The E23K polymorphism in Kir6.2 gene and coronary heart disease

Clinica Chimica Acta 367 (2006) 93 – 97 www.elsevier.com/locate/clinchim

The E23K polymorphism in Kir6.2 gene and coronary heart disease Chenling Xiong a, Fang Zheng a,*, Jun Wan b, Xin Zhou a, Zhinong Yin c, Xiaobo Sun a a

Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, China b Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430064, China c Clinic Lab, Beijing Chuiyangliu Hospital, Beijing, 100022, China

Received 7 September 2005; received in revised form 25 November 2005; accepted 26 November 2005 Available online 7 February 2006

Abstract Background: The G to A mutation in the Kir 6.2, the ATP-sensitive potassium channel subunit, resulted a glutamate (E) to lysine (K) substitution at codon 23, and the A allele was shown to have a relationship with high risk to type 2 diabetes in previous study. Their role in coronary heart disease (CHD) has not been evaluated. We hypothesized that the polymorphism would be associated with increased susceptibility to CHD. Methods: The E23K gene polymorphism of Kir6.2 gene was analyzed by PCR-restriction site polymorphism (PCR-RSP) methods in 101 controls and 119 CHD patients. Serum lipids and C reactive protein concentrations were measured in all subjects. Results: Among the CHD patients, the frequency of the G allele was higher (63.4% vs. 56.9%, P > 0.05) and the frequency of the A allele was lower (36.6% vs. 43.1%, P > 0.05) than among controls. No significant differences were found in allele frequencies between CHD and controls ( P > 0.05), but there were significant differences in GG and combined (GA + AA) genotypes frequencies (42.0% vs. 28.7%, and 58.0% vs. 71.3%, P < 0.050). Conclusions: The E23K gene polymorphism in Kir6.2 gene appeared to be related to high susceptibility to CHD. D 2005 Elsevier B.V. All rights reserved. Keywords: Kir6.2; Gene polymorphism; Coronary heart disease; Susceptibility

1. Introduction The ATP-sensitive potassium channel (KATP) was originally described in guinea pig cardiac cell membrane patches by Noma [1]. From then on, KATP has been detected in various kinds of tissues such as neur cell, pancreatic h cell, a cell, skeletal muscle, renal and liver cells etc. [2– 5]. The classic KATP channels were complexes of 2 subunits, a regulatory sulfonylurea receptor (SUR) subunit belonging to the ATP binding cassette transporter superfamily [6] and an ATP-sensitive and pore-forming inwardly rectifying K+ channel (Kir6.X) subunit that belonged to the Kir6.0 subfamily of the inward rectifier family [7]. The Kir6.X Abbreviations: ApoA-I, apolipoprotein A-I; ApoB, apolipoprotein B; KATP, ATP-sensitive potassium channel. * Corresponding author. Tel.: +86 2761155235; fax: +86 2787336254. E-mail address: [email protected] (F. Zheng). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.11.032

subunits including Kir6.1 and Kir6.2 has 2 transmembrane domains and form the pore, conferring channel sensitivity to ATP and other cell metabolites such as ADP, phosphatidylinositol-4, 5-bisphosphate (PIP2) and long chain fatty acids etc. [8,9]. In the heart, KATP channels were composed of Kir6.2 and SUR2A [10]. The genes encoding human KATP subunit Kir6.2 was located on chromosome 11p15.1 with only an 1173 bp exon. KATP channels played important functions in the coronary heart disease (CHD) such as ischemia preconditioning, regulation of coronary blood flow, adaptation of the cardiac myocytes to stress and resistance of cell apoptosis [11 – 13]. In the pancreatic h cell, KATP channels were composed of Kir6.2 and SUR1 [10]. Initial studies of the polymorphism in Kir6.2 gene focused on its association with type 2 diabetes. More and more reports have pointed out that E23K variant of Kir6.2 was related to type 2 diabetes in whites [14 – 16]. The

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mutation mediated channel overactivity caused undersecretion of insulin and then represented a risk factor of diabetes. Furthermore, recent clinic investigations [17,18] revealed that inhibition of KATP channels by insulin secretagogues during diabetes treatment was considered to increase cardiovascular risk. Therefore the relationship of E23K polymorphism and coronary heart disease is worth noting.

2. Materials and methods 2.1. Human subjects The CHD group included 119 patients (77 males and 42 females, aged 58.6 T 6.7 y old) selected randomly from those who underwent coronary angiography after recent myocardial infarction or angina in Zhongnan Hospital of Wuhan University and Wuhan Asia Heart Hospital. In all cases, CHD was established if > 50% of 1 or more coronary artery had stenosis. The control group consisted of 101 individuals (62 males and 39 females, aged 57.3 T 8.5 years old), randomly selected after medical check-up at Zhongnan Hospital, to screen out those who had a history of chest pain, diabetes, hypertension, and the above general illness. The informed consents were obtained from all subjects. The population was approved by ethic committee of Wuhan University and met the declaration of Helsinki.

449 bp, containing at least 2 BanII recognition site (Fig. 1, panel A, lane 1). There were 3 different genotypes, GG, GA and AA (Fig. 1, panel A). In the homozygous genotype AA where one BanII recognition site was abolished, the digestion of the PCR products occurred in the other 2 BanII recognition sites, 227, 178 and another 44 bp band appeared (Fig. 1, panel A, lane 2). In the homozygous genotype GG where 3 BanII recognition sites were present, the PCR products were completely digested into 4 small fragments: 227, 150, 44 and 28 bp, respectively, (Fig. 1, panel A, lane3). In the heterozygous genotype GA, 1 allele was digested in 2 BanII recognition sites and another allele was completely digested in 3 BanII recognition sites, so that all 5 possible fragments appeared: 227, 178, 150, 44 and 28 bp, respectively, (Fig. 1, panel A, lane 4). The 44 and 28 bp small fragments run out of the polyacrylamide gel and couldn’t be seen. This new protocol produced clear-cut genotyping results. DNA sequences underlying the E23K polymorphism were determined by DNA sequencing. Panel B in Fig. 1 showed the confirmed nucleotide sequences in the GG and AA genotypes. Genotype analysis was determined by electro-

2.2. Design of PCR primers and amplification The primers were designed using Primer Premier 5.0 based on the published human Kir6.2 DNA sequence (GenBank accession: NM_000525). The primer sequences were as follows: forward primer, 5V-GACTCTGCAGTGAGGCCCTA-3V; reverse primer, 5V-AGAAAAGGAAGGCAGACGAGAAG-3V. The primers were synthesized in Shanghai Sangon Biological Engineering Technology And Service Co., LTD( Shanghai, China). Genomic DNA was extracted from peripheral blood leukocytes using improved NaI method [19]. Amplification PCR was carried out with 100 ng of genomic DNA in a volume of 25 Al containing 10 pmol of each primer, 1.5 mmol/l Mg2+, 200 Amol/l of each dNTP and 1.0 U of Taq DNA polymerase. After predenaturation at 95 -C for 5 min, DNA fragments were amplified for 35 cycles by the following steps: denaturation at 95 -C for 30 s, annealing at 60 -C for 30 s and extension at 72 -C for 30 s, following by a final extension step at 72 -C for 10 min. All PCR reactions were performed in a Thermal Cycler 9700 (Applied Biosystems, Foster City, CA). 2.3. Genotyping Then 10 AL PCR product was digested in 20 Al system with 8 U BanII (TaKaRa Biotechnology (Dalian) Co. Ltd, Dalian, China) for 12 h at 37 -C. The PCR product was

Fig. 1. Representative gel patterns of the BanII polymorphism in Kir6.2 gene (panel A) and the confirmation of sequence changes underlying the BanII polymorphism (panel B). Panel A: 10 AL PCR product was digested by BanII and separated on 8% polyacrylamide gel electrophoresis. Lane 1 shows the PCR product, lane 2 is the homozygous AA genotype, lane 3 is the GG genotype and lane 4 is the heterozygous GA genotype. The marker is a 50-bp DNA ladder (MBI Fermentas). Panel B: DNA sequencing were carried out as described in Materials and methods. Copies of the partial chromatograms covering the region of interest are displayed. Panel B. B1 is the sequence for the GG genotype and B2 is that for the AA genotype.

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phoresis on 8.0% polyacrylamide gel at constant voltage of 200 V. The image was captured using a JD801 Video System (Jiangsu JEDA Science-Technology Development Co., Ltd, NanJing, China). A representative genotype pattern was displayed in Fig. 1. The PCR products amplified from homozygous GG and AA genotype samples were sequenced directly in Shanghai Genecore BioTechnologies Co., Ltd. 2.4. Determination of biochemical parameters Blood samples were collected after 12 to 16 h of an overnight fast and plasma were separated by 3000 g centrifugation for 15 min. Total cholesterol (TC) and triglyceride (TG) were assessed by enzymatic colorimetry methods (Diasys Diagnostic Technology Co., Ltd, Germany). The cholesterol contents of high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) were analyzed using homogeneous methods (Daiichi Pure Chemicals Co., Ltd, Tokyo, Japan). C-reactive protein was assayed by using of a high-sensitivity assay (hs-CRP, Dade Behring Inc., Newark, DE). Lp(a), apolipoprotein A-I (ApoA-I), apolipoprotein B (ApoB) were determined by nephelometric immunoassay (Daiichi Pure Chemicals Co., Ltd, Tokyo, Japan; Diasys Diagnostic Technology Co., Ltd, Germany). All of these were measured on an automatic biochemical analyzer (Aeroset, Abbott Diagnostics, Abbott Park, IL). 2.5. Statistical analysis Allele frequencies were calculated by allele counting and the v 2 test was used to apply for Hardy – Weinberg equilibrium and to compare allele and genotype frequencies between the CHD and control group. Quantitative clinical data were compared between subjects with CHD and controls and plasma concentrations of lipids were compared by the unpaired Student’s t-test, while the rank sum test was performed to compare the mean concentrations of plasma Table 1 Characteristics of study groups Characteristics

CHD patients (n = 119)

Controls (n = 101)

Age (Mean T S.D.) Men/Women (n) BMI (kg/m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Diabetes (%) Smoker (%) TC (mmol/l) TG (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) ApoA-I (g/l) ApoB (g/l) Lp(a) (mg/l) CRP (mg/l, media, 25% – 75%)

58.6 T 6.7 77 / 42 24.4 T 3.1 135.2 T 18.6* 83.7 T 13.2* 4.20 21.85* 5.13 T 0.72* 1.94 T 1.33* 1.16 T 0.68 2.69 T 0.57 1.24 T 0.29 0.82 T 0.25* 172.44 T 188.84* 1.02, 0.62 – 2.00*

57.3 T 8.5 62 / 39 23.8 T 3.4 113.3 T 10.3 76.7 T 7.6 0.00 7.92 4.79 T 1.13 1.34 T 0.61 1.23 T 0.26 2.69 T 0.39 1.19 T 0.26 0.69 T 0.14 104.94 T 157.08 0.76, 0.59 – 1.25

* P < 0.01, compared with controls.

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Table 2 Genotype and allele frequencies of the E23K polymorphism of Kir6.2 gene in CHD patients and controls Group CHD (n = 119) Controls (n = 101) v2 P value OR (95%CI)

Genotype (%)

Allele (%)

GG

G

GA + AA

50 (42.0) 69 (58.0) 29 (28.7) 72 (71.3) 4.202 <0.050 1.799 (1.023~3.163)*

A

151 (63.4) 87 (36.6) 115 (56.9) 87 (43.1) 1.940 NS 1.313 (0.895¨1.927)**

* The frequency of GG genotype was compared with that of combined (GA + AA) genotype. ** The frequency of G allele was compared with A allele.

high-sensitive CRP (hsCRP) in different genotype subsets. Results were presented as 95% confidence internal (95% CI). A value of P < 0.05 was considered statistically significant. All data were analyzed using SPSS 11.5.

3. Results The clinical characteristics of the study subjects are shown in Table 1. There was no statistically significant difference between CHD group and controls for age, gender, body mass index (BMI) and diabetes. Comparing with the controls, individuals in the CHD group showed significantly higher blood pressure, prevalence of smoking and plasma concentrations of TC, TG, ApoB, Lp (a), and CRP ( P < 0.01). The distributions of the different genotypes and alleles were consistent with the Hardy – Weinberg equilibrium. Among the CHD patients, the frequency of the G allele was higher (63.4% vs. 56.9%, P > 0.05) and the frequency of the A allele was lower (36.6% vs. 43.1%, P > 0.05) than among controls. No significant differences were found in allele frequencies between CHD and controls ( P > 0.05), but there were significant differences in GG and combined (GA + AA) genotypes frequencies (42.0% vs. 28.7%, and 58.0% vs. 71.3%, P = 0.040). The genotype and allele frequency distributions in patients and controls were shown in Table 2. The risk factors including blood pressure, lipid concentrations and inflammatory factors were compared among subgroups defined by genotypes. But we didn’t find any significant differences ( P > 0.05) among the concentrations of these risk factors in genotype subgroups (Table 3).

4. Discussion The ATP sensitive channels subserve important functions in the coronary heart disease [20]. Normally, the KATP channels in cardiac tissue were closed by high intracellular ATP concentration [21], but were activated in many cases of cardiovascular pathological states such as ischemia, reperfusion, cell stress and apoptosis [11 – 13]. Cellular membrane hyperpolarization in response to activation of KATP channels will promote the synthesis of nitric oxide (NO) and

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Table 3 The levels of serum lipid, blood pressure and CRP in different genotype subgroups of patients and controls Group

n

TG (mmol/l)

TC (mmol/l)

CHD patients 119 GG 50 1.90 T 1.87 5.10 T 0.72 GA 51 1.81 T 0.90 5.13 T 0.71 AA 18 2.03 T 1.15 5.16 T 0.78 Controls 101 GG 29 1.25 T 0.42 4.99 T 1.28 GA 57 1.40 T 0.74 4.73 T 0.93 AA 15 1.43 T 0.33 4.66 T 1.03

HDL-C (mmol/l)

LDL-C (mmol/l)

ApoA (g/l) ApoB (g/l) Lp(a) (mg/l)

Systolic pressure (mm Hg)

Diastolic pressure (mm Hg)

CRP (mg/l,media, 25% – 75%)

1.08 T 0.22 2.75 T 0.76 1.17 T 0.28 0.84 T 0.29 178.88 T 220.88 133.6 T 17.8 81.3 T 9.3 0.95 (0.61 – 1.40) 1.14 T 0.25 2.70 T 0.36 1.26 T 0.30 0.79 T 0.21 162.38 T 166.65 133.5 T 18.5 81.98 T 12.0 0.99 (0.52 – 2.00) 1.45 T 1.71 2.57 T 0.53 1.23 T 0.24 0.84 T 0.30 159.87 T 180.92 129.6 T 20.6 83.3 T 10.5 1.01 (0.62 – 1.73) 1.25 T 0.16 2.73 T 0.38 1.20 T 0.32 0.67 T 0.13 1.25 T 0.24 2.66 T 0.38 1.16 T 0.20 0.70 T 0.15 1.06 T 0.50 2.72 T 0.45 1.19 T 0.23 0.71 T 0.14

other vasorelaxant factors and increases the permeability of the vascular wall. The hyperpolarization-mediated dilation often compensates for loss of other dilator mechanisms such as the endothelium-dependent vasodilation, which may be impaired in the pathologic development of coronary atherosclerosis [22,23]. Additionally, KATP channels activation in the sarcolemma of cardiomyocytes are required for adaptation of the cardial myocytes to stress [13]. The KATP channels activation leads to clamp the cell at the K+ equilibrium potential and brings the cell to rest. Furthermore, the ischemic preconditioning (i.e., a brief period of ischemia lessons the amount of myocardial damage produced by a subsequent prolonged ischemia) induced by KATP channel openers blocks cell apoptosis in ischemic heart [24 – 26]. Suppression of apoptosis is a key event to prevent the pathogenesis and progression of coronary heart disease. Thus, it is important to remember that the KATP channel links the cross-membrane K+ flux and the electrical activity of membrane to the metabolic state of the cell. Any mild mutations or polymorphisms modifying the KATP channel current or activity will be correlated with diseases. In this study, we analyzed the E23K (G Y A) polymorphism in CHD patients and normal controls in Chinese Han population. The present sample size had a statistic power of 0.873 to detect significant polymorphism differences between the two groups (the effect sample size set at 0.2, a = 0.05). The frequencies of combined (GA + AA) genotype were significantly lower in CHD group than in controls, which implicated that the A allele may be a protective factor for CHD. The E23K (G Y A) variant is a missense SNP located within the N-terminus of the Kir6.2 subunit conferring a subsequent negative to positive shift in residue charge. This shift could cause decreased ATP sensitivity and enhance the open probability of the channel [27,28]. With these alterations, K+ efflux increased and cell membrane hyperpolarized, which would have the effect of calcium inflow inhibition and sequentially resulted in decreased insulin secretion [29]. This ‘‘activating’’ variant in Kir6.2 subunit may predispose to diabetes [30]. However, in another aspect, the overactivity of KATP channel could facilitate the cellular membrane polarization and then cellular excitability reduction. These could protect depressed endothelial cells and cardiocytes from cell stress

95.00 T 155.22 114.0 T 12.3 91.90 T 114.44 113.2 T 10.6 57.05 T 39.17 112.7 T 14.8

76.9 T 6.9 76.9 T 7.2 73.1 T12.3

0.77(0.68 – 1.44) 0.73(0.57 – 1.14) 0.96(0.56 – 1.31)

and apoptosis. Hence, the A allele that resulted in a modest increase in KATP channel activity showed a relative protective effect on defending the cells from trauma in atherosclerosis (Fig. 2). In addition, inhibition of cardiovascular KATP channels by insulin secretagogues (sulfonylureas and glinides) will be a benefit for diabetes treatment but considered to increase cardiovascular risk in clinic [17,18]. Furthermore, potassium channel openers cause myocardial preconditioning and decrease the infarction size in animal model, however, in counter effect, they inhibit the insulin release after glucose administration in healthy subjects [31]. So the effect of KATP channels on diabetes and CHD may be contradictory. Compared to G allele, the A allele may increase the channel open probability and enhance the channel activity. It will give rise to the protection of the cardiocytes and endothelial cells whereas predispose to diabetes just as the potassium channel openers. We observed that the frequencies of genotype carrying A allele in controls were higher than those in CHD patients. G to A variant (E to K variant)

ATP sensitivity , open activity

K+ efflux Membrane hyperpolarization

Cellular excitability , Ca2+ inflow

Insulin secretion , glucose level

Severity of ischemia

Cell stress/apoptosis

Substrate supply

Reduce cell injury

Protect endothelial cells from damage

Fig. 2. The possible mechanism of the E23K variant affects on coronary heart disease.

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But we didn’t observe any significant differences among the concentrations of blood lipids, blood pressure and CRP in genotype subgroups in this study, which suggested that the E23K polymorphism might be an independent protective factor for coronary heart disease. However, the molecular mechanism by which the E23K amino acid shifts altered the ability of the KATP channels to sense change in ATP is currently unknown. The X-ray crystallization of the KATP channels may be a need to establish the complete model of the KATP channel at the molecular level. And though we found the significant difference between the frequencies of combined (GA + AA) genotype and GG genotype we didn’t find significant differences between the frequencies of alleles in controls and patients. It was possible due to the small sample size. In order to further identify the protective role of the A allele in coronary heart disease, we could either enlarge the sample size or investigate the allele frequency distributions in CHD patient subgroups regarding clinical symptom stratification.

Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30200107) as well as the funds from Wuhan University School of Medicine.

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