Journal of Diabetes and Its Complications 33 (2019) 178–181 Contents lists available at ScienceDirect Journal of Diabetes and Its Complications journal homepage: WWW.JDCJOURNAL.COM Association of the genetic variant rs2000999 with haptoglobin and diabetic macrovascular diseases in Chinese patients with type 2 diabetes Shiyun Wang a,1,RongZhanga,1,TaoWanga,FengJianga,ChengHua,b,⁎, Weiping Jia a,⁎ a Shanghai Diabetes Institute, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Clinical Center for Diabetes, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai 200233, People's Republic of China b Institute for Metabolic Disease, Fengxian Central Hospital Affiliated to Southern Medical University, 6600 Nanfeng Road, Shanghai 201499, People's Republic of China article info abstract Article history: Aims: The common copy number variant (CNV) in the haptoglobin (Hp) gene may influence the susceptibility to Received 21 November 2017 diabetic macrovascular diseases. We aimed to investigate the relationship of the genetic variant rs2000999, lo- Received in revised form 17 April 2018 cated in the haptoglobin-related protein (HPR) gene, with serum Hp levels and diabetic macrovascular diseases Accepted 7 October 2018 in Chinese type 2 diabetes patients. Available online 11 October 2018 Methods: The Hp CNV and rs2000999 were genotyped in a group of 5457 Chinese patients with type 2 diabetes. Associations of rs2000999 with the common Hp CNV, susceptibility to diabetic macrovascular diseases and re- Keywords: lated metabolic traits were analysed. Furthermore, 886 patients were selected to detect serum Hp levels and to Haptoglobin Copy number variant evaluate the correlation between rs2000999 and serum Hp levels. rs2000999 Results: The genetic variant rs2000999 was not associated with diabetic macrovascular diseases (P = 0.6109), Type 2 diabetes while subjects carrying the A allele had higher levels of low-density lipoprotein cholesterol (P = 0.0578) and a Chinese patients smaller inter-adventitial diameter of the common carotid artery (P = 0.0266). Additionally, rs2000999 exhibited strong association with serum Hp levels (P = 2.03 × 10−21). Conclusions: The genetic variant rs2000999 was not associated with diabetic macrovascular diseases but showed an association with metabolic traits and serum Hp levels in Chinese patients with type 2 diabetes. © 2018 Elsevier Inc. All rights reserved. 1. Introduction polymorphism (SNP) rs2000999 located in the haptoglobin-related protein (HPR) gene has been identified to influence serum cholesterol Type 2 diabetes mellitus (T2DM) remains a concurrent public health levels in the general European population.6 The haptoglobin (Hp) problem worldwide, with considerably increased risk of coronary heart gene, located adjacent to the HPR gene, encodes the Hp protein, which disease, stroke, and peripheral arterial diseases in affected patients.1 As binds haemoglobin (Hb) and prevent Hb-induced oxidative tissue diabetic macrovascular diseases are the major causes of morbidity and damage.7,8 The common copy number variant (CNV) in the Hp gene mortality in patients with T2DM, the risk factors contributing to the ac- (Hp1-1, Hp2-1 and Hp2-2) was reported as an independent risk factor celerated atherosclerosis have long been a major focus.2 The roles of ab- for cardiovascular disease in T2DM.9,10 Furthermore, the results of an- normal lipid metabolism, including the reduction of high-density other GWAS demonstrated that SNP rs2000999 was a strong genetic de- lipoprotein cholesterol (HDL-C) levels and increasing low-density lipo- terminant of serum Hp levels in children with European ancestry.11 protein cholesterol (LDL-C) levels, in diabetic macrovascular diseases However, it is unknown whether SNP rs2000999 was correlated with have been fiercely debated.3 diabetic macrovascular diseases through the influence of circulating In recent years, genome-wide association studies (GWAS) have dis- Hp levels or serum lipids in Chinese populations. covered several loci that are genetic determinants of dyslipidaemia, Therefore, in the present study, we investigated the relationship of with relevance for diabetes and atherosclerosis.4,5 A single nucleotide SNP rs2000999 with circulating Hp levels, lipid profiles and diabetic macrovascular diseases in Chinese patients with type 2 diabetes. Duality of interest: The authors have no conflicts of interest. ⁎ Corresponding authors at: Shanghai Diabetes Institute, Shanghai Key Laboratory of Di- 2. Methods abetes Mellitus, Shanghai Clinical Center for Diabetes, Shanghai Jiao Tong University Affil- iated Sixth People's Hospital, 600 Yishan Road, Shanghai 200233, People's Republic of 2.1. Ethical approval China. E-mail addresses: [email protected] (S. Wang), [email protected] (C. Hu), [email protected] (W. Jia). Ethical approval of this research was granted by the institutional 1 These authors contributed equally to this work. review board of Shanghai Jiao Tong University Affiliated Sixth People's https://doi.org/10.1016/j.jdiacomp.2018.10.006 1056-8727/© 2018 Elsevier Inc. All rights reserved. S. Wang et al. / Journal of Diabetes and Its Complications 33 (2019) 178–181 179 Hospital according to the Helsinki Declaration II. We obtained oral and diabetic macrovascular disease-related quantitative traits, clinical traits written informed consent from all patients. and serum Hp levels were preformed using multiply linear regression under additive models. 2.2. Participants 3. Results A total of 5457 type 2 diabetes patients, including 2953 males and 2504 females from the Shanghai Diabetes Institute Inpatient Database 3.1. Clinical characteristics of Shanghai Jiao Tong University Affiliated Sixth People's Hospital were enrolled in the present study from May 2013 to December 2015. The genotyping results of Hp CNV were as follows: 6.60% Hp1-1 12 Type 2 diabetes was diagnosed according to the 1999 WHO criteria. (N = 360), 35.75% Hp2-1 (N = 1951), 50.10% Hp2-2 (N = 2734), All populations underwent a series of vascular examinations, including 2.36% Hp1-del (N = 129), 5.19% Hp2-del (N = 283). Considering the in- ultrasound of the bilateral common carotid artery to measure intima- terruption of the Hpdel allele, we further examined those patients with media thickness (IMT), inter-adventitial diameter (IAD) and bilateral common Hp CNVs (N = 5045). The genotypes of rs2000999 were in lower limb arteries and computed tomography or magnetic resonance Hardy-Weinberg equilibrium (P = 0.0777) and distributed as follows: imaging to evaluate cerebrovascular diseases. Diabetic macrovascular AA, 7.51% (N = 379); AG, 38.00% (N = 1917); and GG, 54.49% (N = diseases were diagnosed by the evidence of atherosclerosis from 2749). After further analysis, rs2000999 A was in low linkage disequilib- cerebrovascular, carotid arteries, myocardial infarction and lower limb rium with Hp2 in patients without Hpdel (|D′|=0.93,r2 =0.11). 13 arteries. The clinical characteristics of the 5045 patients grouped by rs2000999 genotypes are shown in Table 1.Therewasasignificant difference in sys- 2.3. Clinical measurements tolic blood pressure (P = 0.0315) among different genotypes. The trend of the difference in LDL-C levels was observed (P = 0.0834). After The anthropometric parameters of all the participants were col- adjusting for age, sex, BMI and blood pressure, LDL-C was marginally as- 2 lected. Body mass index (BMI) was assessed by weight (kg)/height sociated with rs2000999 genotypes (P = 0.0578, β ± SE = 0.0060 ± 2 (m ). Systolic and diastolic blood pressures were measured by skilled 0.0032 for the A allele) (Table 2). However, this association was no longer medical workers. Fasting venous blood samples were obtained from statistically significant after adjusting for the common Hp CNV (P = all the participants to test the clinical metabolic parameters. HbA1c 0.6189). Contrarily, the correlation between Hp CNV and LDL-C levels ™ levels were measured via Bio-Rad VARIANT high-performance liquid was significant after adjusting for SNP rs2000999 (P = 0.0003, β ± chromatography methods (Bio-Rad Laboratories, Hercules, CA). Blood SE = 0.0124 ± 0.0034 for Hp2 allele). lipids, including high-density lipoprotein cholesterol (HDL-C), low- density lipoprotein cholesterol (LDL-C), total cholesterol and triglycer- 3.2. Association between rs2000999 and diabetic macrovascular diseases ides, were tested via a 7600-020 Automated Analyser (Hitachi, Tokyo, Japan). We measured Hp levels in a group of 886 serum samples (468/ There was no significant difference in the minor allele frequency of 418 cases/controls of diabetic macrovascular diseases, respectively) SNP rs2000999 between diabetic macrovascular cases and controls via enzyme-linked immunosorbent (ELISA) assay by using commer- (P = 0.5066) among 5045 patients without Hpdel (Fig. 1a). After cially available ELISA kits (R&D Systems, Inc., Minneapolis, USA). adjusting for age, sex, BMI, blood pressure, duration of diabetes, HbA1c, LDL-C and HDL-C levels, we still failed to detect a significant association 2.4. Genotyping between the variant rs2000999 and diabetic macrovascular diseases (P = 0.6109, OR = 0.968 [0.854, 1.097] for the A allele) (Fig. 1b). Further- DNA was extracted from the blood specimens. The SNP rs2000999 more, we analysed the association of rs2000999 with IMT and IAD of the was genotyped by primer extension of the multiplex products with detec- common carotid artery. A significant association was identified between tion through matrix-assisted