The rs10947803 SNP of KCNK17 is associated with cerebral hemorrhage but not ischemic stroke in a Chinese population

Neuroscience Letters 539 (2013) 82–85

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The rs10947803 SNP of KCNK17 is associated with cerebral hemorrhage but not ischemic stroke in a Chinese population Qingfeng Ma a,1 , Yongqin Wang b,d,1 , Yue Shen b,e,f , Xin Liu b,c , Xiaotong Zhu g , Hongye Zhang b , Lisheng Liu b , Xuerui Tan c,∗ , Lefeng Wang h,∗∗ , Xingyu Wang b,c,∗ ∗ ∗ a

Department of Neurology, Xuanwu Hospital, Beijing, China Laboratory of Human Genetics, Beijing Hypertension League Institute, Beijing, China c First Affiliated Hospital, Medical College of Shantou University, Shantou, Guangdong, China d School of Basic Courses, Baotou Medical College, Baotou, Neimenggu, China e Graduate School of Peking Union Medicine College, China Graduate School of Peking Union Medicine College, Beijing, China f Department of Epidemiology, Capital Institute of Pediatrics, Beijing, China g College of Life Sciences, Peking University, Beijing, China h Department of Cardiology, Chaoyang Hospital, Beijing, China b

h i g h l i g h t s  We are the first demonstrate the association of KCNK17 polymorphism with hemorrhagic stroke in Chinese population.  The MAF for KCNK17 polymorphism is higher in Chinese than that of Europeans.  Our finding did not support the association of KCNK17 with ischemic stroke in Chinese population.

a r t i c l e

i n f o

Article history: Received 29 November 2012 Received in revised form 16 January 2013 Accepted 18 January 2013 Keywords: Cerebral hemorrhage Ischemic stroke KCNK17

a b s t r a c t KCNK17 (potassium channel, subfamily K, member17) was first discovered to associate with the pathogenesis of ischemic stroke in the first genome-wide association study. The rs10947803 SNP in KCNK17 is significantly associated with ischemic stroke in Caucasian populations. The aim of the present study was to investigate the association with strokes in the Chinese population. A total of 1364 stroke patients and 1293 controls were examined using a case-control methodology. The rs10947803 SNP in KCNK17 was genotyped by a TaqMan real-time PCR assay. The rs10947803 SNP (A allele) of KCNK17 was significantly associated with cerebral hemorrhage (for AA + AC versus CC, unadjusted odds ratio [OR] = 1.70; 95% confidence interval [CI], 1.08–2.69; P = 0.020). After adjustment for age and sex, the association remained significant for AA + AC versus CC, OR = 1.65; 95% CI, 1.04–2.62; P = 0.033. In addition, the rs10947803 SNP in KCNK17 was not associated with ischemic stroke (for AA + AC versus CC, unadjusted OR = 0.92; 95% CI, 0.81–1.05; P = 0.212, after age- and sex-adjustment, OR = 0.87; 95% CI, 0.72–1.05; P = 0.143). The rs10947803 SNP (A allele) in KCNK17 increases the risk of cerebral hemorrhage but not ischemic stroke in the Chinese population. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

∗ Corresponding author at: First Affiliated Hospital, Medical College of Shantou University, Shantou, Guangdong 515041, China. Tel.: +86 754 8861 1690. ∗∗ Corresponding author at: Department of Cardiology, Chaoyang Hospital, Capital Medical University, 8th Gongtinanlu Road, Beijing 100020, China. Tel.: +86 10 85231937. ∗ ∗ ∗Corresponding author at: Laboratory of Human Genetics, Beijing Hypertension League Institute, 24 Shijingshan Road, Beijing 100043, China. Tel.: +86 10 8868 1564. E-mail addresses: [email protected] (X. Tan), [email protected] (L. Wang), [email protected] (X. Wang). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.01.041

Stroke is the second most common cause of death in developed countries [7,20]. In China, the incidence of stroke is much higher than that of coronary heart disease [2,22]. Stroke in developing countries accounts for nearly 70% of global stroke deaths, and 40% of global stroke deaths occur in China [13]. Stroke is generally classified as ischemic or hemorrhagic. Recent epidemiological studies revealed that hemorrhagic stroke accounts for at least 30% of stroke cases in China, which is significantly higher than the incidence in Western countries [10,17,18,24]. Matarin et al. reported the first phase of a genome-wide association study on ischemic stroke, analyzing more than 400,000 single

Q. Ma et al. / Neuroscience Letters 539 (2013) 82–85

nucleotide polymorphisms (SNPs). They identified the rs10947803 SNP in KCNK17 that was associated with ischemic stroke in the cohorts [14]. The finding was replicated by Domingues-Montanari et al. [5] in a Spanish cohort. However, a study carried out on a Chinese population by Ding et al. did not replicate the association reported in the original GWAS study [4], but due to the relatively small sample size, the results were not conclusive. The minor allele frequencies (MAF) of the rs10947803 SNP in KCNK17 were reported in the HapMap database to be 0.208 in CEU (Utah residents with Northern and Western European ancestry from the CEPH collection), 0.00 in YRT (Yoruba in Ibadan, Nigeria), 0.455 in JPT (Japanese in Tokyo, Japan), and 0.444 in CHB (Han Chinese in Beijing, China). These data suggest that the MAF of this SNP in Asian populations should have a greater power to detect a positive association. We previously conducted a genetic study of stroke in Chinese patients. The types of stroke in the SHINGING cohort included ischemic stroke, cerebral hemorrhage, subarachnoid hemorrhage, transient ischemic attacks (TIA), and stroke with an undetermined cause. Therefore, we tested the association of rs10947803 SNP in KCNK17 with ischemic stroke in the SHINGING cohort. In addition, we tested the rs10947803 SNP in KCNK17 and its association with other types of stroke in the Chinese population. 2. Materials and methods 2.1. Subjects The Stroke Hypertension Investigation in Genetics (SHINING) study was conducted by the Beijing Hypertension League Institute. Between 1997 and 2000, patients and control subjects from 6 geographical regions within China were recruited for the case–control study (70% subjects came from within and near Beijing) [19]. The SHINING study comprised subjects of Chinese Han ethnicity only [21]. Any stroke patients who suffered a stroke within the past 5 years were eligible to participate [19,21]. All patients have medical records with a diagnosis of brain CT/MRI. Patients with ischemia, cerebral hemorrhage, ischemia/hemorrhage, subarachnoid hemorrhage, transient ischemic attacks, and cryptogenic causes were included. Control subjects were selected according to the case–control study criteria (control subjects matched to cases by sex, aged within 3 years, geographic location, and blood pressure category (<140/90, ≥140/90 and ≤180/105, >180/105 mm Hg)) [21]. The data collected included age, sex, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), and hypertension. Hypertension was defined as having a current or past prescription for antihypertensive medication, systolic blood pressure ≥140 mm Hg, or diastolic blood pressure ≥90 mm Hg [19,21]. We obtained written informed consent from all study participants, and the study was approved by the ethics committees of the Beijing Hypertension League Institute. 2.2. Genotyping A total of 3119 participants (1559 stroke cases and 1560 controls) were recruited for the SHINING study, and of these, 2657 participants (1364 stroke cases and 1293 controls) had DNA samples available. The rs10947803 SNP of the KCNK17 gene was genotyped by TaqMan Real Time PCR (Assay ID: AHY9X1K, Applied Biosystems Inc.) using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). The genotyping call rate was 98.0%. 2.3. Statistical analysis Clinical data regarding continuous variables were expressed as the mean ± SD, and univariate associations were explored with frequency tables and Pearson’s 2 tests for independent proportions.

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Table 1 Characteristics of study participants.

Number of subjects Agea (years) Sex (% male) BMIa (kg/m2 ) SBPa (mm Hg) DBPa (mm Hg) Hypertension (% yes) a

Stroke patients

Controls

P-value

1364 60.7 ± 10.3 59.5 24.3 ± 3.0 144.3 ± 23.6 86.9 ± 12.9 77.8

1293 61.9 ± 10.5 60.0 25.1 ± 3.3 144.4 ± 23.6 86.4 ± 13.0 79.4

0.002 0.769 <0.0001 0.917 0.279 0.326

Continuous variables were expressed as the mean ± standard deviations.

Continuous variables were compared with t-tests or appropriate non-parametric tests, depending on their distribution. The deviation from the Hardy–Weinberg equilibrium in cases, controls, and total samples were assessed using an exact 2 goodness-of-fit test [9]. The analyses concerning the entire study group were subsequently stratified into two major types of stroke, ischemic stroke and cerebral hemorrhage. In each stratum, cases were compared with those of the corresponding control groups. Odds ratios in the dominant model with corresponding 95% confidence intervals (CIs) and adjusted odds ratios for age and sex were determined by logistic regressions with allele frequency and genotypes, using age and sex as the independent variables [11]. Power calculations were performed using the QUANTO software program (Version 1.2.3). Data were analyzed using SAS statistical software (version 9.2, SAS Institute Inc). P < 0.05 was used to indicate statistically significant differences. 3. Results The characteristics of the study participants are shown in Table 1. The means of age and BMI were lower in cases than those in controls. The SBP and DBP and the rates of hypertension in cases were not significantly different from those in controls. The percentage of ischemic stroke and cerebral hemorrhage were 73.5% and 12.5%, respectively, in our study. The rs10947803 SNP in KCNK17 was genotyped in the SHINGING cohort. The SNP was tested to be in Hardy-Weinberg equilibrium (P > 0.05) in cases and controls. The associations between the rs10947803 SNP and different types of stroke are shown in Table 2. The A allele of the SNP of rs10947803 increased the risk of cerebral hemorrhage in the Chinese population, with an unadjusted odds ratio = 1.70; 95% CI, 1.08–2.69; P = 0.020 after adjusting for age and a sex odds ratio = 1.65; 95% CI, 1.04–2.62; P = 0.033. We failed to observe the association of rs10947803 in KCNK17 with other types of stroke in the present study cohort. For ischemic stroke, the unadjusted odds ratio was 0.86 with 95% CI from 0.72 to 1.04, P = 0.113; after ageand sex-adjustment, the odds ratio was 0.87; 95% CI, 0.72–1.05; P = 0.143 under a dominant model (Table 3). The associations between the rs10947803 SNP and ischemic stroke from different populations, including those considered in the present study, are shown in Table 4. There is a discrepancy in the relationship between rs10947803 genotypes and ischemic stroke in different populations. We calculated the unadjusted OR and P value under the allele frequency model according to data reported in previously published studies. In the work of Matarin et al. [14] and Domingues-Montanari et al. [5], the unadjusted OR of Caucasian populations for ischemic stroke were 1.77 (95% CI, 1.32–2.38; P = 0.0001) and 1.37 (95% CI, 1.04–1.81; P = 0.024) under an allele frequency model. The unadjusted OR was 0.91 (95% CI, 0.79–1.06; P = 0.219) in the study of Hu et al. in southern China, which is similar to that determined in the present study (the unadjusted odds ratio = 0.92; 95% CI, 0.81–1.05; P = 0.210) under an allele frequency model.

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Q. Ma et al. / Neuroscience Letters 539 (2013) 82–85

Table 2 Case–control study showing associations between the SNP (rs10947803) in KCNK17 and stroke. Samples

Screening Stroke Ischemic stroke Cerebral hemorrhage IH stroke‡ Subarachnoid hemorrhage TIA Undetermined a † ‡

H–Wa

Case CC

AC

AA

Sum

515 387 50 28 5 33 12

618 444 93 17 6 39 19

192 143 23 7 3 11 5

1325 974 166 52 14 83 36

0.764 0.395 0.052 0.115 0.640 0.922 0.560

H–Wa

Control CC

AC

AA

Sum

472 343 66 15 4 27 17

623 461 75 22 8 40 17

184 143 15 7 2 11 6

1279 947 156 44 14 78 40

0.348 0.558 0.339 0.821 0.533 0.532 0.611

MAF

Unadjusted (AA + AC vs. CC)

Case

Control

Odds ratio (95% CI)

P-value†

0.378 0.375 0.419 0.298 0.429 0.367 0.403

0.387 0.394 0.337 0.409 0.429 0.397 0.363

0.92 (0.79–1.08) 0.86 (0.72–1.04) 1.70 (1.08–2.69) 0.44(0.19–1.01) 0.72 (0.15–3.54) 0.80 (0.42–1.52) 1.48 (0.58–3.78)

0.302 0.113 0.020 0.054 0.686 0.500 0.411

H–W indicates the P value of Hardy–Weinberg equilibrium. P values were assessed by 2 test. IH stroke indicates patients with both ischemic stroke and cerebral hemorrhage.

Table 3 Odds ratios for the incidence of ischemic stroke and cerebral hemorrhage. Samples

Genotype of SNP

Ischemic stroke

Cerebral hemorrhage

a †

Number of cases

Number of controls

Age- and sex-adjusteda Odds ratio (95% CI)

P-value†

CC AC AA AC + AA

387 444 143 587

343 461 143 604

1.00 0.87 (0.71–1.05) 0.94 (0.82–1.08) 0.87 (0.72–1.05)

0.151 0.409 0.143

CC AC AA AC + AA

50 93 23 116

66 75 15 90

1.00 1.59 (0.98–2.57) 1.42 (0.97–2.07) 1.65 (1.04–2.62)

0.058 0.069 0.033

Multiple logistic regression was performed with adjustment for age and sex. P values were assessed by 2 test.

4. Discussion In this study, we tested rs10947803 SNP in KCNK17 with different types of stroke in a Chinese population. Our results revealed that the rs10947803 SNP in KCNK17 is associated with cerebral hemorrhage but not with ischemic stroke in the Chinese population. This association remains significant under a dominant model after adjusting for age and sex. To our knowledge, this is the first report of the positive association of rs10947803 SNP in KCNK17 with hemorrhagic stroke, albeit without multi-testing adjustment. The results warrant further validation in other cohorts with a larger sample size and in other populations. Patients with TIA, subarachnoid hemorrhage, and cryptogenic stroke were also included in the analysis (Table 2.) but did not show any positive association. This may stem from the small sample sizes for the above-mentioned types of stroke included in the present study. The association between the SNP and IH stroke (patients with both ischemic stroke and cerebral hemorrhage) reached borderline statistical significance (show in Table 2); the unadjusted odds ratio = 0.44; 95% CI, 0.19–1.02; P = 0.054. This result could be due to the unknown etiology of patients with ischemic stroke and cerebral hemorrhage or due to the small sample size. Further investigation with a greater sample size and a clearly defined phenotype is required to evaluate the association.

Data from Matarin et al. [14], Domingues-Montanari et al. [5], and our group indicate that the frequencies of the risk allele for the rs10947803 SNP within KCNK17 in the Caucasian population differ from those of the Chinese population, with 0.18 vs. 0.39 for the MAF, respectively. Therefore, a study conducted on the Chinese population should have a greater power to detect an association between rs10947803 and all types of stroke. The rs10947803 SNP in KCNK17 was not associated with ischemic stroke in our study, which is similar to the results previously reported for the Chinese population by Ding et al., who observed that the SNP (C allele) also failed to show association with ischemic stroke in the Chinese Han population (716 thrombosis stroke, 407 lacunar infarction and 557 normal control subjects) under an additive (adjusted OR = 1.08, 95% CI = 0.91–1.28, P = 0.404) and dominant model (adjusted OR = 1.04, 95% CI = 0.81–1.33, P = 0.756) after adjustment for confounding factors [4]. In the present study, the power to detect positive association with MAF = 0.39 under an additive model is greater than 0.99, assuming the odds ratio of 1.77 reported in the study of Matarin et al. The result that the rs10947803 in KCNK17 is not associated with ischemic stroke in the Chinese population differs significantly from that for European populations (Table 4). One possible explanation for the absence of association in the Chinese population may be that the rs10947803 in KCNK17 is a marker of

Table 4 Odds ratio for ischemic stroke with the rs10947803 polymorphism in different populations. Study

Sophie17 Matarin11 Hu Ding18 Present study

Population

White White Chinese Chinese

Case

449 249 1123 974

Control

254 268 557 947

Risk allele

A A A A

MAF

Allele frequency

Case

Control

Unadjusted OR (95%CI)

P-value†

0.23 0.28 0.37 0.38

0.18 0.18 0.39 0.39

1.37 (1.04–1.81) 1.77 (1.32–2.38) 0.91 (0.79–1.06) 0.92 (0.81–1.05)

0.024 0.0001 0.219 0.210

MAF, minor allele frequency; OR, odds ratio. The unadjusted OR and P values under an allele frequency model were not given directly in the published studies but calculated from the data collected from them. † P-values were assessed by 2 test.

Q. Ma et al. / Neuroscience Letters 539 (2013) 82–85

adjacent functional gene(s), which may exhibit different linkage disequilibrium in different ethnic groups. Hypertension is a common risk factor for ischemic stroke and cerebral hemorrhage. The growing evidence suggests that the function of several types of vascular K+ channels play a role in major cardiovascular diseases, such as chronic hypertension, diabetes, and atherosclerosis [16]. The current study revealed that the negative association of rs10947803 with ischemic stroke may be due to the matching of blood pressure between cases and controls. To address this notion, we tested the association between the SNP (rs10947803) in KCNK17 and hypertension in controls, and rs10947803 SNP in KCNK17 was not associated with hypertension (2 = 0.614, P = 0.736) in the Chinese population. KCNK17 is located in chromosome 6p21.2–p21.1 in humans. The rs10947803 SNP is located in the fourth intron of the gene, 1134 bp from exon 4 [8]. KCNK17 is a member of the 2-pore domain superfamily of background K+ channels, which are open at all membrane potentials and contribute to the cellular resting membrane potential, and its gating is variously regulated by free fatty acids, membrane tension, G protein-generated signaling, and extracellular pH. KCNK17 transcripts are widely expressed in humans. The highest expression is in the liver, lungs, placenta, pancreas, small intestine, and aorta [3]. To date, the mechanism of how the rs10947803 variant affecting funtions of KCNK17 has remained unknown. The protein encoded by the KCNK17 gene, called TALK-2 or TALK-4, belongs to the family of potassium channel proteins containing two pore-forming P domains [3]. This channel is an open rectifier that primarily passes outward current under physiological K+ concentrations. It participates in ion fluxes necessary for cell volume regulation as well as in metabolic acidosis and hypotension caused by the secretion of HCO3− [1,6]. This gene is activated at alkaline pH. In addition, K2P 1.1 is activated by activators of protein kinase C (PKC) and is inhibited by intracellular acidosis [12]. Recently, channels of the TASK-1 family (KCNK3) have been shown to present a neuroprotective function in cerebral ischemia [15,23]. Considering their function and localization in a wide range of tissues, KCNK genes may play an important role in cerebral hemorrhage in humans. Summary Our study showed that the rs10947803 SNP in KCNK17 was significantly associated with cerebral hemorrhage but not ischemic stroke in the Chinese population. Future studies with larger sample sizes are required to investigate the association with cerebral hemorrhage in the Chinese populationas well as in other populations. Functional studies of this potassium channel gene and protein might help to elucidate the mechanisms of KCNK17 in different types of stroke. Sources of funding This study was supported by the Beijing Hypertension League Institute, in part through an unrestricted educational grant from F. Hoffmann-La Roche and the National Infrastructure Program of Chinese Genetic Resources (2005DKA21300). Disclosures None. Acknowledgments We thank Jian Li, Wei Zhang, and Yawei Li for their technical assistance.

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