (reference)	1 (reference)
AG	1.38 (1.13-1.70)	1.44 (1.17-1.77)
AA	1.45 (1.05-2.02)	1.56 (1.12-2.18)
*P for trend*	0.002	<0.001

Genotype	<65 years	≥ 65 years	Pfor interaction
GG	1 (reference)	1 (reference)	0.332
AG	1.59 (1.17-2.17)	1.31 (1.00-1.72)
AA	1.96 (1.22-3.14)	1.22 (0.79-1.90)
Pfor trend	0.001	0.102
Data are presented as mean ± standard error. Model I, adjusted age and sex. Model II, further adjusted for BMI, smoking, diabetes, hypertension, total cholesterol, HDL cholesterol, and log-transformed triglycerides. Adjusted for age, diabetes, and total cholesterol.*