Int J Clin Exp Pathol 2016;9(8):8575-8585 www.ijcep.com /ISSN:1936-2625/IJCEP0026579 Original Article Genetic polymorphisms of INSIG2 were associated with coronary artery disease in Uygur Chinese population in Xinjiang, China Dilare Adi1,2*, Yun Wu3*, Xiang Xie1,2, Gulinaer Baituola1,2, Fen Liu2, Ying-Ying Zheng1,2, Yi-Ning Yang1,2, Xiao-Mei Li1,2, Ding Huang1,2, Xiang Ma1,2, Bang-Dang Chen2, Min-Tao Gai2, Xiao-Cui Chen2, Zhen-Yan Fu1,2, Yi-Tong Ma1,2 1Department of Cardiology, First Affiliated Hospital of Xinjiang Medical University, Urumqi, People’s Republic of China; 2Xinjiang Key Laboratory of Cardiovascular Disease Research, Urumqi, People’s Republic of China; 3Department of General Medicine, First Affiliated Hospital of Xinjiang Medical University, Urumqi, People’s Republic of China. *Equal contributors. Received February 24, 2016; Accepted May 21, 2016; Epub August 1, 2016; Published August 15, 2016 Abstract: Background: Dyslipidemia is a major and independent risk factor for the development of Coronary artery disease (CAD). The protein which is encoded by insulin induced gene2 (INSIG2) plays an important role in the media- tion of the feedback control of cholesterol synthesis, lipogenesis and glucose homeostasis. The aim of the present study was to assess the association between the human INSIG2 gene and CAD in Han Chinese and Uygur Chinese population of Xinjiang, China. Methods: A total of 832 CAD patients (334 Han, 498 Uygur) and 919 controls (346 Han, 573Uygur) were selected for the present Case-control study. Three tagging SNPs (rs1261829, rs21613329 and rs17047757) of INSIG2 gene were genotyped using TaqMan® assays from Applied Biosystems following the manufacturer’s instructions and analyzed in an ABI 7900HT Fast Real-Time PCR System. Results: In the Uygur Chinese population, for total, men and women the rs17047757 was associated with CAD by analyses of a domi- nant model (all, P < 0.001) and the difference remained significant after multiple adjustment in a dominant model (all, P < 0.001). This relationship was also observed in rs2161829 for total and women by analyses of a recessive model (for total: P = 0.002; for women: P = 0.001, respectively) the difference remained significant after multiple adjustment in a recessive model (for both, P = 0.001). However, this relationship was not observed in this three tagging SNPs before and after multiple adjustment in Han Chinese population. Conclusion: Our results indicated that both rs17047757 and rs21613329 in the INSIG2 gene were associated with CAD in Uygur Chinese population in Xinjiang, China. Keywords: Genetics, INSIG2 gene, single nucleotide polymorphism, coronary artery disease, case-control study Introduction Cholesterol is essential component of mamma- lian cell membranes and it plays important Coronary artery disease (CAD) is one of the roles in the biosynthesis of steroid hormones leading causes of disability and mortality world- and the maintenance of membrane integrity wide [1], the etiology and pathogenesis of CAD [5]. Whole-body cholesterol homeostasis refle- are that of a multi-factorial disorder that results cts a balance between dietary uptake, endoge- from both genetic and environmental risk fac- nous synthesis, reverse cholesterol transport tors. Dyslipidemia is a major and independent and removal from the body via biliary and intes- risk factor for the development of CAD and tinal excretion. There are several genes such as accounts for approximatively 50% of CAD cases insulin induced gene (INSIG1 and INSIG2) that in the population [2, 3]. Accumulated eviden- are involved in the feedback control of lipid syn- ces suggest that heritable factors range from thesis at the transcriptional levels. INSIG is not 40%~60% for the variation in concentration only one of the endoplasmic reticulum proteins and components of the plasma lipids [4]. (ER), but also a kind of oxysterol-binding pro- INSIG2 gene and coronary artery disease teins [6, 7] and plays an important role in the Declaration of Helsinki. Written informed con- mediation of the feedback control of cholester- sent was obtained from each participant for ol synthesis, lipogenesis, glucose homeostasis collection and analysis of relevant clinical data. [5, 8]. Studies conducted by Yabe D et al in vitro showed that when sterols are present in the Subjects cell ,INSIG2 blocks further cholesterol synthe- sis [6]; and studies in vivo also have demon- A total of 1451 unrelated Han Chinese and strated that over expression or down regulation Uygur Chinese subjects (680 Han, 771 Uygur) of INSIG2 could significantly affect Cholesterol who lived in Xinjiang Uygur Autonomous Region homeostasis and body weight of the animals of China were included in this study. We recruit- [9, 10]. Krapivner et al showed that INSIG2 is ed 832 cases (334 Han, 498 Uygur) with CAD also expressed in adipocytes and this expres- from The First Affiliated Hospital of Xinjiang Medical University between January 2009 and sion involved in adipocyte metabolism and October 2013. Patients underwent coronary body weight regulation [11]. angiography and diagnosed with CAD based on Human INSIG2 is a ~21.5 Kb gene was identi- the evidence of at least > 50% stenos is in one fied by Yabe et al and mapped on the long arm major coronary artery. Patients with congenital of chromosome 2, localized to band p14.1, and heart disease, cardiomyopathy, valvular dis- contains 225 amino acids [12]. Since Herbert ease and multiple organ failure syndrome were et al [13] discovered in a genome-wide associa- excluded from this group. A total of 919 control tion study that genetic variation of rs7566605 subjects (346 Han, 573 Uygur) were randomly in the upstream of the INSIG2 gene associat- selected from the Cardiovascular Risk Survey ed with BMI, a several studies have explored (CRS) in Xinjiang, northwest of China. The genetic polymorphisms of INSIG2 gene with detailed description of the study population related metabolic traits and CAD, but studies and the methods were described previously of the association between genetic polymor- [14, 15]. Briefly, the CRS consisted of 14,618 phisms of the INSIG2 gene and cardiovascular subjects (5,757 Hans, 4,767 Uygurs and 4,094 disease in diverse ethnicities remain controver- Kazakhs) and was a multiple-ethnic, communi- sial. Several studies have found that genetic ty-based, cross-sectional study was designed polymorphisms of INSIG2 is not only associat- to investigate the prevalence and risk factors ed with CAD but also related to the major risk for cardiovascular diseases and to determine factors of CAD, namely, overweight, obesity, the genetic and environmental contributions to hypercholesterolemia, diabetes while others atherosclerosis, CAD, and cerebral infarction of the Chinese Han, Uygur, and Kazakh popula- have suggested that genetic polymorphisms of tions in the Xinjiang northwest of China from INSIG2 was not associated with CAD or the risk October 2007 to March 2010. Individuals with factors of the CAD. However, the relationship myocardial infarction, CAD, coronary stenting, between genetic polymorphisms of the INSIG2 multiple organ failure syndrome, and those gene and CAD in Han and Uygur Chinese popu- whose data were incomplete were excluded lation of Xinjiang Uygur Autonomous Region from control group. Data and information about northeast of China is remains unknown. traditional risk factors of CAD (including hyper- The aim of the present study is to determine tension, diabetes mellitus, and dislipidemia) the relationship between genetic polymorphism were collected from all participants. Hyperten- of INSIG2 gene and coronary artery disease in sion was defined as systolic blood pressure Han Chinese and Uygur Chinese population of (SBP) ≥ 140 mmHg and/or diastolic blood pres- China. sure (DBP) ≥ 90 mmHg, and/or taking antihy- pertensive medication. Diabetes mellitus was Material and methods defined on the basis of the World Health Organization (WHO) criteria (fasting plasma glu- Ethical approval cose level ≥ 7.0 mM and/or self-reported cur- rent treatment with anti-diabetes medication). This study was approved by the ethics commit- Hyperlipidemia was defined as a total plasma tee of the First Affiliated Hospital of Xinjiang cholesterol > 6.22 mmol or plasma triglycer- Medical University (Xinjiang, China) and was ides > 2.26 mmol and/or the current use of conducted according to the standards of the lipid-lowering drugs [16]. 8576 Int J Clin Exp Pathol 2016;9(8):8575-8585 INSIG2 gene and coronary artery disease Genotyping in this case-con- trol study was performed by using an Applied Biosystems (ABI, Foster City, CA) TaqMan 7900 system. SNPs primers and probes were provided by ABI Assay-on-demand (http:// myscience.appliedbiosyste- ms.com). Thermal cycling was performed using the Applied Biosystems 7900HT Fast Re- al-Time PCR system (Applied Biosystems). PCR amplifica- Figure 1. Structure of the human INSIG2 gene. The gene consists of seven tion was performed using 2.5 exons (boxes) separated by six introns (lines; intergenic regions). Arrows indi- μL of TaqMan Universal Ma- cate the locations of single-nucleotide polymorphisms (SNPs). kbp, kilobase ster Mix, No AmpErase UNG pairs. (2×) (Applied Biosystems) in a 5 μL final reaction volume, Anthropometric and biochemical variables along with 2 ng DNA, 2.375 μL ultrapure water, measurement 0.079 μL Tris-EDTA (TE) buffer (1×), 0.046 μL TaqMan SNP Genotyping Assay Mix (40×) con- Weight and height were measured in a stan- taining a 331.2 nmol/L final concentration of dard method, and body mass index (BMI) was primers and a 73.6 nmol/L final concentration calculated. After 5 min of rest, blood pressure of the probes. The thermal cycling conditions was measured three times within 10 min and were as follows: 50°C for 2 min; 95°C for 10 the median value was used in the statistical min; 50 cycles of 95°C for 15 s; and 60°C for 1 analysis. Smoking and drinking was self-report- min.