D polymorphism in Brazilian hypertensive elderly

Science & Sports (2015) 30, 51—55

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BRIEF NOTE

The association between exercise capacity and angiotensin converting enzyme I/D polymorphism in Brazilian hypertensive elderly Association entre la capacité d’exercice et polymorphisme de l’enzyme de conversion I/D chez des personnes âgées hypertendues brésiliennes C. Veloso Carvalho a,b, M.F. Merli a, V.S. Probst a,c, K. Barros Parron Fernandes a,c, S.M. Maciel d, R.C. Poli-Frederico a,∗,b a

Master program in rehabilitation sciences UEL-UNOPAR, avenue Paris 675 Jd Piza, CEP 86041-140. Cx. P. 401, Londrina, PR, Brazil b Laboratory of molecular biology, Universidade Norte do Paraná (UNOPAR). avenue Paris 675 Jd Piza, CEP 86041-140. Cx. P. 401, Londrina, PR, Brazil c Centre of research in health sciences, Universidade Norte do Paraná (UNOPAR). avenue Paris 675 Jd Piza, CEP 86041-140. Cx. P. 401, Londrina, PR, Brazil d Department of pediatric dentistry, dental school, Universidade Estadual de Maingá (UEM), Maringá, Paraná, Brazil Received 23 August 2013; accepted 14 April 2014 Available online 16 October 2014

KEYWORDS Hypertension; Genetic polymorphism; ACE; Exercise; Elderly

∗

Summary Introduction. — To evaluate the association of the ACE I/D polymorphism with exercise capacity and history of hypertension in physically independent elderly. Summary of facts and results. — The study sample consisted of 57 health elderly and 57 hypertensive elderly. Genotyping for ACE I/D was performed by polymerase chain reaction. The 6-Minute Walk Test evaluates the subject’s functional exercise capacity and was performed in accordance with the guidelines of the American Thoracic Society. The influence of parameters on the risk of hypertension was assessed by analysis of logistic regression.

Corresponding author. E-mail addresses: [email protected], [email protected] (R.C. Poli-Frederico).

http://dx.doi.org/10.1016/j.scispo.2014.04.008 0765-1597/© 2014 Elsevier Masson SAS. All rights reserved.

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C. Veloso Carvalho et al. The significance level for all analyses was P ≤ 0.05. The elderly with the ACE DD genotype were 1.37 (95%CI: 1.09—1.71) more likely to be hypertensive than carriers of the allele I (P = 0.006). The hypertensive group exhibited a significantly higher BMI than the normotensive group (OR = 1.16; 95%CI: 1.02—1.33), as well as, elderly with impaired exercise capacity were 1.24 more likely to be hypertensive than those with more preserved exercise capacity (95%CI: 1.09—1.41). Conclusion. — The physically independent elderly carriers of the DD genotype showed an impaired exercise capacity and consequently an increased risk of hypertension. © 2014 Elsevier Masson SAS. All rights reserved.

MOTS CLÉS Hypertension ; Polymorphisme génétique ; ACE ; Exercice ; Personnes âgées

Résumé Introduction. — Évaluer l’association du polymorphisme ACE I/D avec la capacité d’exercice et l’histoire de l’hypertension chez les personnes âgées physiquement indépendantes. Synthèse des faits et résultats. — L’échantillon de l’étude comprenait 57 personnes âgées en bonne santé et 57 personnes âgées hypertendues. Le génotypage pour ACE I/D a été réalisée par réaction en chaîne par polymérase. Le test de marche de 6 minutes évalue la capacité d’exercice fonctionnel de l’échantillon et a été réalisé en conformité avec les lignes directrices de l’American Thoracic Society. L’influence des paramètres sur le risque d’hypertension artérielle a été évaluée par l’analyse de régression logistique. Le niveau de signification pour toutes les analyses était p ≤ 0,05. Les personnes âgées avec l’ACE génotype DD étaient plus susceptibles d’être hypertendues à 1,37 fois plus (IC95 % : 1,09—1,71) que les porteurs de l’allèle I (p = 0,006). Le groupe hypertendu présentait un IMC significativement plus élevé que le groupe normotendu (OR = 1,16 ; IC95 % : 1,02—1,33), et, les personnes âgées avec une capacité d’exercice affaiblie avaient 1,24 fois plus de chances d’être hypertendues que ceux qui ont la capacité d’exercice plus préservée (IC95 % : 1,09—10,41). Conclusion. — Les porteurs âgées physiquement indépendantes du génotype DD ont montré une capacité d’exercice affaiblie et par conséquent un risque accru d’hypertension. © 2014 Elsevier Masson SAS. Tous droits réservés.

1. Introduction

2. Material and methods

ACE insertion/deletion (I/D) polymorphism has been the major target for genetic investigations of hypertension. An I/D polymorphism of the ACE gene consists of the absence and presence of a 287 bp DNA fragment. Individuals who are homozygous for the deletion allele (DD) have serum ACE levels higher than those who are homozygous for the insertion allele (II) [1]. In addition, accumulated evidence suggests that the D allele is associated with a higher proportion of fast-twitch or type 2 muscle fibers, thus individuals with the D allele tend to perform better in resistance training but poorer in aerobic physical activity than those with the I allele. This genetic influence may also account for some of the propensity to be physically active [2]. Thus, it could be possible that, given an ACE I/D genotype, subjects might be more likely to become physically active and, consequently this in turn would lead to protection against hypertension. Hence and, considering that there are few reports in the literature regarding polymorphism genetic and exercise capacity in older adults, the aim of this study was to evaluate the association of the ACE I/D polymorphism with functional exercise capacity and history of hypertension in physically independent elderly.

2.1. Subjects A case-control study age and sex matched was performed between 57 subjects in the hypertensive group and 57 subjects in the normotensive group (mean age 69.2 ± 5.7). All subjects agreed to participate and signed a written informed consent. The project was approved by the Ethics Committee of the UNOPAR (PP/0253/11). Obesity was diagnosed on the basis of the most commonly used definitions, established by the World Health Organization (WHO). The blood pressure was obtained in the right arm after the participant sat quietly for ≥ 5 minutes. Hypertension was diagnosed according to the diagnostic standard of hypertension set by WHO/ISH in 1999 and if these individuals reported a previous diagnosis of hypertension and if were being treated with antihypertensive medications.

2.2. 6-Minute Walk Test (6MWT) The 6MWT evaluates the subject’s functional exercise capacity and the analysis was based on the percentage of values

Hypertension, exercise and ACE I/D polymorphism predicted. The more preserved exercise capacity group (n = 91) was composed of individuals who achieved 80% or more of the predicted value on the 6MWT and, the impaired exercise capacity group (n = 12) composed of individuals who achieved less than 80% of the predicted value on the 6MWT.

2.3. Determination of ACE I/D polymorphism (rs4646994) DNA was extracted using a commercially available kit (QIAmp DNA Blood Midi Kit—Qiagen) according to the manufacturer’s instructions. The following primer pair was used for PCR amplification of genomic DNA samples: forward 5 CTG GAG ACC ACT CCC ATC CTT TCT3 and reverse 5 GAT GTG GCC ATC ACA TTC GTC AGAT3 (Invitrogen, Carlsbad, CA). Amplification reactions were carried out with 100 ng of genomic DNA in a total volume of 25 ␮L, containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1 ␮M of each primer, 200 ␮M of each dNTPs, 1.5 mM MgCl2, and 1 U Taq DNA polymerase (Invitrogen). The reaction was incubated for 5 min at 95 ◦ C, followed by 30 cycles of 1 minute each at 95 ◦ C/58 ◦ C and 72 ◦ C. The 190 bp deletion allele and the 490 bp insertion allele were identified by Syber-safe (Invitrogen) staining.

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2.4. Statistical analysis Differences in distributions of genotypes as well as frequencies of alleles for the polymorphism were analyzed by Fisher exact tests and Chi2 test among the groups. Hardy-Weinberg equilibrium was tested by a Chi2 test. The differences between the groups were further analyzed through of the simple and multiple logistic regression models with adjustment for age, gender, exercise capacity, genotypes and BMI. P-value < 0.05 were considered as statistically significant.

3. Results Clinical and demographic characteristics of all 114 subjects are presented in Table 1. The hypertensive group exhibited a significantly higher BMI (P = 0.027) than the normotensive group. It was found a significant association between exercise capacity and hypertension. The elderly that presented impaired exercise capacity were hypertensives, while those that had high exercise capacity were normotensive (␹2 = 6.58; P = 0.013). The results of the genetic polymorphism analysis showed a higher frequency of DD genotype (49.1%), followed by

Table 1 Demographic characteristics, body mass index, 6-Minute Walk Test and distribution of angiotensin converting enzyme I/D genotypic and allelic frequencies in hypertensives and normotensives Brazilian elderly. Variables

Hypertensives

Normotensives

P-values

Gender Male/Female

21/36

21/36

n.s.

Age (years) 60—64 65—70 ≥ 71

12 (50%) 21 (50%) 24 (50%)

12 (50%) 21 (50%) 24 (50%)

n.s.

BMI (Kg/m2 ) Mean ± SD Underweight Normal weight Overweight Obesity

28.56 ± 4.51 07 (12,3) 20 (35.1) 09 (15.8) 21 (36.8)

25.78 ± 4.02 15 (23.3) 24 (42.1) 10 (17.5) 08 (14.0)

0.027a

6MWT Preserved exercise capacity Impaired exercise capacity

40 (80.0) 10 (20.0)

51 (96.2) 02 (3.8%)

0.013b

Genotypes II ID DD

02 (03.5) 23 (40.4) 32 (56.1)

08 (14.0) 25 (43.9) 24 (42.1)

0.043c

Alleles I D

27 (23.7) 87 (76.3)

41 (35.9) 73 (64.1)

0.042d

BMI: body mass index; 6MWT: 6-Minute Walk Test; n.s.: no significant. Statistical significance was determined by ␹2 among two groups. a ␹2 = 9.15. b ␹2 = 6.58. c ␹2 = 4.82. d ␹2 = 5.21.

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C. Veloso Carvalho et al.

ID (42.1%), and II (8.8%) genotype. The genotypic frequencies determined in this study did not deviate from those predicted by the Hardy-Weinberg Equilibrium (␹2 test; P = 0.949). The ACE I/D polymorphism displayed significant differences in the allelic and genotypic frequencies between hypertensive and normotensive groups (Table 1). The allele frequencies of ACE I/D with D allele, and the DD genotype were higher in the hypertensive group (␹2 = 5.21, P = 0.042; ␹2 = 4.82, P = 0.043, respectively). Results from simple logistic regression (Table 2) showed that the presence of DD genotype was associated with the elderly’ hypertension experience. Elderly positive for this genotype were more likely to have hypertension than those who were negative (OR = 1.31, 95%CI: 1.04—1.64; P = 0.018). Body mass index was also significantly associated with the outcome variable (OR = 1.29, 95%CI: 1.09—1.53; P = 0.004), as well as it was found association between exercise capacity and hypertension. The elderly that had impaired exercise capacity showed 1.27 (95%CI: 1.11—1.46; P = 0.005) more likely to have hypertension that those who had preserved exercise capacity. In the multiple logistic regression (Table 2), the BMI (P = 0.05), the 6MWT (P = 0.001) and the DD genotypes (P = 0.006) remained significant, indicating that these variables were independently related to the elderly’ hypertension experience. Elderly that presented high BMI had 1.16 (95%CI: 1.01—1.33) times more likely to have hypertension, as well as those that had impaired exercise capacity 1.24 (95%CI: 1.09—1.41) and, those elderly who harbored ID and DD genotypes had, respectively, 1.28 (95%CI: 1.02—1.61) and 1.37 (95%CI: 1.09—1.71) times more likely to have hypertension than their counterparts.

Table 2

4. Discussion In the present study, it was investigated the association of the ACE I/D polymorphism with functional exercise capacity and history of hypertension in a Brazilian elderly population. Some studies have proposed that the DD genotype increases the incidence of essential hypertension [3]. Individuals who are homozygous for the deletion allele (DD) have serum ACE levels higher than those who are homozygous for the insertion allele (II) [1]. Our results showed that the Brazilian elderly were 28% and 37% more likely to develop hypertension among heterozygous and D homozygous subjects respectively (OR = 1.28; 95%CI = 1.02—1.61 and OR = 1.37; 95%CI = 1.09—1.71, respectively). We found a significant association between exercise capacity and hypertension, being the hypertensive elderly the ones with impaired exercise capacity. The general beneficial effects of training on vascular function in subjects with hypertension are well documented. Several studies have shown improved vascular function in skeletal muscle after training in hypertensive individuals [4]. Thus, individuals with preserved exercise capacity seem to have a protective effect against hypertension. Furthermore, the adaptive responses to physical training may be regulated largely by the variation of gene expression. In summary, this study has provided further evidence that DD genotype in the ACE gene, higher BMI, and impaired exercise capacity may act independently on the prevalence of EH in physically independent Brazilian elderly. Considering that little is known about the genetic basis of ACE I/D to hypertension in Brazilian elderly, the analysis of genetic polymorphism in a sample from this population represents important information concerning hypertension in Brazil.

Simple and multiple logistic regressions of exploratory variables on the hypertension history of Brazilian elderly.

Exploratory variables

Unadjusted OR (95%CI)

P-values

Gender Female Male

1 1.00 (0.83—1.21)

1

Elderly age 60—64 years 65—70 years ≥ 71 years

1 1.00 (0.78—1,28) 1.00 (0.78—1.23)

1 1

BMI Normal weight Underweight Overweight Obesity

1 1.09 (0.91—1.30) 1.09 (0.88—1.35) 1.29 (1.09—1.53)

6MWT Preserved exercise capacity Impaired exercise capacity

1 1.27 (1.11—1.46)

Genotypes II ID DD

1 1.23 (0.93—1.51) 1.31 (1.04—1.64)

BMI: body mass index; 6MWT: 6-Minute Walk Test.

Adjusted OR (95%CI)

P-values

—

—

—

—

0.348 1.02 0.004

1 0.96 (0.80—1.15) 1.00 (0.85—1.24) 1.16 (1.01—1.33)

0.644 0.783 0.05

0.005

1 1.24 (1.09—1.41)

0.001

0.072 0.018

1 1.28 (1.02—1.61) 1.37 (1.09—1.71)

0.035 0.006

Hypertension, exercise and ACE I/D polymorphism

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Disclosure of interest

References

The authors declare that they have no conflicts of interest concerning this article. Funding source: RCPF is supported by Fundac ¸ão Nacional de Desenvolvimento do Ensino Superior Particular (FUNADESP, Brazil).

[1] Cambien F, Alhenc-Gelas F, Herbeth B, Andre JL, Rakotovao R, Gonzales MF, et al. Familial resemblance of plasma angiotensin converting enzyme level: the Nancy Study. Am J Hum Genet 1988;43:774—80. [2] Simonen RL, Rankinen T, Perusse L, Rice T, Rao DC, Chagnon Y, et al. Genome wide linkage scan for physical activity levels in the Quebec Family study. Med Sci Sports Exerc 2003;35:1355—9. [3] Ali A, Alghasham A, Ismail H, Dowaidar M, Settin A. ACE I/D and eNOS E298D gene polymorphisms in Saudi subjects with hypertension. J Renin Angiotensin Aldosterone Syst 2013;14(4):348—53. [4] McGowan CL, Visocchi A, Faulkner M, Verduyn R, Rakobowchuk M, Levy AS, et al. Isometric handgrip training improves local flow-mediated dilation in medicated hypertensives. Eur J Appl Physiol 2006;98:355—62.

Acknowledgments The authors would like to thank the university students who helped with the data collection of the study. We are also grateful to all professors from the Centre of Research in Health Sciences, who contributed to the development of the study.