Indian J Med Res 132, October 2010, pp 379385 Association of aldosterone synthase (CY11B2 C344) gene polymorphism & susceptibility to essential hypertension in a south Indian amil population S. Rajan,. Ramu, G. Umamaheswaran & C. Adithan Department of harmacology, Jawaharlal Institute of ostgraduate Medical Education & Research, ondicherry, India Received March 30, 2009 Background & objective: Reninangiotensin aldosterone system (RAAS) plays an important role in the regulation of blood pressure. Aldosterone, synthesized by aldosterone synthase in the adrenal cortex is encoded by the CY11B2 gene. In this casecontrol study we examined the association between CY11B2 C344 polymorphism and essential hypertension in south Indian amil population. Methods: he study was conducted in 406 hypertensive cases and 424 healthy controls from amil population. Genotyping was performed by CRrestriction fragment length polymorphism method. Statistical analysis was performed by logistic regression analysis. Results: he 344 homozygous variant genotype (OR1.8; 95% CI: 1.12.8; =0.02) and allele (=0.007) were found to be significantly associated with hypertension. In gender based analysis, the risk was significantly higher in male hypertensives (OR1.8; 95% CI: 3.6, =0.05) but not in female subjects. Interpretation & conclusion: A significant association between CY11B2 gene polymorphism and essential hypertension was observed and the risk was confined to male subjects in south Indian amil population. Key words Aldosterone CY11B2 genotype hypertension polymorphism Essential hypertension, a major risk factor for cardiovascular disease, is a multifactorial and polygenic disorder, predisposed by genetic and environmental factors 1. he reninangiotensinaldosterone system (RAAS) is one of the key modulators of blood pressure in essential hypertension 2. Aldosterone hormone, secreted by the adrenal cortex of the adrenal gland, is chiefly concerned with waterelectrolytes balance. Aldosterone is synthesized by the aldosterone synthase enzyme 3, which is encoded by the CY11B2 gene located on chromosome 8q22 46. Several polymorphisms have been identified in the CY11B2 gene 7. Among them, the promoter region C344 polymorphism (rs id 1799998) is the most widely 379
380 INDIAN J MED RES, OCOBER 2010 studied as it persuades the binding of steroidogenic factor1, the transcriptional regulatory protein 8. his polymorphism either increases aldosterone to renin ratio (ARR) in essential hypertensives or decreases aldosterone production, leading to sodium wasting and decreased excretion of potassium 910. Studies on C344 polymorphism have shown positive 6,1114 as well as negative association 1518 with hypertension and other cardiovascular parameters. hese studies were conducted extensively in Caucasians and Orientals 5,6,9,1216,1820 but studies in Indian population are rare. A casecontrol study on hypertension in highlanders with high salt intake is perhaps the only association study on CY11B2 gene C344 polymorphism in Indian population 21. Hence, we investigated the association between aldosterone synthase (CY11B2 C344) gene polymorphism and susceptibility to essential hypertension in south Indian amil population. Material & Methods Study population: he study was conducted from June 2005 to April 2008 in 406 unrelated essential hypertensive patients (200 males and 206 females) aged 3060 yr who were residents of amil nadu and ondicherry for at least three generations. he subjects were selected randomly and the sample size was calculated using Sower and sample size calculation version 3.0 software. hey were recruited from the outpatient clinics of hypertension and internal medicine of Jawaharlal Institute of ostgraduate Medical Education & Research (JIMER) hospital, uducherry in south India. atients, who were receiving antihypertensive medications for more than 3 months or newly diagnosed hypertensive patients with systolic blood pressure (SB) more than 140 mmhg and/or diastolic blood pressure (DB) more than 90 mmhg on two or more consecutive visits were considered as hypertensives 22. atients with history of diabetes mellitus, hyperlipidaemia, liver or renal disease, congestive cardiac failure and recent episode of myocardial infarction were excluded. atients with pregnancy and lactation and receiving medications for other indications that could affect blood pressure were also excluded. he control group consisted of 424 (183 males and 241 females, aged between 2560 yr) unrelated healthy volunteers. hese subjects had no personal or family history of hypertension and other cardiovascular diseases in firstdegree relatives and had SB <130 mmhg and DB <85 mmhg. Healthy volunteers from the health camp and patients who visited the outpatient clinics with minor illness without hypertension, diabetes mellitus, hyperlipidaemia and family history of hypertension in previous records were recruited as controls. None of the subjects in the control group was receiving antihypertensive therapy, treatment for heart disease or hormonereplacement therapy during the time of the study. lasma lipid profile and blood glucose level were measured after overnight fasting in both hypertensives and normotensives to rule out diabetes and hyperlipidaemia. All the participants were interviewed using standardized questionnaire with regard to their lifestyle, smoking, alcohol consumption and drug intake. he questionnaire was prepared according to the British Hypertension Society guidelines by the clinicians in the department of medicine and hypertension clinic, JIMER and Institute ethics committee approval was obtained. In all subjects, height was measured to the nearest centimeter and weight to the nearest 0.1 kg which were used for calculation of BMI (kg/m 2 ). Blood pressure was measured two minutes apart three times in the right arm using standard sphygmomanometer after the subjects rested for 10 min and the average reading was recorded. he study was approved by the institutional ethics committee and written informed consent was obtained from all the participants. Genotyping: Five milliliters of venous blood was collected from the participants using ethylene diamine tetra acetic acid (EDA) as an anticoagulant and the genomic DNA was extracted using phenolchloroform method. he CY11B2 C344 polymorphism was detected using CRRFL method 5. CR reaction was performed in mastercycler gradient (Eppendorf, AG, Hamburg, Germany) using 25 µl reaction mixture containing 100 ng of DNA, 200 µm of dns, 0.2 µm of each primers (Alpha DNA, Montreal, Canada) (Sense5 CAGGAGGAGACCCCAGGAC 3 ; antisense5 CCCCA CCCGCAGCCC3 ), 1.5 mm of MgCl 2 and 0.5 U of aq DNA polymerase. his was subjected to 35 cycles with initial denaturation at 94 C for 5 min, cyclic denaturation at 94 C for 60 sec, followed by annealing at 67 C for 60 sec, extension at 72 C for 60 sec and a final extension at 72 C for 5 min. he amplification was checked in horizontal gel electrophoresis unit (Apelex, Massy cedex, France) using 1 per cent agarose gel followed by restriction digestion of the 538 bp CR product with HaeIII endonuclease (New England Biolabs inc., IA, USA) for 2 h at 37 C. he digested product was analyzed in vertical electrophoresis system (Shelton scientific inc., USA) using 8 per cent polyacrylamide gel electrophoresis (AGE) which resulted in 203, 138,
RAJAN et al: CY11B2 OLYMORHISM IN AMIL OULAION 381 126, 71 bp for wild type, 274, 203, 138, 126 and 71 bp for heterozygous variant and 274, 138 and 126 bp for homozygous variant 23. Statistical analysis: Statistical analysis was done using the SSS, version 13 (SSS Inc., Chicago, Illinois, USA). he demographic details of cases and controls with continuous variables were compared using Student unpaired t test while categorical variables, allele and genotype frequencies between hypertensives and normotensives, and HardyWeinberg equilibrium was calculated using chisquare analysis. he association between genotypes and hypertension risk was analyzed by calculating the odds ratio (OR) and 95 per cent confidence interval. Logistic regression analysis was used to adjust the potential confounders for hypertension such as age, smoking and alcohol consumption with the low risk genotype designated as reference category. <0.05 was considered as statistically significance. Results he mean age was significantly higher in controls when compared to cases (<0.001). here was a significant difference in SB, DB, smoking, alcohol intake, total cholesterol, LDL, and VLDL cholesterol level among the cases and controls (able I). Confounding factors that revealed significant difference, except B, were taken for multiple logistic regression analysis. able I. Demographic details and biochemical parameters of the study subjects arameters (n=406) (n=424) Sex (Male : Female) 200 : 206 183 : 241 Age (yr) 44.1 ± 0.4 ** 47.3 ± 0.4 BMI (kg/m 2 ) 23.0 ± 0.3 23.1 ± 0.2 SB (mm Hg) 152.6 ± 0.8 ** 118.0 ± 0.5 DB (mm Hg) 97.2 ± 0.5 ** 78.1 ± 0.3 Smoking Yes 71 (17.5%) ** 34 (8.0%) No 335 (82.5%) 390 (92%) Alcohol Yes 109 (26.8%) * 84 (19.8%) No 297 (73.2%) 340 (80.2%) Serum total cholesterol 175.6 ± 1.4 * 170.2 ± 1.6 (mg/dl) Serum triglycerides 118.0 ± 2.3 115.6 ± 2.5 (mg/dl) Serum HDL (mg/dl) 40.6 ± 0.4 41.1 ± 0.5 Serum LDL (mg/dl) 110.6 ± 1.3 * 106.6 ± 1.4 Serum VLDL (mg/dl) 24.3 ± 0.5 * 22.5 ± 0.4 Values are expressed as mean ± SEM or numbers and percentages * <0.05, ** <0.001 compared to controls he different restriction fragments of CY11B2 C344 gene variant are shown in the Fig. he homozygous variant genotype / was significantly higher in hypertensive cases when compared to the controls (OR1.8; 95% CI: 1.12.8, =0.02) after adjusting the confounding factors. he variant allele was also higher in the cases when compared to the controls (66.6 vs. 60.1%, =0.007) (able II). he observed and expected genotype frequencies were in concordance with the HardyWeinberg equilibrium. Genderwise analysis showed, the frequency of homozygous variant genotype / (OR1.8; 95% CI: 3.6, =0.05) and variant allele (66.1 vs 58.2%) was significantly higher in male cases when compared to male controls, but there was no difference among the female cases and controls (able III). Meta analysis on C344 polymorphism among major ethnics: A meta analysis comprising 24 studies including the present study showed that the overall prevalence of C and allele amounted to 39 and 61 per cent respectively in 15,059 subjects (able IV). Further, the risk of hypertension was estimated by combining 10 casecontrol studies, including the present study. he odds ratio for hypertension was 1.4 (95% CI: 1.21.7, <0.0001) and 1.2 (95% CI: 1.4, <0.05) for C/ heterozygous and homozygous genotype respectively as compared to CC homozygous genotype 6,1214,21,2326. Fig. Representative AGE picture of CY11B2 C344 polymorphism. Lanes 1 2 log DNA ladder; Lane 2 Undigested CR product; Lanes 3 and 6 Homozygous mutant (); Lanes 4 and 7 Wild type (CC), Lanes 5 and 8 Heterozygous (C).
382 INDIAN J MED RES, OCOBER 2010 CY11B2 Genotype: C/C C/ / Allele: C able II. Genotype and allele frequencies of CY11B2 C344 polymorphism among the cases and controls (n=406) 43 (10.6) 185 (45.6) 178 (43.8) 271 (33.4) 541 (66.6) (n=424) 56 (13.2) 226 (53.3) 142 (33.5) 338 (39.9) 510 (60.1) Unadjusted OR ( 95% CI ) 1.1 (0.71.7) 1.6 (2.6) 1.3 (1.11.6) Values in parenthesis indicate percentages Odds ratio adjusted for confounding factors (age, sex, smoking and alcohol consumption) value 0.8 0.04 0.007 Adjusted OR ( 95% CI ) 1.2 (0.92.0) 1.8 (1.12.8) value 0.4 0.02 CY11B2 C344 Genotypes: CC C Alleles: C able III. Gender specific distribution of aldosterone synthase CY11B2 C344 gene polymorphism among the study subjects Male subjects (n = 200) 22 (11) 91 (45.5) 87 (43.5) 33.9 66.1 (n= 183) 31 (17.0) 91 (49.7) 61 (33.3) 41.8 58.2 OR 1.4 (0.82.6) 2.0 (1.13.8) OR * 0.3 0.04 1.4 (41.9) 0.02 1.3 (0.72.5) 1.8 (3.6) 0.5 0.05 Female subjects (n = 206) 21 (10.2) 94 (45.6) 91 (44.2) 33.0 67.0 (n=241) 25 (10.4) 135 (56.0) 81 (33.6) 38.4 61.6 OR 0.8 (0.41.5) 1.3 (0.72.6) 1.3 (0.951.7) OR * 0.6 0.4 0.1 0.8 (0.41.7) 1.4 (0.72.8) 0.6 0.4 Values in parenthesis indicate percentage * Odds ratio according to genotypes were estimated after adjusting the confounding variables for smoking and alcohol consumption in male subjects and for age in female subjects Discussion Hypertension, a major risk factor for cardiovascular and renal disease, is predicted to increase cardiovascular deaths by 111 per cent in India by 2020, much more than in any other Asian country 1. Hypersecretion of aldosterone was authenticated to cause hypertension. he variations in CY11B2 gene, influences the aldosterone synthesis 27. In our study, the homozygous variant genotype and variant allele was significantly higher in hypertensive cases than the controls. Several studies have investigated the association between CY11B2 polymorphism and essential hypertension among different ethnic groups, with conflicting results 6,11,14. A casecontrol study conducted among Indians living in highland, with smaller sample size, did not find significant association between C344 polymorphism and hypertension. he study showed a significant association with BMI but the subjects had mean BMI below 25 kg/m 2 which is not considered as a risk factor for hypertension 21. Another Indian study reported that allele was highly prevalent in highlanders as compared to lowlanders of north India 28. In accordance with this study, ours showed a higher prevalence of allele in both hypertensives and normotensives. A casecontrol study conducted in French and Scottish Caucasian population showed that the variant allele was highly prevalent in hypertensive cases and significantly associated with hypertension 6,12. When the influence of C344 polymorphism was analyzed separately for male and female participants, variant allele was found to be significantly higher and the homozygous genotype / had a marginal significant association in male hypertensive cases, but not in the female subjects. Further, the present study did not show any genesex interaction. A study conducted in Chinese population did not find any genderspecific association with hypertension, whereas a study done in Australian population showed that female hypertensive cases with C allele were at increased risk of hypertension 24,25. he underlying cause to develop hypertension was limited to female cases, suggesting that this polymorphism could interact with the Y
RAJAN et al: CY11B2 OLYMORHISM IN AMIL OULAION 383 able IV. Summary of the influence of CY11B2 C344 polymorphism on various ethnic populations Author opulation Cardiovascular disorder Sample size (case/control) % allele frequency Associated allele Study type och et al 2 Spanish EH 66 64 NS Crosssectional Kupari et al 5 Finnish Healthy volunteers 84 51 C Healthy controls Brand et al 6 French EH 380/293 56/49 * Case control Nicod et al 9 Swiss EH 141 53 Crosssectional Matsubara et al 11 Japanese EH 1242 65 Crosssectional Davies et al 12 Scottish EH 138/138 60/53 * Case control sukada et al 13 Japanese EH 250/221 66/73 * C Case control ang et al 14 Chinese EH 271/267 78/86 * C Case control Schunkert et al 15 Germans LVH 562 58 NS Crosssectional iret et al 16 French IDC 433/401 57/54 * NS Case control Yamagishi et al 17 Japanese B 2823 67 NS Crosssectional Hengstenberg Germans MI 606 56 NS Crosssectional et al 18 Stella et al 19 Italians EH 210 54 Crosssectional Barbato et al 20 Rajput et al 21 Whites Africans Asians Indians (Highlanders) A, ARR,B 456 441 416 55 79 56 Crosssectional EH 100/190 64/70 * C Case control Komiya et al 23 Japanese EH 73/134 66/63 * C Case control Kumar et al 24 Australian EH 146/291 48/58 * C Case control Gu et al 25 Chinese EH 503/503 70/68 * NS Case control amaki et al 26 Japanese Hypertension 255/227 74/68 * C Case control Russoa et al 30 Italian Healthy volunteers 811 52 Cross sectional Lim et al 32 Whites (English) Hypertension 325 53 Cross sectional rasad et al 33 Indians DM 196 64 Cross sectional CRI (DM) 225 73 Hautanen et al 34 Finnish MI 141/270 48/53 * C Case control resent study South Indians (amil population) EH 406/424 67/60 * Case control EH, essential hypertension; ARR, aldosterone renin ratio; B, blood pressure; LVH, left ventricular hypertrophy; A, plasma aldosterone; CRI, chronic renal insuffiency; DM, diabetes mellitus; IDC, idiopathic dilated cardiomyopathy; MI, myocardial infarction. NS indicates that the study was not significant for either or C allele; * indicates the percentage of allele frequency for case group/control group of case control study chromosome 24. Our study suggests that the risk was confined to male hypertensives and the reason for this has to be further evaluated. A larger communitybased study in Japanese population revealed that C344 polymorphism was not associated with blood pressure levels in either sex 17. Variations in CY11B2 C344 have been shown to be associated with increased blood pressure levels, plasma and urinary levels of aldosterone, and plasma renin levels 6,21,29. We could not analyze the mean blood pressure and plasma aldosterone levels in cases since many of our subjects were on antihypertensive medication which might influence the study. When the blood pressure level was analyzed in healthy volunteers, significant difference was not observed among the genotypes (data not shown). However, the results of our study are in agreement with the Caucasian study that did not show significant association between blood pressure and C344 genotypes 29. here was a linear increase in diastolic blood pressure in 344 allele carriers with increase in age in the Italian population 30. Another study conducted in elderly Caucasian population reported that systolic blood pressure was higher in genotypes, compared to CC genotypes 31. Association of genetic polymorphisms with complex polygenic disorders such as hypertension is further strengthened by haplotype and linkage disequilibrium analysis. CY11B2 C344
384 INDIAN J MED RES, OCOBER 2010 polymorphism was found to be in linkage disequilibrium with other CY11B2 Int2W/C and K173R polymorphisms 21. Aldosteronerenin ratio was found to be significantly higher in hypertensive cases with haplotype combination of variant alleles 344 and Int2C when compared to other haplotype combinations 9. Interethnic variation exists in the polymorphism of C344 polymorphism. A metaanalysis was carried out to examine the prevalence of C344 polymorphism among different ethnics (able IV). here is significant heterogeneity among the reports, with some studies showing association with allele and others with C allele. he difference could be attributed to variations in environmental factors, apart from the differences in the selection of cases and controls, sample size, age, BMI, and other ecological factors. 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