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Clinical Chemistry 51: 952-956, 2005. First published February 25, 2005; 10.1373/clinchem.2004.046276
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(Clinical Chemistry. 2005;51:952-956.)
© 2005 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Impact of {alpha}ENaC Polymorphisms on the Risk of Ischemic Cerebrovascular Events: A Multicenter Case–Control Study

Kety Hsieh1, Wolfgang Lalouschek2, Martin Schillinger3, Georg Endler1, Manuela Reisinger1, Michael Janisiw1, Wilfried Lang2, Suzanne Cheng4, Oswald Wagner1,a and Christine Mannhalter1,a

1 Clinical Institute for Medical and Chemical Laboratory Diagnostics, 2 University Clinic of Neurology, Clinical Department of Clinical Neurology, and 3 University Clinic of Internal Medicine II, Clinical Department of Angiology, Medical University Vienna, Vienna, Austria.
4 Department of Human Genetics, Roche Molecular Systems, Inc., Alameda, CA.

aThese authors shared responsibility for the project and should both be considered corresponding authors. Address for correspondence: Clinical Institute of Medical and Chemical Laboratory Diagnostics, AKH-Wien, Waehringer Guertel 18-20, 1097 Vienna, Austria. Fax 43-1-40400-2097; e-mail christine.mannhalter{at}meduniwien.ac.at or oswald.wagner{at}meduniwien.ac.at.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Amiloride-sensitive epithelial sodium channels (ENaCs) are important candidates in the development of hypertension, which is a major risk factor for stroke. Two single-nucleotide polymorphisms (SNPs) in the gene that encodes the ENac {alpha}-subunit ({alpha}ENaC) have been identified. We evaluated those SNPs for a possible association with ischemic cerebrovascular events (ICEs).

Methods and Results: We genotyped 1399 patients with ICEs [median age, 70 years; interquartile range, 58–78 years; 745 (53%) men] and 1076 control individuals without vascular disease [47 (39–58) years; 557 (52%) men] for the SNPs Trp493Arg and Ala663Thr. The SNP frequencies at nucleotide 3977 (Trp493Arg) in the {alpha}ENaC gene were significantly different in patients and controls. Carriers of 493Arg had a 1.78-fold increased risk (95% confidence interval, 1.02–3.12) for ICEs compared with Trp/Trp carriers. Interaction analysis revealed that the relative risk was even higher in women in the lowest age tertile [adjusted odds ratio, 3.26 (1.10–9.72)].

Conclusions: Carriers of the 493Arg allele are at increased risk for ICEs compared with Trp/Trp carriers. The effect is independent of traditional vascular risk factors and is particularly evident in younger women. The Trp493Arg variant in {alpha}ENaC may represent an important candidate genetic susceptibility factor in the development of ICEs.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Stroke represents one of the leading causes of morbidity and mortality in industrialized countries. Twin and family studies, together with observations in animal models, have shown that familial predisposition, in addition to established risk factors such as hypertension, smoking, diabetes, obesity, and age, contributes to the risk of stroke (1). Identification and characterization of gene variants that mediate this genetic predisposition may allow improved therapy and prevention (2)(3)(4)(5). The role of individual mutations has been controversial. This is related in part to the fact that genetic influences in stroke are polygenic and that stroke comprises several different phenotypes. The effect of a genetic alteration may depend on a permissive background. The presence of a genetic variant could become phenotypically evident only in combination with modulating exogenous factors such as smoking, diet, or obesity, among others, and may increase the risk of disease.

The results of epidemiologic and animal studies have indicated that genetic influences play a role in the pathogenesis of hypertension (6), which is the major risk factor for stroke. Amiloride-sensitive epithelial sodium channels (ENaCs) 1 are responsible for the rate-limiting step of sodium reabsorption in the distal nephron and are therefore important candidates in the development of hypertension. We evaluated the association of 2 common single-nucleotide polymorphisms (SNPs) in the {alpha}-subunit of the ENaC ({alpha}ENaC; SCNN1A; OMIM 600228) with ischemic cerebrovascular events (ICEs).


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patients and controls
Between October 1998 and December 2001, a total of 3516 Caucasian patients were recruited for the Vienna Stroke Registry database (7). Of these patients, 1397 with ICEs from 8 hospitals in Vienna could be included in the study. At the time of this study, 731 of 3516 clinical questionnaires (20%) had not been completed; 992 (28%) patients had not provided informed consent or blood or DNA samples; for 145 (4%) patients, the diagnosis of cerebrovascular event had not been confirmed; and for 251 (7%) individuals, the cerebrovascular event was not ischemic. Importantly, the included patients were not significantly different from patients who could not be included with respect to age and sex distributions and stroke severity. The control group comprised 1078 individuals without a personal and/or familial history of arterial diseases, all participants of an official healthcare program in the city of Vienna. All control individuals were of Caucasian origin, and all individuals gave written informed consent. The study complied with the Declaration of Helsinki and was approved by the local ethics committee.

The detailed clinical questionnaire included documentation of each participant’s medical history, with special reference to vascular diseases, including family history, risk factors, and medications. Results of technical and laboratory investigations, information regarding clinical severity as measured by validated scales, and the outcome and etiology of the event were documented (8).

definitions
Patients with ICEs had either transient ischemic attacks or ischemic strokes verified clinically by a neurologist. Intracerebral hemorrhage was excluded by neuroradiology in all patients. Arterial hypertension was defined as either a history of arterial hypertension or blood pressure values above 160/95 mmHg 1 week after the event. Hypertension was also considered present in patients receiving antihypertensive medication. Diabetes mellitus was assumed when fasting blood glucose concentrations were >1500 mg/L or if a history of diabetes was reported (including patients receiving diabetes medication). This glucose concentration was chosen because the acute-phase reaction in stroke patients can be associated with increased blood glucose even in the absence of diabetes. Hyperlipidemia was defined as fasting total serum cholesterol >2400 mg/L and was assumed to be present in all patients receiving lipid-lowering medications.

genotyping assays
Two common SNPs in {alpha}ENaC, Trp493Arg (9) and Ala663Thr (10), were tested as described previously. Briefly, DNA was extracted with the Puregene® DNA Isolation Kit (Gentra System Inc.). Genotyping was performed by PCR amplification followed by allele-specific hybridization essentially as reported previously (11).

statistical analysis
Continuous data are reported as the median and interquartile range (25th–75th percentiles). Discrete data are reported as counts and frequencies. {chi}2 tests or, if appropriate, exact tests were used to compare groups of categorical data. Groups of continuous data were compared by the Mann–Whitney U-test or, as appropriate, the Kruskal–Wallis test.

The SNPs were entered in a multivariate binary logistic regression model adjusting for potentially confounding effects of other baseline variables. These were selected for the final model if they had either a clinically/biologically plausible relationship with the outcome or were distributed unevenly between the genotypes, indicated by a P value <0.20. We used a hierarchical modeling strategy to test separately and jointly for an effect of demographic variables and cardiovascular risk factors. Primary interactions with demographic variables and vascular risk factors were assessed by multiplicative interaction terms and log likelihood-ratio {chi}2 tests. The linearity of the logit assumption was checked for continuous predictor variables, and an analysis of residuals was performed. Regression analysis and overall model fit were performed according to standard procedures (12). The Cox–Snell R2 was used to assess the variability explained by each model. Stratified analyses were adjusted for the abovementioned baseline variables.

A 2-sided P value ≤0.05 was considered statistically significant. Calculations were performed with SPSS (Ver. 10.0; SPSS Inc.). Haplotype analyses were performed with a Bayesian method using PHASE (Ver. 2.0.2) (13).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
analyses of demographic data
The demographic data and baseline characteristics for patients and controls are given in Table 1 . The patient population was significantly older and had a higher prevalence of vascular risk factors than controls. Consequently, we adjusted for these differences by multivariate logistic regression analysis.


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  		Table 1. Demographic data and clinical characteristics of patients with ischemic stroke and controls.

analyses of genotyping
In our cohort, the frequency of carriers of the rare 493Arg allele was significantly higher in patients [n = 80 (6%)] than in controls [n = 31 (3%); Table 2 ], whereas the frequency of the Thr663Ala variant was similar in both groups. Because the screening model indicated increased odds for stroke in the presence of 1 Arg allele, all additional statistical analyses were performed assuming a dominant effect (Table 3 ). Baseline variables were evenly distributed between groups according to the Trp493Arg genotype (Table 4 ). The distributions of both variants were as expected for the Hardy–Weinberg proportions. Haplotype analyses did not reveal additional significant information (data not shown), only that the double mutant 493Arg/663Thr allele was slightly more frequent in patients (2.4%) than in controls (1.4%; P = 0.03), which is consistent with the significantly higher frequency of the 493Arg allele in cases. All other haplotypes were equally distributed among both groups.


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  		Table 2. Prevalence of {alpha}ENaC 493 Arg>Trp polymorphism in 1399 patients with ICE and 1076 controls.


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  		Table 3. Screening procedure: Logistic regression analyses assessing the risk for stroke after adjustment for age (in tertiles), sex, and history of hypertension.


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  		Table 4. Demographic data and cardiovascular risk factors in 1399 patients grouped according to the {alpha}ENaC 493 genotype.

association of TRP493ARG variant and ice
After adjustment for age (in tertiles), sex, hypertension, diabetes mellitus, hyperlipidemia, smoking, and body mass index, 493Arg carriers had a 1.78-fold increased risk for ICE [95% confidence interval (95% CI), 1.02–3.12; P = 0.04] compared with 493Trp/Trp carriers by multivariate analysis (Table 5 ).


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  		Table 5. Logistic regression analyses assessing the risk for ICE according to the {alpha}ENaC 493 genotype.

The interactions between the Trp493Arg variant, age, sex, and the risk of stroke were significant. Carriers of the 493Arg allele in the lowest age tertile (<52 years) had a markedly increased risk [odds ratio (OR) = 2.88; 95% CI, 1.39–5.96; P = 0.004], whereas for individuals in the middle (52–69 years) and upper (>69 years) tertiles, we found no significant association between Trp493Arg and risk of stroke. Hypertension, diabetes, hyperlipidemia, and smoking did not interact significantly with the 493Arg allele. Female carriers of 493Arg exhibited a 2.06-fold increased risk for stroke (95% CI, 1.14–3.73; P = 0.016), whereas we observed no significant difference in males (OR = 1.52; 95% CI, 0.88–2.62; P = 0.14; data not shown). Considering age and sex together, male carriers of 493Arg in the lowest age tertile had a 2.66-fold increased risk for ICE (95% CI, 1.0–7.06; P = 0.05), and female carriers of 493Arg in the lowest age tertile had a 3.26-fold increased risk for ICE (95% CI, 1.10–9.72; P = 0.034). In the middle and upper age tertiles, we found no associations (for men, P = 0.16 and 0.64; for women, P = 0.13 and 0.43, respectively).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ENaCs are heteromultimeric proteins formed by the association of 3 homologous subunits: {alpha}, ß, and {gamma}. ENaCs are responsible for the rate-limiting step of sodium reabsorption and therefore are important candidates in the development of hypertension (14). The {alpha}-subunit appears to be the conducting unit because it alone can generate functional channels. Furthermore, this subunit has been found to be part of the amiloride-binding site. Previously, genetic variations have been identified in the gene coding for {alpha}ENaC. Some of these variants have been associated with essential hypertension and with the development of ICE (13)(14).

The frequent Thr663Ala variant is situated in the COOH terminus of the protein, which shows considerable interspecies variability (15). These interspecies differences in the 20 C-terminal amino acid residues also seem to explain species differences for regulatory proteins such as cystic fibrosis transmembrane conductance regulator. In contrast to the 663Thr allele, {alpha}ENaC activity was considerably lower in the presence of the 663Ala allele in a Xenopus oocyte model. At present, little is known about the Trp493Arg substitution, which is located at the C-terminal portion of the extracellular loop (9). Possibly, substitution of the polar Arg by the nonpolar Trp could also has functional effects on ENaC activity, but detailed studies of this variant have not been published.

In our study on 2475 Caucasian individuals, we found that the Trp493Arg variant was associated with ICE. Interestingly, the Thr663Ala genotype, which has been reported to represent a risk factor for stroke in a Chinese population (16), was equally frequent among patients and controls of Caucasian origin. The association of Trp493Arg with stroke was independent of the presence of hypertension and other established vascular risk factors, but the effect of this polymorphism depended on age and sex. Stratification for age revealed a significant risk of ICE in the lowest age tertile. Considering age and sex together, young female 493Arg carriers (<52 years) had a greater than 3-fold increased risk of ICE, but young male 493Arg carriers had a greater than 2-fold increased risk of stroke. One might speculate that the stronger effect of the Trp493Arg polymorphism in women is related to the mineralocorticoid effects of estrogen, which are similar to the effects of aldosterone, which is the most potent stimulus for sodium reabsorption in the cortical collecting tubule and the distal colon (17)(18). Interactions between genotype and sex are not uncommon. Recently, it was shown that expression of the angiotensinogen gene is modulated by female sex hormones (19). The decreasing effect of the Trp493Arg polymorphism with age may be explained by a more pronounced role of other risk factors of ICE in older patients, e.g., hypertension, diabetes mellitus, and hyperlipidemia.

One limitation of this study is that the design of our study was of observational nature. We therefore can demonstrate only an association between the 493Arg allele and ICE; the causality of this genetic variant has to be determined in a prospective study. The controls were not population based, but the samples were collected during different times of the year within a 2-year period in 6 different health centers located in areas of Vienna with different sociodemographic characteristics. Age and the presence of vascular risk factors were different in the patients and controls; therefore, all analyses were performed after appropriate statistical adjustments. We must also mention that although we tested for biologically plausible primary interactions, we cannot exclude secondary interactions.

In conclusion, we present data suggesting that 493Arg represents a risk factor for ICE in Caucasians, especially in younger persons (<52 years). The effect of the Trp493Arg variant seems to be independent of other vascular risk factors, including hypertension. Further studies confirming these results seem indicated.


   Acknowledgments
 
This study was supported by funds from Austrian National Bank Project 9488 and by a grant from the "Virologie Fonds zur Förderung der molekularen Grundlagenforschung in der Medizin". S. Cheng is employed by Roche Molecular Systems, which provided research assays for genotyping under collaboration at no cost.


   Footnotes
 
1 Nonstandard abbreviations: ENaC, epithelial sodium channel; SNP, single-nucleotide polymorphism; ICE, ischemic cerebrovascular event; 95% CI, 95% confidence interval; and OR, odds ratio.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sharma P. Genes for ischaemic stroke: strategies for their detection. J Hypertens 1996;14:277-285.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  2. Hoppe C, Cheng S, Grow M, Silbergleit A, Klitz W, Trachtenberg E, et al. A novel multilocus genotyping assay to identify genetic predictors of stroke in sickle cell anaemia. Br J Haematol 2001;114:718-720.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  3. Takami S, Imai Y, Katsuya T, Ohkubo T, Tsuji I, Nagai K, et al. Gene polymorphism of the renin-angiotensin system associates with risk for lacunar infarction. The Ohasama study. Am J Hypertens 2000;13:121-127.[CrossRef][Medline] [Order article via Infotrieve]
  4. Sierra C, Coca A, Gomez-Angelats E, Poch E, Sobrino J, de la Sierra A. Renin-angiotensin system genetic polymorphisms and cerebral white matter lesions in essential hypertension. Hypertension 2002;39:343-347.[Abstract/Free Full Text]
  5. Zee RY, Cook NR, Cheng S, Reynolds R, Erlich HA, Lindpaintner K, et al. Polymorphism in the P-selectin and interleukin-4 genes as determinants of stroke: a population-based, prospective genetic analysis. Hum Mol Genet 2004;13:389-396.[Abstract/Free Full Text]
  6. Mizutani K, Sugimoto K, Okuda T, Katsuya T, Miyata T, Tanabe T, et al. Kynureninase is a novel candidate gene for hypertension in spontaneously hypertensive rats. Hypertens Res 2002;25:135-140.[CrossRef][Medline] [Order article via Infotrieve]
  7. . Vienna Stroke Registry. Vienna Stroke Study Group. The Vienna Stroke Registry—objectives and methodology. The Vienna Stroke Study Group. Wien Klin Wochenschr 2001;113:141-147.[Web of Science][Medline] [Order article via Infotrieve]
  8. Lalouschek W, Lang W, Mullner M. Current strategies of secondary prevention after a cerebrovascular event: the Vienna Stroke Registry. Stroke 2001;32:2860-2866.[Abstract/Free Full Text]
  9. Schaedel C, Marthinsen L, Kristoffersson AC, Kornfalt R, Nilsson KO, Orlenius B, et al. Lung symptoms in pseudohypoaldosteronism type 1 are associated with deficiency of the {alpha}-subunit of the epithelial sodium channel. J Pediatr 1999;135:739-745.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  10. Ludwig M, Bolkenius U, Wickert L, Marynen P, Bidlingmaier F. Structural organisation of the gene encoding the {alpha}-subunit of the human amiloride-sensitive epithelial sodium channel. Hum Genet 1998;102:576-581.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  11. Cheng S, Grow MA, Pallaud C, Klitz W, Erlich HA, Visvikis S, et al. A multilocus genotyping assay for candidate markers of cardiovascular disease risk. Genome Res 1999;9:936-949.[Abstract/Free Full Text]
  12. Hosmer D, Lemeshow S. Applied logistic regression, 1st ed 1989:135-175 John Wiley & Sons New York. .
  13. Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001;68:978-989.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  14. Warnock DG. The epithelial sodium channel in hypertension. Curr Hypertens Rep 1999;1:158-163.[Medline] [Order article via Infotrieve]
  15. Yan W, Samaha FF, Ramkumar M, Kleyman TR, Rubenstein RC. Cystic fibrosis transmembrane conductance regulator differentially regulates human and mouse epithelial sodium channels in Xenopus oocytes. J Biol Chem 2004;279:23183-23192.[Abstract/Free Full Text]
  16. Zhao Y, Ma LY, Liu YX, Wang XY, Liu LS, Lindpaintner K. [Relationship between {alpha}-ENaC gene Thr663Ala polymorphism and ischemic stroke]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2001;23:499-501.[Medline] [Order article via Infotrieve]
  17. Kemendy AE, Kleyman TR, Eaton DC. Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia. Am J Physiol 1992;263:C825-C837.
  18. Schafer JA, Hawk CT. Regulation of Na+ channels in the cortical collecting duct by AVP and mineralocorticoids. Kidney Int 1992;41:255-268.[Web of Science][Medline] [Order article via Infotrieve]
  19. Williams GH, Fisher ND, Hunt SC, Jeunemaitre X, Hopkins PN, Hollenberg NK. Effects of gender and genotype on the phenotypic expression of nonmodulating essential hypertension. Kidney Int 2000;57:1404-1407.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]



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