Renin angiotensin system polymorphisms in patients with metabolic syndrome (MetS)

European Journal of Internal Medicine 21 (2010) 414–418

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European Journal of Internal Medicine j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j i m

Original article

Renin angiotensin system polymorphisms in patients with metabolic syndrome (MetS) Lucia Maria Procopciuc a,⁎, Adela Sitar-Tăut b, Dana Pop b, Dan-Andrei Sitar-Tăut c, Ileana Olteanu a, Dumitru Zdrenghea b a b c

Department of Medical Biochemistry, “Iuliu Hatieganu” University of Medicine and Pharmacy Cluj-Napoca, Romania Department of Cardiology, Clinical Rehabilitation Hospital, “University of Medicine and Pharmacy”, Cluj-Napoca, Romania Department of Business Information Systems, “Babes-Bolyai” University, Cluj-Napoca, Romania

a r t i c l e

i n f o

Article history: Received 15 February 2010 Received in revised form 26 April 2010 Accepted 1 June 2010 Available online 1 July 2010 Keywords: Metabolic syndrome RAS polymorphisms PCR-RFLP

a b s t r a c t Background: The genes associated with hypertension could be genetic risk factors for metabolic syndrome (MetS). Aim: To determine the frequency of M235T and T174M-AGT, I/D-ACE and A1166C-AGTR1 in hypertensive patients with MetS and to evaluate the relationship between these polymorphisms and central obesity and dyslipidemia, respectively. Materials and methods: We performed AGT, AGTR1 and ACE genotyping in 56 hypertensive women (24 with MetS) and 71 normotensive women using PCR-RFLP methods and PCR, respectively. Results: Hypertensive patients carrying the mutated TT235, MM174 and DD genotypes had an 1.53 (p = 0.56), 1.78 (p = 0.52) and 1.28 (p = 0.78)-fold increased risk to develop MetS. Hypertensive carriers of both mutated TT235 and MM174 or TT235 and D/D or TT235 and CC + AC genotypes had an 8.15 (p = 0.04), 4.83 (p = 0.04) and 10.53 (p = 0.05)-fold increased risk to develop MetS. Hypertensive patients with MetS and TT, D/D or CC genotypes had higher body mass index compared to hypertensive patients without MetS (p ≤ 0.05 for all the genotypes). Hypertensive patients with MetS and TT235, MM174, D/D or CC1166 genotypes had higher triglyceride levels, lower HDL-cholesterol levels and higher waist circumference compared to hypertensive patients without MetS (p ≤ 0.05, except for the association between CC1166 and HDL-cholesterol level). Conclusions: The effect of the T174M, I/D and A1166C polymorphisms on MetS may depend on the M235T polymorphism. Among hypertensive subjects with MetS, the presence of TT235, MM174, DD and CC1166 genotypes could be a risk factor for central obesity and dyslipidemia. © 2010 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

1. Introduction Metabolic syndrome (MetS) is a clustering of risk factors that predispose an individual to cardiovascular morbidity and mortality, representing a primary cause of morbidity and mortality in the industrialized world, exceeding in terms of frequency infections, traumas and smoking-related diseases [1–3]. MetS, called syndrome X by Reaven in 1988, is a multifactorial disorder associated with an increased risk for type 2 diabetes, but also for cardiovascular, neurological, immunological, renal, endocrine diseases [4]. Definitions of metabolic syndrome are very debatable, being known until now at least six variants. Although insulin resistance is considered a major pathological influence, it has been included in metabolic syndrome's definition only by World Health Organization (WHO) and European Group for the study of Insulin Resistance [5–7]. In the same time, International Diabetes Federation (IDF) definition

⁎ Corresponding author. 8/14 Padis Street, Cluj-Napoca, Romania. Tel.: +40 724335730. E-mail address: [email protected] (L.M. Procopciuc).

considered waist circumference as a mandatory component, but NCEP (ATP III-National Cholesterol Education Program-Adult Treatment Panel III) proposed for definition, three criteria from five (including waist circumference) [1,8]. It is a lack of diagnostic concordance, only about 30% of people appear to be diagnosable by most definitions [1]. In the present article, metabolic syndrome was defined (according to the European guidelines on cardiovascular disease prevention) as the cluster of three out of five factors among abdominal obesity (N102 cm in men, N88 cm in women), impaired fasting glycaemia (fasting plasma glucose 100–125 mg/dl or previously diagnosed type 2 diabetes), high blood pressure (N130/85 mm Hg or treatment of previously diagnosed hypertension), low HDL-colesterol (b40 mg/dl in male, b50 mg/dl in women) and high triglycerides (≥150 mg/dl) [3]. The risk for disease increases over time as the number of metabolic syndrome characteristics accumulates, being important for the physicians to recognize it and to diagnose it (even using genetic determination) [9,10]. Epidemiological data obtained by Yusuf et al. in 2005 in a study conducted in 27,000 participants from 52 countries indicate a prevalence of 26% for MetS [11]. The prevalence of the metabolic syndrome is higher in women. In a study performed by Ford in 2002, the

0953-6205/$ – see front matter © 2010 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ejim.2010.06.001

L.M. Procopciuc et al. / European Journal of Internal Medicine 21 (2010) 414–418 Table 1 Clinical characteristics for the three studied groups: hypertensive patients with MetS, hypertensive patients without MetS, control subjects. p

415

Buraczynska [22] confirming this association. Moreover, the components of RAS were detected in some tissues, such as kidney, heart, brain or adipose tissue, with an important role in gene expression, cell growth. Activation of these components may determine obesity-associated hypertension, dyslipidemia and diabetes. [23–27].

Clinical characteristics

Control Hypertensive Hypertensive patients without subjects patients with MetS (MetS+) MetS (MetS−)

Number Age, years Environment Urban Rural SBP (mmHg) DBP (mmHg) BMI (kg/m2) Waist circumference (cm) Glucose level (mg/dl) Total cholesterol (mg/dl) Triglycerides (mg/dl) HDL-cholesterol (mg/dl) Dyslipidemia, no (%) Central obesity, no (%) Diabetes mellitus, no (%)

24 48 ± 4.25

32 47.03 ± 4.7

71 46.66 ± 5.48

1.1. Objective

17 (70.83%) 7 (29.16%) 138.33 ± 14.19 86.45 ± 10.15 29.62 ± 4.34 97.41 ± 9.23

19 (59.37%) 13 (40.62%) 140.3 ± 15.18 88.44 ± 5.3 26.76 ± 2.2 87.43 ± 9.51

50 (70.42%) 21 (29.57%) 114.85 ± 6.81 73.38 ± 7.16 26.88 ± 1.98 83.33 ± 7.98

The purpose of this study was: 1. to determine the frequency of RAS polymorphisms, including M235T and T174M-AGT, I/D-ACE and A1166C-AGTR1, in hypertensive patients 2. To evaluate the relationship between the candidate genes of the RAS and the components of MetS, central obesity and dyslipidemia, we examined their possible role as a risk factor in hypertensive patients with MetS.

97.25 ± 27.04

99.03 ± 29.85

91.54 ± 20.2

0.81

251.83 ± 42.58

223.34 ± 42.3

0.02

161.7 ± 75.06

123.65 ± 45.25

40.5 ± 7.61

51.9 ± 9.63

197.05 ± 36.87 119.71 ± 48.91 51.59 ± 7.98

b 0.01

16 (66.66%)

13 (40.62%)

14 (19.71%)

0.03

21 (87.5%)

14 (43.75%)

12 (16.9%)

9 (37.5%)

2 (6.25%)

–

0.61 0.39 0.05 b 0.01

2. Design and methods 0.034

b 0.01 0.005

MetS + patients with metabolic syndrome; Met — patients without metabolic syndrome; p — comparative analysis between hypertensive patients with and without MetS; p b 0.05 means statistically significance.

prevalence of MetS varies from 37% of Afro-American men to 57% of Afro-American women [12]. The prevalence was also higher in Turkey women, 40%. The prevalence varied with obesity and age, around 6.7% in patients aged 20–29 years and increases to 42% in patients older than 70 years [13]. Metabolic syndrome results from the interaction between genetic susceptibility risk factors, such as the factors involved in the blood pressure regulation (renin angiotensin system-RAS genes), lipid metabolism (APOE, APOC3, ARCP3 encoding adiponectin) or obesity (the gene of melanocortin-4 receptor, the leptin gene or the ones involved in the control of brain levels of gama-aminobutyric acid) [14– 17]. RAS could have a role in the pathogenesis of MetS because of the implication in the development of hypertension and insulin resistance. The results regarding the association between RAS genes including angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensin II type I receptor (AGTR1) and hypertension are controversial, the studies made by Engeli [18], Jeunemaitre [19], Tsai [20], Zhang [21] and

2.1. Studied groups We performed AGT, ACE, AGTR1 genotyping in 56 hypertensive women, 24 with MetS (mean age 48 ± 4.25 years) and 32 without MetS (mean age 47.03 ± 4.7 years), taking into account the known risk factors for MetS. The control group consisted of 71 normotensive women (mean age 46.66 ± 5.48 years). The patients were selected on the basis of inclusion criteria. Individuals using blood pressure medications or oral hypoglycemic agents were considered to meet the selection criteria for high blood pressure and high fasting glucose concentration, respectively. Assessed parameters: body mass index (BMI), waist circumference (WC), systolic and diastolic blood pressure (SBP, DBP) were noted. Lipid and glycemic parameters (concentration of total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, glucose) were determined by standard biochemical methods. All the participants in the study gave their informed consent. 2.2. Methods After DNA isolation from blood leukocytes, M235T, T174M, and A1166C were identified using the PCR amplification reaction followed by enzymatic digestion with restriction endonucleases, (PCR–RFLP analysis), methods described by Rupert [28] and Caulfield [29]. PCR reaction was carried out to detect the insertion (I)/deletion (D) polymorphism in the ACE gene using the method described by Rupert [28]. 2.2.1. Statistical analysis Differences in the distribution of genotypes between hypertensive patients with or without MetS and between all hypertensive patients and the control group were determined by the Fisher exact test. Odds

Table 2 Distribution of mutated RAS genotypes in hypertensive patients with and without MetS and in control subjects. Polymorphisms

M235T

T174M

I/D

A1166C

Genotypes

TT TM T allele MM MT M allele D/D I/D D allele CC AC C allele

Patients with essential hypertension All, no (%)

MetS+, no (%)

MetS−, no (%)

18 28 64 13 13 39 26 25 77 7 15 29

9 (37.5) 11 (45.8) 29 (0.6) 7 (29) 6 (25) 20 (0.41) 12 (50) 10 (41.7) 34 (0.7) 3 (12.5) 7 (29.2) 13 (0.27)

9 (28.1) 17 (53.1) 35 (0.54) 6 (18.8) 7 (21.9) 19 (0.29) 14 (43.8) 15 (46.9) 43 (0.67) 4 (12.5) 8 (25) 16 (0.25)

(32.1) (50) (0.57) (23.2) (23.2) (0.34) (46.4) (44.6) (0.68) (12.5) (26.8) (0.25)

Controls, no (%)

OR, 95%CI

p

MetS+ vs. MetS− 9 27 45 7 4 18 19 32 70 5 18 28

(12.7) (38) (0.31) (9.8) (5.6) (0.12) (26.8) (45.1) (0.49) (7) (25.3) (0.19)

1.53 [0.5–4.74] 0.74 [0.2–2.15] 1.26 [0.59–2.7] 1.78 [0.5–6.22] 1.19 [0.34–4.1] 1.69 [0.77–3.7] 1.28 [0.44–3.7] 0.8 [0.27–2.35] 1.18 [0.52–2.6] 1 [0.2–4.95] 1.23 [0.3–4.05] 1.18 [0.4–3.41]

OR, 95%CI

p

All vs. controls 0.56 0.78 0.56 0.52 1 0.23 0.57 0.78 0.83 1 0.76 0.79

3.2 1.62 2.87 2.7 5.58 3.82 2.37 0.98 2.2 1.88 1.07 1.4

[1.33–7.8] [0.8–3.31] [1.71–4.8] [1.02–7.5] [1.7–18.1] [2.04–7.2] [1.12–4.9] [0.48–1.98] [1.34–3.8] [0.56–6.3] [0.48–2.39] [0.78–2.5]

b 0.01 0.2 b 0.01 0.05 b 0.01 b 0.01 0.02 1 b 0.01 0.36 1 b 0.01

MetS+ patients with metabolic syndrome; MetS− patients without metabolic syndrome; OR — odds ratio; 95%CI–95% confidence interval; p b 0.05 is statistically significant.

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Table 3 Mutated genotype interaction on the risk for MetS. Genotypes

OR, 95%CI p Hypertensive Hypertensive patients without MetS+ vs. MetS− patients with MetS (MetS+) MetS (MetS−)

TT235 + MM17 5/24 TT235 + DD 8/24 TT235 + (AC + CC) 6/24

1/32 3/32 1/32

8.15, [0.88–75.24] 0.04 4.83, [1.12–20.82] 0.04 10.53, [1.15–92.81] 0.05

OR — odds ratio; 95%CI–95% confidence interval; p less than 0.05 is statistically significant.

ratio (OR) and 95% confidence interval (95%CI) were also calculated. A p value less than 0.05 was considered to be significant. Differences in anthropometric and biochemical parameters between hypertensive patients with and without MetS were determined using Student's test.

3. Results The clinical characteristics of the studied groups were presented in Table 1. Compared to subjects without MetS, abdominal obesity was more obvious in patients with MetS who had a higher BMI and waist circumference. Higher levels for the total cholesterol and triglycerides and a lower level for the HDL-cholesterol were noted in hypertensive patients with MetS compared to hypertensive patients without MetS. The genotype frequencies for the AGT, ACE and AGTR1 genes are presented in Table 2. The genotypes distribution of the M235T, T174M, I/D and A1166C polymorphisms differed between hypertensive patients with and without MetS. Statistically significant differences between the hypertensive and control groups were found in the genotype distribution for AGT and ACE polymorphisms.

4. Discussion The RAS system represents an important regulator of blood pressure [30,31]. Each component of RAS could be a potential candidate for development of hypertension [19,20,32,33]. Some of these genes, probably associated with the susceptibility of hypertension, are those encoded AGT, ACE or AGTR1. The T235 and D alleles were associated with hypertension and coronary heart disease in Caucasian, Chinese, or Indian populations [32–35]. The meta-analysis made by Zafarmand in 2008 showed increased risk for cardiovascular disease in patients with M235T variant of the AGT gene [36]. In one study made by Freitas in 2007, an increased risk to develop resistant hypertension in patients with both TT235 and (AC + CC) genotypes was detected [37].

In the present study, the risk for development of hypertension was 2.87 (pb 0.01), 3.82 (pb 0.01), 2.2 (p= 0.02) and 1.4 (pb 0.01) in individuals carrying at least one T235, M174, D or C1166 allele (Table 2). The metabolic syndrome is a disorders in which hypertension is one of the risk factors. Unfortunately, there are only few results regarding the distribution of RAS polymorphisms in patients with MetS. Some of these confirm the association, but not all of them [38–40]. The study made by Hamada [41] confirmed that the ACE-I/D polymorphism influence the metabolic profiles in patients with metabolic syndrome after administration of low-energy diets. In our studied population, the frequency of mutated TT235, MM174, D/D and CC genotypes were different in the group of patients with MetS compared with the frequencies in the group of patients without MetS. Hypertensive patients carrying the mutated TT235, MM174 and DD genotypes had a 1.53, 1.78 and 1.28-fold increased risk to develop MetS, but the results were not statistically significant (all p values were greater than p 0.05). The risk to develop MetS in hypertensive patients carrying at least one T235, M174 and D allele was 1.26, 1.69 and 1.18, respectively. In all cases p was greater than 0.05 (Table 2). Our results are in agreement with those obtained by Pacholczyk [39]. We also tested the association of the M235T polymorphism with T174M, or I/D, or A116C polymorphisms on the risk for MetS in hypertensive carriers. Hypertensive carriers of both mutated TT235 and MM174, TT235 and D/D, or TT235 and (AC + CC) genotypes had a 8.15 (p = 0.04), 4.83 (p = 0.04) and 10.53 (p = 0.05)-fold increased risk to develop MetS. In MetS, the genes associated with hypertension is clustered together with the gene associated with obesity, dyslipidemia and insulin resistance, but the mechanism by which RAS influences the lipid metabolism are not very well establish. As is sustained by Bettinaglio [42], Feng [43], Thomas [44] and Baroudi [45], activation of AGT and ACE could determine dyslipidemia and diabetes. Giachelletti [46], Thomas [44], Hainault [47] and Prat-Larquemin [48] established that in obese patients the adipocytes had the ability to secrete the AGT and ACE proteins, suggesting the possibility that M235T polymorphism of the AGT gene or the ACE insertion/deletion polymorphism could have an influence on lipid metabolism in adipocytes. For these reasons, AGT, ACE and AGTR1 could be good candidate genes for causing MetS. In the studies made by Strazzullo [50] and Niemiec [49] there was an association between T235 allele and hypercholesterolemia and between the DD genotype and abdominal adiposity. However, other authors did not confirm the results, Kaetsu showing in 2006 no significant association between AGT M235T and TC [51]. In our study, there is an association between BMI and the mutated genotypes for the M235T, I/D and A1166C as a risk factors for MetS. Hypertensive patients with MetS carrying the TT235, MM174, D/D or

Table 4 Association between RAS polymorphisms and the components of the MetS, central obesity (BMI and waist circumference) and dyslipidemia (tryglicerides and HDL-cholesterol).

M235T

T174M

I/D

A1166C

Mutated genotypes and components of the MetS

Hypertensive patients with MetS

Hypertensive patients without MetS

TT and BMI (kg/m2) TT and WC (cm) TT and TG (mg/dl) TT and HDL-C (mg/d MM and BMI (kg/m2) MM and WC (cm) MM and TG (mg/dl) MM and HDL-C (mg/dl) DD and BMI (kg/m2) DD and WC (cm) DD and TG (mg/dl) DD and HDL-C (mg/dl) (CC + AC) and BMI (kg/m2) (CC + AC) and WC (cm) (CC + AC) and TG (mg/dl) (CC + AC) and HDL-C (mg/dl)

30.34 ± 5.3 99.88 ± 9.87 213.66 ± 79.97 38.88 ± 8.44 27.69 ± 1.89 94.42 ± 8.69 216.42 ± 100.55 40.14 ± 9.37 30.41 ± 4.62 98.25 ± 8.86 208.91 ± 69.25 39.5 ± 8.44 31.09 ± 4.94 98.22 ± 8.64 224.11 ± 74.03 40.77 ± 8.96

26.51 ± 1.85 88. ± 9.47 116.88 ± 59.5 57.77 ± 10.77 27.28 ± 1.71 87.5 ± 6.05 107.83 ± 26.13 50.83 ± 10.14 27.31 ± 2.29 87.27 ± 8.21 88.9 ± 22.72 54.36 ± 2.8 26.53 ± 1.88 87.83 ± 12.45 99.3 ± 26.39 64 ± 31.55

p 0.05 0.02 0.01 b0.01 0.68 0.03 0.028 0.07 0.05 b0.01 b0.01 b0.01 0.02 0.03 b0.01 0.02

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CC + AC genotypes had higher triglyceride, lower HDL-cholesterol levels and higher WC than those without MetS. In all cases p was lower than 0.05 except for the association between the MM174 genotype and HDL-cholesterol (Table 3). The results were in agreement with those obtained by Strazzullo [50] who showed that the DD genotype is associated with higher BMI [50]. The results obtained in our population support the conclusion that the molecular variants of AGT, ACE and AGTR1 are associated with hypertension. No statistical differences in the individual RAS polymorphism distribution were observed between hypertensive patients with and without MetS. The effect of the T174M, I/D and A1166C polymorphisms on MetS may depend on the M235T polymorphism. Among hypertensive subjects with MetS, the presence of TT235, MM174, DD and CC1166 genotypes could be a risk factor for central obesity and dyslipidemia (Table 4). 5. Learning points • Molecular variants of AGT, ACE and AGTR1 are associated with hypertension. • The effect of the T174M, I/D and A1166C polymorphisms on MetS may depend on the M235T polymorphism. • Among hypertensive subjects with MetS, the presence of TT235, MM174, DD and CC1166 genotypes could be a risk factor for central obesity and dyslipidemia. Acknowledgements This paper was supported by Research Project No. 947, ID_2246/ 2009 Code, part of PN II Program financed by the Romanian Ministry of Education, Research and Innovation-The National University Research Council. References [1] Day C. Metabolic syndrome, or what you will: definitions and epidemiology. Diab Vasc Dis Res 2007;4:32–8. [2] Grundy SM. Metabolic syndrome: connecting and reconciling cardiovascular and diabetes worlds. J Am Coll Cardiol 2006;47(6):1093–100. [3] Wilkin TJ, Voss LD. Metabolic syndrome: maladaptation to a modern world. J R Soc Med 2004;97(11):511–20. [4] Vega GL. Obesity, the metabolic syndrome, and cardiovascular disease. Am Heart J 2001;142:1008–16. [5] Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes of diabetes mellitus, provisional report of a WHO consultation. Diabet Med 1998;15: 539–53. [6] WHO Consultation. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus. Geneva: Geneva: World Health Organisation; 1999. Report No. 9.2. [7] Balkau B, Charles MA. Comment on the provisional report from the WHO consultation. European Group for the Study of Insulin Resistance (EGIR). Diabet Med 1999;16:442–3. [8] Grundy SM, Brewer Jr B, Cleeman JI, et al. Definition of metabolic syndrome. Circulation 2004;109:433–8. [9] Expert Panel of Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive summary of the third report of The National Cholesterol Education program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. (Adult treatment Panel III). JAMA 2001;285(19):2486–97. [10] Smith Jr SC. Multiple risk factors for cardiovascular disease and diabetes mellitus. Am J Med 2007;120(3 Suppl 1):S3–S11. [11] Yusuf S, Hawken S, Ounpuu S, et al. INTERHEART Study Investigators. Obesity and the risk of myocardial infarction in 27, 000 participans from 52 countries: a casecontrol study. Lancet 2005;366(9497):1640–9. [12] Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome among US adults: findings from the Third National Health and Nutrition Examination Survey. JAMA 2005;287:356–9. [13] Gu D. Body weight and mortality among men and women in China. JAMA 2006;295 (7):776–83. [14] Bray GA. The Metabolic Syndrome and Obesity. Humana Press Inc; 2005. pg 98. [15] Hegele RA, Pollex RL. Genetic and physiological insights into the metabolic syndrome. Am J Physiol Regul Integr Comp Physiol 2005;289:663–9. [16] Pollex RL, Hanley AJ, Zinman B, Harris SB, Khan HM, Hegele RA. Metabolic syndrome in aboriginal Canadians: prevalence and genetic associations. Atherosclerosis 2006;184:121–9.

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