- Email: [email protected]
T Polymorphism in the Connexin 37 Gene and Essential Hypertension
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Heart, Lung and Circulation (2014) xx, 1–6 1443-9506/04/$36.00 http://dx.doi.org/10.1016/j.hlc.2014.02.016
ORIGINAL ARTICLE
Association Between 1019C/T Polymorphism in the Connexin 37 Gene and Essential Hypertension Suxia Guo 1*, Weixiang Chen, Ying Yang, Zhenyu Yang 1*, Minghua Cao Department of Cardiology, Affiliated People’s Hospital of Nanjing Medical University in Wuxi and People’s Hospital of Wuxi City, Wuxi, Jiangsu, 214023 P.R.China Received 15 January 2014; received in revised form 11 February 2014; accepted 17 February 2014; online published-ahead-of-print xxx
Objective
To investigate the association between the CX 37 1019C/T polymorphism and the susceptibility to essential hypertension (EH).
Methods
A total of 1126 cases of EH were diagnosed in the People’s Hospital of Wuxi City, China. A control group consisted of 874 healthy people, i.e., non-EH patients. All cases were genotyped by DNA sequencing.
Results
Polymorphism C1019T on the Connexin37 gene was found in the whole population. The distribution of three genotype frequencies in both groups was in accordance with the Hardy-Weinberg equilibrium. The frequency of the CX37 C allele was higher in EH patients (57.4% vs. 42.1%, x2=92.5, P<0.01) compared to the control group. The frequency of C carriers (CC+TC) was 80.5% in EH patients compared to 66.7% in the control (x2=49.0, P<0.01). EH risk was significantly increased in carriers of C the allele (CC+TC) over that in the TT homozygote (OR=2.06, 95% CI: 1.682.52). Subsequent stratified analyses demonstrate that a significant difference exists in the frequency of C carriers between male EH patients and controls (79.2% vs. 69.1%, x2=13.4, P<0.01) and in female EH patients and the control group (81.8% vs. 64.4%, x2=38.7, P<0.01). The carriers of the C allele had higher EH risk compared with the TT homozygote without sex differences (male: OR=1.71, 95% CI: 1.282.27; female: OR=2.48, 95%CI: 1.853.31).
Conclusion
The C allele in the CX37 gene might be associated with the susceptibility to EH in population of Wuxi, China.
Keywords
Essential hypertension Connexin37gene Genotype Allele frequency C1019T
Introduction Essential hypertension (EH) is a worldwide, common form of hypertension disease and a risk factor in heart disease, cerebrovascular disease and chronic kidney disease. Recent work suggests that the pathogenesis is poorly understood and that the primary cause is unknown in 90–95% of cases [1]. Heredity is estimated to account for 30-50% of disease cases so efforts are ongoing to elucidate genetic links. Large meta-analyses and multi-centre studies prove robust genetic associations with hypertension [6–10]. Genetic
variations in endothelial genes such as endothelin-1 and endothelial nitric oxide synthase may contribute to essential hypertension–and both relate to blood pressure (BP) regulation [2–5]. However, another study showed that hypertension links to genetic factors proved more challenging. It recommended that further research into the pathogenesis of EH was needed as its results contradicted other study’s results which demonstrated positive genetic linkages. Connexins are a protein family encoded by at least 20 different mammalian genes expressed in a variety of tissues
*Corresponding authors at: Department of Cardiology, Affiliated People’s Hospital of Nanjing Medical University in Wuxi and People’s Hospital of Wuxi City, Wuxi, Jiangsu, 214023 P.R.China, China., Email: [email protected] 1
Suxia Guo and Weixiang Chen have contributed equally to this study. So Suxia Guo and Weixiang Chen should be regarded as first joint authors.
© 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier Inc. All rights reserved.
Please cite this article in press as: Guo S, et al. Association Between 1019C/T Polymorphism in the Connexin 37 Gene and Essential Hypertension. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.02.016
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[11–13]. They form trans-membrane channels called gap junctions that connect neighbouring cells and allow passive diffusion of small connexin molecules. Connexin37 is mainly expressed in the vascular endothelium which is involved in growth, injured tissue regeneration and endothelial cells’ aging, suggesting that changes in connexin37 are associated with diseases following intima injury [14–16]. Boerma initially linked a single nucleotide polymorphism (SNP) in the human connexin37 gene with thickening of the carotid in a Swedish male population [17] by observing the C allele overrepresented in individuals with atherosclerotic plaques. The C SNP allele was also associated with coronary artery disease (CAD) in Taiwan, northern China and Switzerland [18–20]. Two other study results show the T SNP allele to be a risk factor for acute myocardial infarction (AMI) particularly in Japanese and Caucasian males [21,22], while most gene polymorphism-association studies suggest that the C1019T SNP in the human connexin37 gene is associated with CAD and myocardial infarction (MI) in various populations. Whether polymorphism can be an influence in EH is the subject of this study as is the relationship between C1019T polymorphism in the connexin37 gene, EH and CAD. Study shows it is associated with CAD but relationships to hypertension have not been reported even though 60-70% of CAD patients are also diagnosed with hypertension. The present study explores the association between the C1019T polymorphism in the connexin37 gene and EH.
Methods A total of 1126 Han Chinese diagnosed with essential hypertension between 2008 and 2011 were studied and compared with a control group of 874 patients without EH using a patient cohort from the People’s Hospital of Wuxi City, China. Cohort inclusion criteria included: 1) persons over 60 years of age; 2) diagnosed with EH according to the European Society of Hypertension and the European Society of Cardiology classification schemes [23]; 3) hypertension diagnosed as systolic blood pressure 140 mmHg (1 mmHg = 0.133 kPa) and / or diastolic blood pressure 90 mmHg and 4) excluded secondary hypertension diagnoses. An additional 874, over 60 year-old individuals, characterised as healthy and without hypertension or a family history of such after biochemical and clinical examinations, were used as a control population. No test or control groups included blood relatives. At the same time, except for severe acute and chronic infections, cancer, liver and kidney dysfunction and other systemic diseases, coronary artery disease, valvular heart disease, primary cardiomyopathy, cerebrovascular accident and diabetes. The ethics committee of the People’s Hospital of Wuxi City approved the study. All participants gave written consent. Each study measured right arm blood pressure after resting for half an hour three times at different times, as
the average of three measurements values per measured object.
Genotyping DNA was purified from samples of whole white blood cells stored frozen at 20 8C. Purification was performed using a DNA Purification kit (Promega Inc., Madison, WI, USA) according to the manufacturer’s instructions. A polymerase chain reaction (PCR) test that was designed and produced by Ying-Jun Biological Company, Shanghai, China was conducted with the upstream primer, 50 -CCTCCTCAGACCCTTACACGG-30 and downstream primer, 50 -CATCCCAGGCAGCCAGACT-30 . A 20 mL reaction volume was used for PCR containing 10 mL 2mix (including Mg2+, dNTPs and Taq DNA polymerase), 1.0 mL upstream primer (10 pmol), 1.0 mL downstream primer (10 pmol), 2.0 mL genomic DNA template (up to 4.0 mL, according to the concentration) and 6.0 mL ddH2O. The reaction began with denaturation at 94 8C for 5 min, followed by 35 cycles of denaturation at 95 8C (30 s), annealing at 60 8C (30 s), extension at 72 8C (30 s) and a final extension at 72 8C (7 min). Genotype analysis was performed using DNA sequencing completed by Ying-Jun Biological Company.
Statistical analysis Allele frequencies were calculated by allele counting. Analyses for possible deviations of the genotype distribution from that expected for a population in Hardy-Weinberg equilibrium were performed with the x2 test. Data are presented as the mean SD or number (proportion, %). Continuous variables with a Gaussian distribution, as determined by the ShapiroWilk test, were compared by one-way analysis of variance (ANOVA) or t-tests. Categorical values were compared by the x2 test. Continuous variables with a non-Gaussian distribution were compared by the Mann-Whitney U test. Analyses were performed using the SPSS statistical software (Version 13.0, SPSS Inc, Chicago, IL USA). P<0.05 was considered to indicate a statistically significant difference.
Results The distribution of clinical and procedure-associated factors, including age, gender, body mass index (BMI) and fasting blood glucose, were not observed to be significantly different in either the control or test groups. However, hypertension patients had systolic blood pressure, diastolic blood pressure, an alcohol consumption and smoking history that were significantly higher than the control group (Table 1).
Connexin37 C1019T genotyping CX37 gene sequencing results identified three genotypes: CC, TT, and TC (Fig. 1).
CX37 genotype and allele frequency The frequency of the connexin37 C allele was higher in EH patients (57.4% vs. 42.1%, P<0.01) compared to the control
Please cite this article in press as: Guo S, et al. Association Between 1019C/T Polymorphism in the Connexin 37 Gene and Essential Hypertension. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.02.016
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Association between 1019C/T Polymorphism
Table 1 Comparison of patient clinical data.
D
Control group
EH group
P value
(n=874)
(n=1126)
(x2)
P
Age (years)
69.56.1
72.38.6
1.32
0.21
Gender (male/female)
433/441
567/559
0.13
0.71
1.17
0.29
BMI
23.92.2
23.72.1
Systolic blood pressure (mmHg)
121.612.2
156.414.4D
13.6
Diastolic blood pressure (mmHg)
79.47.2
95.98.7D
11.2
Fasting blood glucose (mmol/L)
5.20.7
5.30.8
History
672/202
987/139D
40.3
0.00
Smoking (yes/no) Drinking (yes/no)
537/337
862/264D
53.5
0.00
1.69
0.02 0.03 0.18
P<0.05, others P>0.05.
group. The frequency of C carriers (CC+TC) was 80.5% in the EH patients and 66.7% in the control (P<0.01). The EH risk was significantly increased in carriers of the C allele (CC+TC) compared with TT homozygotes (OR, 2.06; 95% CI: 1.682.52), Table 2.
CX37 genotype and allele frequency between sexes Stratified analysis demonstrated significant differences in the frequency of C carriers between the male EH patients and
Figure 1 Gene sequencing diagram. (A) CC type. (B) TT type. (C) TC type.
Please cite this article in press as: Guo S, et al. Association Between 1019C/T Polymorphism in the Connexin 37 Gene and Essential Hypertension. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.02.016
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Table 2 Gene polymorphism results in the EH and control groups (%). Group
EH group
Genotype
Allele
CC
CT
TT
CC+TC
C
T
386(34.3)
520(46.2)
220(19.5)
906(80.5)
1292(57.4)
960(42.6)
152(17.4)
431(49.3)
291(33.3)
583(66.7)
735(42.1)
1013(58.0)
(n=1126) Control group (n=874) x2
89.6
P
0.00
OR(95%CI)
49.0
92.5
0.00a
0.00
2.06(1.682.52)
1.86(1.64-2.11)
Note: compared with TT genotype.
Table 3 Gene polymorphism results in the male EH and control groups (%). Group
EH group (n=567) Control group
Genotype
Allele
CC
CT
TT
CC+TC
C
T
182(32.1)
267(47.1)
118(20.8)
449(79.2)
631(55.6)
503(44.4)
68(15.7)
231(53.4)
134(31.0)
299(69.1)
367(42.4)
499(57.6)
(n=433) x2
38.3
P
0.00
OR(95%CI)
13.4
34.6
0.00a
0.00
1.71(1.282.27)
1.71(1.43-2.04)
Note: compared with TT genotype.
Table 4 Gene polymorphism results in female EH and control groups (%). Group
EH group
Genotype
Allele
CC
CT
TT
CC+TC
C
T
204(36.5)
253(45.3)
102(18.3)
457(81.8)
661(59.1)
457(40.9)
84(19.1)
200(45.4)
157(35.6)
284(64.4)
368(41.7)
514(58.3)
(n=559) Control group (n=441) x2 P
54.7 0.00
OR(95%CI)
38.7
26.4
0.00a
0.00
2.48(1.85-3.31)
2.02(1.69-2.42)
Note: compared with TT genotype.
controls (79.2% vs. 69.1%, x2=13.4, P<0.01), and between female EH patients and controls (81.8% vs. 64.4%, x2=38.7, P<0.01). Carriers of the C allele had higher EH risk compared with TT homozygote without sex differences (male: OR=1.71, 95% CI: 1.282.27; female: OR=2.48, 95%CI: 1.853.31), Tables 3 and 4.
Discussion Connexins form transmembrane channels called gap junctions which connect neighbouring cells and allow the passive diffusion of small molecules below 1KDa. 6 CX formed the half channel on one side of the membrane, and the other half of the
Please cite this article in press as: Guo S, et al. Association Between 1019C/T Polymorphism in the Connexin 37 Gene and Essential Hypertension. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.02.016
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adjacent channel on the cell membrane docking, assembled to form a complete channel [24]. Studies show this plays an important role in the pathogenesis of hypertension. Normal vascular tone and blood pressure depend on vasodilation and contraction to balance blood pressure and tissue perfusion. Hypertension is the imbalance of the vasomotor response to where vasorelaxation is weakened. Vasorelaxation depends on the vascular endothelium, acetylcholine (Ach), bradykinin, adenosine, blood flow shear stress and other stimuli, endothelial cell activation, and the release of vasodilator substances to diffuse subendothelial smooth muscle cells, causing vascular diastole. Known endothelial vasodilators are nitric oxide (NO), prostacyclin (PGI2) and endothelium-dependent hyperpolarising factor (EDHF). EDHFs role in hypertension on resistance arteries and small artery vasodilation is particularly notable. Animal experiments in rats [25] showed spontaneously hypertensive resistance arteries EDHF-mediated vasodilation was significantly reduced, and with prolonged duration of hypertension such weakening the more aggravating, but in normotensive rats no such change in the regulation of blood pressure proved EDHF plays an important role. It is still unclear and controversial as to the identity of all EHDF-like vasodilatory factors. However a key consensus is that external factors which stimulate blood vessels, activate their respective receptors, alter endothelial cell Ca2+ concentration, and thereby activate the small, medium conductivitytype calcium-dependent potassium channels, which affect the intracellular K+ outflow and thus endothelial cell hyperpolarisation and signal transduction leading to hyperpolarisation of vascular smooth muscle cells causing vasodilation. Involvement of endothelial cell gap junctions with smooth muscle cells has been identified by studies using non-specific gap junction inhibitors such as the uncoupler 18a-and 18bglycyrrhetinic acid to block endothelium-dependent hyperpolarisation, ultimately affecting multiple vascular beds and diastolic pressure [26–28]. Thus, gap junctions involved EDHF mediated vasodilation in the incidence of hypertension and the development process is essential. Connexins are members of a family of proteins encoded by at least 20 different mammalian genes expressed in vasculature such as vascular endothelium and smooth muscle cells. In an animal model [29], compared with normotensive mice, spontaneously hypertensive mice aortic endothelial CX37 expression decreased 59% (P<0.001), after carvedilol treatment for a week, CX37 decline was completely reversed, while spontaneously hypertensive mice blood pressure returned to normal. It was suggested that induced connexin37 expression may be an indicator of blood pressure control. The human connexin37 gene maps to chromosome lp35.1 and encodes 333 amino acids which form the connexin37 protein. Although it is not clear which allele is the more closely associated, the majority of gene polymorphismassociation studies have detected that the C1019T SNP in the human connexin37 gene is associated with coronary heart disease in various populations. However, whether such a
polymorphism is related to essential hypertension has not reported. The present study of 1,126 EH patients and 874 healthy controls gene polymorphism, reports that the C allele frequency distribution in the EH group was significantly higher than the normal control group. In addition, we find that this correlated with significantly increased risk of EH, however gender subgroup analysis suggests that this correlation is not gender related. We presently suggest that genetic polymorphisms resulting in encoded CX37 protein tertiary structure abnormalities, contribute to altered formation of gap junction channels. Consequently, vasodilatory dysfunction that contributes to higher blood pressure involves greater hyperpolarisation of endothelial cells and abnormal function of smooth muscle cells. Further studies are needed to examine CX37-related effects on these cells.
References [1] Larsson E, Wahlstrand B, Hedblad B, Hedner T, Kjeldsen SE, Melander O, et al. Hypertension and genetic variation in endothelial-specific genes. PLOS ONE 2013;8(4):e62035. [2] Asai T, Ohkubo T, Katsuya T, Higaki J, Fu Y, Fukuda M, et al. Endothelin-1 gene variant associates with blood pressure in obese Japanese subjects: the Ohasama Study. Hypertension 2001;38:1321–4. [3] Glenn CL, Wang WY, Morris BJ. Different frequencies of inducible nitric oxide synthase genotypes in older hypertensives. Hypertension 1999;33: 927–32. [4] Rutherford S, Johnson MP, Curtain RP, Griffiths LR. Chromosome 17 and the inducible nitric oxide synthase gene in human essential hypertension. Hum Genet 2001;109:408–15. [5] Tiret L, Poirier O, Hallet V, McDonagh TA, Morrison C, McMurray JJ, et al. The Lys198Asn polymorphism in the endothelin-1 gene is associated with blood pressure in overweight people. Hypertension 1999;33:1169–74. [6] Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 2007;447:661–78. [7] Ehret GB, Munroe PB, Rice KM, Bochud M, Johnson AD, Chasman DI, et al. International Consortium for Blood Pressure Genome-Wide Association Studies (2011) Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk. Nature 478: 103–109. [8] Saxena R, Voight BF, Lyssenko V, Burtt NP, de Bakker PI, Chen H, et al. Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science 2007;316:1331–6. [9] Levy D, Ehret GB, Rice K, Verwoert GC, Launer LJ, et al. Genomewide association study of blood pressure and hypertension. Nat Genet 2009;41:677–87. [10] Newton-Cheh C, Johnson T, Gateva V, Tobin MD, Bochud M, Coin L, et al. Genome-wide association study identifies eight loci associated with blood pressure. Nat Genet 2009;41:666–76. [11] Saez JC, Berthoud VM, Branes MC, Martinez AD, Beyer EC. Plasma membrane channels formed by connexins: their regulation and functions. Physiol Rev 2003;83:1359–400. [12] Fang JS, Angelov SN, Simon AM, Burt JM. Cx37 deletion enhances vascular growth and facilitates ischemic limb recovery. Am J Physiol Heart Circ Physiol 2011;301:H1872–81. [13] Kanady JD, Dellinger MT, Munger SJ, Witte MH, Simon AM. Connexin37 and Connexin43 deficiencies in mice disrupt lymphatic valve development and result in lymphatic disorders including lymphedema and chylothorax. Dev Biol 2011;354:253–66. [14] Yeh HI, Chang HM, Lu WW, Lee YN, Ko YS, Severs NJ, et al. Agerelated alteration of gap junction distribution and connexin expression in rat aortic endothelium. J Histochem Cytochem 2000;48:1377–89. [15] Pfenniger A, Wong C, Sutter E, Cuhlmann S, Dunoyer-Geindre S, Mach F, et al. Shear stress modulates the expression of the atheroprotective protein Cx37 in endothelial cells. J Mol Cell Cardiol 2012;53:299–309. [16] Fang JS, Angelov SN, Simon AM, Burt JM. Cx40 is required for, and cx37 limits, postischemic hindlimb perfusion, survival and recovery. J Vasc Res 2012;49:2–12. [17] Boerma M, Forsberg L, Van Zeij L, Morgenstern R, De Faire U, Lemne C, et al. A genetic polymorphismin connexin 37 as a prognostic marker for atherosclerotic plaque development. J Intern Med 1999;246:211–8.
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[18] Yeh HL, Chou Y, Liu HF, Chang SC, Tsai CH. Connexin37 gene polymorphism and coronary artery disease in Taiwan. Int J Cardiol 2001;81:251–5. [19] Han YL, Xi SY, Zhang XL, Yan CH, Yang Y, Kang J. Association of C1019T polymorphism in the Connexin 37 gene and coronary artery disease in Chinese Han population. Zhonghua Yi Xue Za Zhi 2007;87:100–4 (In Chinese). [20] Wong CW, Christen T, Pfenniger A, James RW, Kwak BR. Do allelic variants of the connexin37 1019 gene polymorphism differentially predict for coronary artery disease and myocardial infarction. Atherosclorosis 2007;191:355–61. [21] Yamada Y, Izawa H, Ichihara S, Takatsu F, Ishihara H, Hirayama H, et al. Prediction of the risk of myocardial infarction from polymorphisms in candidate genes. N Engl J Med 2002;347:1916–23. [22] Listi F, Candore G, Lio D, Russo M, Colonna-Romano G, Caruso M, et al. Association between C1019T polymorphism of connexin37 and acute myocardial infarction: a study in patients from Sicily. Int J Cardiol 2005;102:269–71. [23] Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL, et al. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure: the JNC 7
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report. JAMA 2003; 289: 2560-2572. [PMID: 12748199 http://dx.doi.org/ 10.1001/jama.289.19.2560]. Kumar NM, Gilula NB. The gap junction communication channel. Cell 1996;84. 381–318. Mori Y, Ohyanagi M, Koida S, Ueda A, Ishiko K, Iwasaki T. Effects of Endothelium-Derived Hyperpolarizing Factor and Nitric Oxide on Endothelial Function in Femoral Resistance Arteries of Spontaneously Hypertensive Rats. Hypertens Res 2006;29(3):187–95. Sandow SL, Bramich NJ, Bandi HP, Rummery NM, Hill CE. Structure, function, and endothelium-derived hyperpolarizing factor in the caudal artery of the SHR and WKY rat. Arterioscler Thromb Vasc Biol 2003;23:822–8. Coleman HA, Tare M, Parkington HC. EDHF is not K+ but may be due to spread of current from the endothelium in guinea pig arterioles. Am J Physiol 2001;280:H2478–83. Sandow SL, Tare M, Coleman HA, Hill CE, Parkington HC. Involvement of myoendothelial gap junctions in the actions of endotheliumderived hyperpolarizing factor. Circ Res 2002;90:1108–13. Yeh HI, Lee PY, Su CH, Tian TY, Ko YS, Tsai CH. Reduced expression of endothelial connexins 43 and 37 in hypertensive rats is rectified after 7day carvedilol treatment. Am J Hypertens 2006;19(2):129–35.
Please cite this article in press as: Guo S, et al. Association Between 1019C/T Polymorphism in the Connexin 37 Gene and Essential Hypertension. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.02.016
Heart, Lung and Circulation (2014) xx, 1–6 1443-9506/04/$36.00 http://dx.doi.org/10.1016/j.hlc.2014.02.016
ORIGINAL ARTICLE
Association Between 1019C/T Polymorphism in the Connexin 37 Gene and Essential Hypertension Suxia Guo 1*, Weixiang Chen, Ying Yang, Zhenyu Yang 1*, Minghua Cao Department of Cardiology, Affiliated People’s Hospital of Nanjing Medical University in Wuxi and People’s Hospital of Wuxi City, Wuxi, Jiangsu, 214023 P.R.China Received 15 January 2014; received in revised form 11 February 2014; accepted 17 February 2014; online published-ahead-of-print xxx
Objective
To investigate the association between the CX 37 1019C/T polymorphism and the susceptibility to essential hypertension (EH).
Methods
A total of 1126 cases of EH were diagnosed in the People’s Hospital of Wuxi City, China. A control group consisted of 874 healthy people, i.e., non-EH patients. All cases were genotyped by DNA sequencing.
Results
Polymorphism C1019T on the Connexin37 gene was found in the whole population. The distribution of three genotype frequencies in both groups was in accordance with the Hardy-Weinberg equilibrium. The frequency of the CX37 C allele was higher in EH patients (57.4% vs. 42.1%, x2=92.5, P<0.01) compared to the control group. The frequency of C carriers (CC+TC) was 80.5% in EH patients compared to 66.7% in the control (x2=49.0, P<0.01). EH risk was significantly increased in carriers of C the allele (CC+TC) over that in the TT homozygote (OR=2.06, 95% CI: 1.682.52). Subsequent stratified analyses demonstrate that a significant difference exists in the frequency of C carriers between male EH patients and controls (79.2% vs. 69.1%, x2=13.4, P<0.01) and in female EH patients and the control group (81.8% vs. 64.4%, x2=38.7, P<0.01). The carriers of the C allele had higher EH risk compared with the TT homozygote without sex differences (male: OR=1.71, 95% CI: 1.282.27; female: OR=2.48, 95%CI: 1.853.31).
Conclusion
The C allele in the CX37 gene might be associated with the susceptibility to EH in population of Wuxi, China.
Keywords
Essential hypertension Connexin37gene Genotype Allele frequency C1019T
Introduction Essential hypertension (EH) is a worldwide, common form of hypertension disease and a risk factor in heart disease, cerebrovascular disease and chronic kidney disease. Recent work suggests that the pathogenesis is poorly understood and that the primary cause is unknown in 90–95% of cases [1]. Heredity is estimated to account for 30-50% of disease cases so efforts are ongoing to elucidate genetic links. Large meta-analyses and multi-centre studies prove robust genetic associations with hypertension [6–10]. Genetic
variations in endothelial genes such as endothelin-1 and endothelial nitric oxide synthase may contribute to essential hypertension–and both relate to blood pressure (BP) regulation [2–5]. However, another study showed that hypertension links to genetic factors proved more challenging. It recommended that further research into the pathogenesis of EH was needed as its results contradicted other study’s results which demonstrated positive genetic linkages. Connexins are a protein family encoded by at least 20 different mammalian genes expressed in a variety of tissues
*Corresponding authors at: Department of Cardiology, Affiliated People’s Hospital of Nanjing Medical University in Wuxi and People’s Hospital of Wuxi City, Wuxi, Jiangsu, 214023 P.R.China, China., Email: [email protected] 1
Suxia Guo and Weixiang Chen have contributed equally to this study. So Suxia Guo and Weixiang Chen should be regarded as first joint authors.
© 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier Inc. All rights reserved.
Please cite this article in press as: Guo S, et al. Association Between 1019C/T Polymorphism in the Connexin 37 Gene and Essential Hypertension. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.02.016
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[11–13]. They form trans-membrane channels called gap junctions that connect neighbouring cells and allow passive diffusion of small connexin molecules. Connexin37 is mainly expressed in the vascular endothelium which is involved in growth, injured tissue regeneration and endothelial cells’ aging, suggesting that changes in connexin37 are associated with diseases following intima injury [14–16]. Boerma initially linked a single nucleotide polymorphism (SNP) in the human connexin37 gene with thickening of the carotid in a Swedish male population [17] by observing the C allele overrepresented in individuals with atherosclerotic plaques. The C SNP allele was also associated with coronary artery disease (CAD) in Taiwan, northern China and Switzerland [18–20]. Two other study results show the T SNP allele to be a risk factor for acute myocardial infarction (AMI) particularly in Japanese and Caucasian males [21,22], while most gene polymorphism-association studies suggest that the C1019T SNP in the human connexin37 gene is associated with CAD and myocardial infarction (MI) in various populations. Whether polymorphism can be an influence in EH is the subject of this study as is the relationship between C1019T polymorphism in the connexin37 gene, EH and CAD. Study shows it is associated with CAD but relationships to hypertension have not been reported even though 60-70% of CAD patients are also diagnosed with hypertension. The present study explores the association between the C1019T polymorphism in the connexin37 gene and EH.
Methods A total of 1126 Han Chinese diagnosed with essential hypertension between 2008 and 2011 were studied and compared with a control group of 874 patients without EH using a patient cohort from the People’s Hospital of Wuxi City, China. Cohort inclusion criteria included: 1) persons over 60 years of age; 2) diagnosed with EH according to the European Society of Hypertension and the European Society of Cardiology classification schemes [23]; 3) hypertension diagnosed as systolic blood pressure 140 mmHg (1 mmHg = 0.133 kPa) and / or diastolic blood pressure 90 mmHg and 4) excluded secondary hypertension diagnoses. An additional 874, over 60 year-old individuals, characterised as healthy and without hypertension or a family history of such after biochemical and clinical examinations, were used as a control population. No test or control groups included blood relatives. At the same time, except for severe acute and chronic infections, cancer, liver and kidney dysfunction and other systemic diseases, coronary artery disease, valvular heart disease, primary cardiomyopathy, cerebrovascular accident and diabetes. The ethics committee of the People’s Hospital of Wuxi City approved the study. All participants gave written consent. Each study measured right arm blood pressure after resting for half an hour three times at different times, as
the average of three measurements values per measured object.
Genotyping DNA was purified from samples of whole white blood cells stored frozen at 20 8C. Purification was performed using a DNA Purification kit (Promega Inc., Madison, WI, USA) according to the manufacturer’s instructions. A polymerase chain reaction (PCR) test that was designed and produced by Ying-Jun Biological Company, Shanghai, China was conducted with the upstream primer, 50 -CCTCCTCAGACCCTTACACGG-30 and downstream primer, 50 -CATCCCAGGCAGCCAGACT-30 . A 20 mL reaction volume was used for PCR containing 10 mL 2mix (including Mg2+, dNTPs and Taq DNA polymerase), 1.0 mL upstream primer (10 pmol), 1.0 mL downstream primer (10 pmol), 2.0 mL genomic DNA template (up to 4.0 mL, according to the concentration) and 6.0 mL ddH2O. The reaction began with denaturation at 94 8C for 5 min, followed by 35 cycles of denaturation at 95 8C (30 s), annealing at 60 8C (30 s), extension at 72 8C (30 s) and a final extension at 72 8C (7 min). Genotype analysis was performed using DNA sequencing completed by Ying-Jun Biological Company.
Statistical analysis Allele frequencies were calculated by allele counting. Analyses for possible deviations of the genotype distribution from that expected for a population in Hardy-Weinberg equilibrium were performed with the x2 test. Data are presented as the mean SD or number (proportion, %). Continuous variables with a Gaussian distribution, as determined by the ShapiroWilk test, were compared by one-way analysis of variance (ANOVA) or t-tests. Categorical values were compared by the x2 test. Continuous variables with a non-Gaussian distribution were compared by the Mann-Whitney U test. Analyses were performed using the SPSS statistical software (Version 13.0, SPSS Inc, Chicago, IL USA). P<0.05 was considered to indicate a statistically significant difference.
Results The distribution of clinical and procedure-associated factors, including age, gender, body mass index (BMI) and fasting blood glucose, were not observed to be significantly different in either the control or test groups. However, hypertension patients had systolic blood pressure, diastolic blood pressure, an alcohol consumption and smoking history that were significantly higher than the control group (Table 1).
Connexin37 C1019T genotyping CX37 gene sequencing results identified three genotypes: CC, TT, and TC (Fig. 1).
CX37 genotype and allele frequency The frequency of the connexin37 C allele was higher in EH patients (57.4% vs. 42.1%, P<0.01) compared to the control
Please cite this article in press as: Guo S, et al. Association Between 1019C/T Polymorphism in the Connexin 37 Gene and Essential Hypertension. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.02.016
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Table 1 Comparison of patient clinical data.
D
Control group
EH group
P value
(n=874)
(n=1126)
(x2)
P
Age (years)
69.56.1
72.38.6
1.32
0.21
Gender (male/female)
433/441
567/559
0.13
0.71
1.17
0.29
BMI
23.92.2
23.72.1
Systolic blood pressure (mmHg)
121.612.2
156.414.4D
13.6
Diastolic blood pressure (mmHg)
79.47.2
95.98.7D
11.2
Fasting blood glucose (mmol/L)
5.20.7
5.30.8
History
672/202
987/139D
40.3
0.00
Smoking (yes/no) Drinking (yes/no)
537/337
862/264D
53.5
0.00
1.69
0.02 0.03 0.18
P<0.05, others P>0.05.
group. The frequency of C carriers (CC+TC) was 80.5% in the EH patients and 66.7% in the control (P<0.01). The EH risk was significantly increased in carriers of the C allele (CC+TC) compared with TT homozygotes (OR, 2.06; 95% CI: 1.682.52), Table 2.
CX37 genotype and allele frequency between sexes Stratified analysis demonstrated significant differences in the frequency of C carriers between the male EH patients and
Figure 1 Gene sequencing diagram. (A) CC type. (B) TT type. (C) TC type.
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Table 2 Gene polymorphism results in the EH and control groups (%). Group
EH group
Genotype
Allele
CC
CT
TT
CC+TC
C
T
386(34.3)
520(46.2)
220(19.5)
906(80.5)
1292(57.4)
960(42.6)
152(17.4)
431(49.3)
291(33.3)
583(66.7)
735(42.1)
1013(58.0)
(n=1126) Control group (n=874) x2
89.6
P
0.00
OR(95%CI)
49.0
92.5
0.00a
0.00
2.06(1.682.52)
1.86(1.64-2.11)
Note: compared with TT genotype.
Table 3 Gene polymorphism results in the male EH and control groups (%). Group
EH group (n=567) Control group
Genotype
Allele
CC
CT
TT
CC+TC
C
T
182(32.1)
267(47.1)
118(20.8)
449(79.2)
631(55.6)
503(44.4)
68(15.7)
231(53.4)
134(31.0)
299(69.1)
367(42.4)
499(57.6)
(n=433) x2
38.3
P
0.00
OR(95%CI)
13.4
34.6
0.00a
0.00
1.71(1.282.27)
1.71(1.43-2.04)
Note: compared with TT genotype.
Table 4 Gene polymorphism results in female EH and control groups (%). Group
EH group
Genotype
Allele
CC
CT
TT
CC+TC
C
T
204(36.5)
253(45.3)
102(18.3)
457(81.8)
661(59.1)
457(40.9)
84(19.1)
200(45.4)
157(35.6)
284(64.4)
368(41.7)
514(58.3)
(n=559) Control group (n=441) x2 P
54.7 0.00
OR(95%CI)
38.7
26.4
0.00a
0.00
2.48(1.85-3.31)
2.02(1.69-2.42)
Note: compared with TT genotype.
controls (79.2% vs. 69.1%, x2=13.4, P<0.01), and between female EH patients and controls (81.8% vs. 64.4%, x2=38.7, P<0.01). Carriers of the C allele had higher EH risk compared with TT homozygote without sex differences (male: OR=1.71, 95% CI: 1.282.27; female: OR=2.48, 95%CI: 1.853.31), Tables 3 and 4.
Discussion Connexins form transmembrane channels called gap junctions which connect neighbouring cells and allow the passive diffusion of small molecules below 1KDa. 6 CX formed the half channel on one side of the membrane, and the other half of the
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adjacent channel on the cell membrane docking, assembled to form a complete channel [24]. Studies show this plays an important role in the pathogenesis of hypertension. Normal vascular tone and blood pressure depend on vasodilation and contraction to balance blood pressure and tissue perfusion. Hypertension is the imbalance of the vasomotor response to where vasorelaxation is weakened. Vasorelaxation depends on the vascular endothelium, acetylcholine (Ach), bradykinin, adenosine, blood flow shear stress and other stimuli, endothelial cell activation, and the release of vasodilator substances to diffuse subendothelial smooth muscle cells, causing vascular diastole. Known endothelial vasodilators are nitric oxide (NO), prostacyclin (PGI2) and endothelium-dependent hyperpolarising factor (EDHF). EDHFs role in hypertension on resistance arteries and small artery vasodilation is particularly notable. Animal experiments in rats [25] showed spontaneously hypertensive resistance arteries EDHF-mediated vasodilation was significantly reduced, and with prolonged duration of hypertension such weakening the more aggravating, but in normotensive rats no such change in the regulation of blood pressure proved EDHF plays an important role. It is still unclear and controversial as to the identity of all EHDF-like vasodilatory factors. However a key consensus is that external factors which stimulate blood vessels, activate their respective receptors, alter endothelial cell Ca2+ concentration, and thereby activate the small, medium conductivitytype calcium-dependent potassium channels, which affect the intracellular K+ outflow and thus endothelial cell hyperpolarisation and signal transduction leading to hyperpolarisation of vascular smooth muscle cells causing vasodilation. Involvement of endothelial cell gap junctions with smooth muscle cells has been identified by studies using non-specific gap junction inhibitors such as the uncoupler 18a-and 18bglycyrrhetinic acid to block endothelium-dependent hyperpolarisation, ultimately affecting multiple vascular beds and diastolic pressure [26–28]. Thus, gap junctions involved EDHF mediated vasodilation in the incidence of hypertension and the development process is essential. Connexins are members of a family of proteins encoded by at least 20 different mammalian genes expressed in vasculature such as vascular endothelium and smooth muscle cells. In an animal model [29], compared with normotensive mice, spontaneously hypertensive mice aortic endothelial CX37 expression decreased 59% (P<0.001), after carvedilol treatment for a week, CX37 decline was completely reversed, while spontaneously hypertensive mice blood pressure returned to normal. It was suggested that induced connexin37 expression may be an indicator of blood pressure control. The human connexin37 gene maps to chromosome lp35.1 and encodes 333 amino acids which form the connexin37 protein. Although it is not clear which allele is the more closely associated, the majority of gene polymorphismassociation studies have detected that the C1019T SNP in the human connexin37 gene is associated with coronary heart disease in various populations. However, whether such a
polymorphism is related to essential hypertension has not reported. The present study of 1,126 EH patients and 874 healthy controls gene polymorphism, reports that the C allele frequency distribution in the EH group was significantly higher than the normal control group. In addition, we find that this correlated with significantly increased risk of EH, however gender subgroup analysis suggests that this correlation is not gender related. We presently suggest that genetic polymorphisms resulting in encoded CX37 protein tertiary structure abnormalities, contribute to altered formation of gap junction channels. Consequently, vasodilatory dysfunction that contributes to higher blood pressure involves greater hyperpolarisation of endothelial cells and abnormal function of smooth muscle cells. Further studies are needed to examine CX37-related effects on these cells.
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Please cite this article in press as: Guo S, et al. Association Between 1019C/T Polymorphism in the Connexin 37 Gene and Essential Hypertension. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.02.016