Departments of 1Biochemistry and 2Medicine, Era’s Lucknow Medical College & Hospital, Lucknow, India *Corresponding Author e-mail: tasleem24@gmail.com ارتباط تعدد األشكال اجلينية ألنزمي حتويل األجنيوتنسني واجللوتاثيون س-ترانسفرييز مع مؤشر كتلة اجلسم بني اهلنود الشماليني املصابني بارتفاع ضفط الدم �سليحة ر�سوي، �سيد ت�سليم ر�سا، زيبا �سديقي، �سانيا عبا�ض، فرزانة مهدي abstract: Objectives: This study aimed to examine the association of angiotensin-converting enzyme (ACE) and glutathione S-transferase (GST) gene polymorphisms with body mass index (BMI) in hypertensive North Indians. Methods: This case-control study was carried out between May 2013 and November 2014 at the Era’s Lucknow Medical College & Hospital, Lucknow, India, and included 378 subjects divided into three groups. One group constituted 253 hypertensive individuals (sustained diastolic blood pressure of >90 mmHg and systolic blood pressure of >140 mmHg) who were subcategorised according to normal (<25 kg/m2) or high (≥25 kg/m2) BMI. The third group consisted of 125 age-, gender- and ethnically-matched normotensive controls with a normal BMI. Gene polymorphisms were evaluated by polymerase chain reaction. The genotypic and allelic frequency distribution among both groups were analysed. Results: A significant difference was found between GST theta 1-null and GST mu 1-positive genotype frequencies among the hypertensive overweight/obese individuals and controls (P = 0.014 and 0.033, respectively). However, no difference was observed in the frequency of ACE polymorphisms. ACE insertion/insertion genotype (P = 0.006), insertion and deletion alleles (P = 0.007 each) and GST theta 1-null and GST theta 1-positive genotypes (P = 0.006 each) were found to differ significantly between hypertensive cases and controls, regardless of BMI. Conclusion: ACE and GST gene polymorphisms were not associated with BMI but were significantly associated with hypertension among the studied group of North Indians. Keywords: Angiotensin Converting Enzyme; Glutathione Transferase; Genetic Polymorphisms; Hypertension; Body Mass Index; Obesity; India. واجللوتاثيون )ACE( الأجنيوتن�سني حتويل لأنزمي اجليني الأ�سكال تعدد ارتباط درا�سة اإىل الدرا�سة هذه هدفت الهدف: امللخ�ص: �ض-تران�سفرييز )GST( مع موؤ�رس كتلة اجل�سم بني الهنود ال�سماليني ذوي مر�ض �سفط الدم املرتفع. الطريقة: اأجريت هذه الدرا�سة املقارنة بني احلالت وجمموعة التحكم بني مايو 2013 ونوفمرب 2014 يف كلية ايرا�ض لكناو الطبية وم�ست�سفى، لكناو، ولية اوتار برادي�ض، الهند، و�سملت 378 �سخ�ض مت تق�سيمهم اإىل ثالث جمموعات. ت�سكل املجموعة الأوىل 253 من الأفراد امل�سابني بارتفاع �سغط الدم )�سغط الدم النب�ساطي امل�ستمر 90> مم زئبق و�سغط الدم النقبا�سي 140> مم زئبق( الذين مت تق�سيمهم بعد ذلك ح�سب كتلة اجل�سم اىل )25< كجم/ م2( و)25≤ كجم/م2( وتاألفت املجموعة الثالثة من 125 متماثلي العمر واجلن�ض والعرق و �سغط الدم و موؤ�رس كتلة ج�سم طبيعيني. مت تقييم اجلينات متعددة الأ�سكال عن طريق تفاعل البلمرة املت�سل�سل. وقد مت حتليل توزيع الرتدد الوراثي واأليلية بني املجموعتني. النتائج: وجد فرق كبري بني وترية النمط اجليني ل GST ثيتا 1 امل�سفر و GST ميوالإيجابي بني الأفراد امل�سابني بارتفاع �سغط الدم و يعانون من .ACE على التوايل(. ومع ذلك، مل يالحظ اأي اختالف يف تعدد اأ�سكال ،P = 0.033 و P = 0.014( زيادة الوزن/ال�سمنة وال�سوابط الأ�سحاء GST ثيتا 1-ا امل�سفر و GST لكل منهما( و P = 0.007( واإدراج وحذف الأليالت ،)P = 0.006( ACE بالن�سبة لالإدراج/احلذف اجليني ل ثيتا 1 الإيجابي )P = 0.006 لكل منهما( وجد اأن هناك اختالف اح�سائي بني حالت ارتفاع �سغط الدم واملجموعة ال�سابطة بغ�ض النظر عن موؤ�رس كتلة اجل�سم. اخلال�صة: مل يرتبط تعدد الأ�سكال اجليني ل ACE و GST مع موؤ�رس كتلة اجل�سم لكن ارتبط اإح�سائيا مع ارتفاع �سغط الدم يف جمموعة البحث من الهنود ال�سماليني. اجل�سم؛ كتلة موؤ�رس الدم؛ �سغط ارتفاع اجلينية؛ الأ�سكال تعدد تران�سفريز؛ اجللوتاثيون لالأجنيوتن�سني؛ املحول الإنزمي الكلمات: مفتاح ال�سمنة؛ الهند. Association of Angiotensin-Converting Enzyme and Glutathione S-Transferase Gene Polymorphisms with Body Mass Index among Hypertensive North Indians Saliha Rizvi,1 *Syed T. Raza,1 Zeba Siddiqi,2 Shania Abbas,1 Farzana Mahdi1 Advances in Knowledge - To the best of the authors’ knowledge, this is the first study to investigate the effect of single nucleotide polymorphisms in angiotensin- converting enzyme (ACE) and glutathione S-transferase (GST) genes on body mass index (BMI) in hypertensive North Indians. clinical & basic research Sultan Qaboos University Med J, November 2015, Vol. 15, Iss. 4, pp. e477–485, Epub. 23 Nov 15 Submitted 16 Jan 15 Revisions Req. 5 Apr & 16 Jun 15; Revisions Recd. 22 Apr & 19 Jun 15 Accepted 2 Jul 15 doi: 10.18295/squmj.2015.15.04.006 Association of Angiotensin-Converting Enzyme and Glutathione S-Transferase Gene Polymorphisms with Body Mass Index among Hypertensive North Indians e478 | SQU Medical Journal, November 2015, Volume 15, Issue 4 Hypertension and obesity are emer-ging global health concerns.1 Many epidemiological studies have shown an association between these medical conditions.2,3 Hypertension predisposes individuals to future weight gain as compared to their normotensive counterparts, which means that even hypertensive individuals at a normal weight are at significantly higher risk of becoming obese.4 The body mass index (BMI) is used to classify individuals according to various weight categories. A high BMI is associated with an increased burden of diabetes, hypertension and cardiovascular diseases.1 Aside from environmental and lifestyle factors, BMI is also influenced by genetic factors. Research has confirmed the involvement of different genetic polymorphisms in the metabolism and appetite of predisposed individuals, thus causing them to become overweight or obese.1 More than 41 loci on the human genome have been linked with the development of obesity.1 The angiotensin-converting enzyme (ACE) is part of the renin-angiotensin system, which is responsible for regulating blood pressure and the balance of fluids and salts inside the body. Engeli et al. suggested that components of the renin-angiotensin system might be involved in the development of obesity and associated hypertension.5 The insertion/deletion polymorphism occurs in intron 16 of the ACE gene due to the pre- sence or absence of a 278 base pairs (bp) Arthrobacter luteus repetitive sequence.6 This polymorphism has been found to drastically affect ACE activity where the deletion/deletion genotype increases ACE activity while the insertion/insertion genotype decreases activity.7 A study conducted in Pakistan showed the association of the ACE gene polymorphism with obesity, suggesting a role for the deletion allele in adipogenesis and adipocyte metabolism.8 Hirata et al. confirmed the role of oxidative stress in linking obesity with hypertension.9 Systemic oxidative stress levels increase obesity, which may lead to the development of obesity-related hypertension. Glutathione S-transferase (GST) plays a major role in combating reactive oxygen species, thus protecting the body against its harmful effects.10 Evidence also indicates the involvement of oxidative stress with the pathogenesis of hypertension.9 Among humans, the gene coding for GST mu 1 (GSTM1) and GST theta 1 (GSTT1) are located on chromosomes 1p13.3 and 22, respectively. Previous studies have demonstrated that GST polymorphisms were not associated with BMI in diabetic North Indians or with weight gain in schizophrenic Koreans.11,12 Since there is a close link between hypertension and obesity, this study sought to investigate the association of ACE and GST gene polymorphisms with BMI among a selected group of hypertensive North Indians. Methods This case-control study was carried out between May 2013 and November 2014 at the Era’s Lucknow Medical College & Hospital, Lucknow, India. A total of 253 subjects with either a recent diagnosis or history of hypertension were recruited from the Department of Medicine at Era’s Lucknow Medical College & Hospital. These patients mostly resided in Lucknow and adjoining areas, including Hardoi and Barabanki. A diagnosis of hypertension was based on sustained diastolic blood pressure of >90 mmHg and systolic blood pressure of >140 mmHg. Hypertensive patients were subsequently subcate- gorised according to BMI into normal weight (BMI <25 kg/m2) or overweight/obese (≥25 kg/m2) groups. As a control group, 125 age-, gender- and ethnically-matched normotensive individuals of normal weight (BMI <25 kg/m2) were recruited for inclusion in the study. Patients who visited the outpatient clinics for minor illnesses and with no history of major clinical diseases were recruited as controls. Subjects in either group with a history of smoking or evidence of cancer, organ dysfunction or metabolic disease were excluded. The sample size calculations were based on the proportion of ACE genotypes among the cases and controls using the following formula: Based on a 95% level of significance and 80% expected power (20% type 2 error), the minimum sample size was 103 in each group. Subject to the N = (Zα + Zβ)2/{ln (1-e)}*[{(1-p1)/p1} + (1-p2)/p2}] - While ACE and GST gene polymorphisms were not found to be associated with BMI among this study population, they were significantly associated with hypertension. Application to Patient Care - The results of this study could be helpful in understanding the impact of genetic polymorphisms on BMI and on an individual’s predisposition to hypertension. - Additionally, these findings could contribute in the identification of novel factors leading to the development of a high BMI; this would assist in the prevention and cure of various related metabolic diseases. Saliha Rizvi, Syed T. Raza, Zeba Siddiqi, Shania Abbas and Farzana Mahdi Clinical and Basic Research | e479 availability of cases, this was then increased so that the power would increase correspondingly. Clinical data were collected from each subject, including gender, age, alcohol consumption, waist-to- hip ratio (WHR), weight, height and family history. Quetelet’s equation (weight in kg/height in m2) was used to calculate the BMI of each participant. WHR was calculated by dividing the waist circumference at the narrowest point above the hip by the hip circumference at its greatest gluteal protuberance. Serum triglycerides were measured using the glycerol phosphate oxidase-peroxidase-amidopyrine method while serum cholesterol was measured by the cholesterol oxidase-peroxidase method. High- density lipoprotein cholesterol was measured by the immunoinhibition method using the Erba XL-300 fully automated chemistry analyser (Erba Diagnostics Mannheim GmbH, Mannheim, Germany). Very low- density lipoprotein levels were estimated by dividing measured triglycerides by 5 mg/dL units. Low-density lipoprotein cholesterol levels were estimated using the Friedewald formula.13 The DNA extraction was performed using 3 mL of venous blood collected from all participants in 0.5 M ethylenediaminetetraacetic acid tubes. A stan- dard phenol-chloroform extraction method was used to isolate genomic DNA from the blood samples.14 The DNA concentration was determined by a spectrophotometer and samples were stored at -20 °C. ACE insertion/deletion polymorphisms (rs464994) were analysed by genomic DNA amplification using polymerase chain reaction (PCR) technology.15 PCR amplification was conducted in a total volume of 20 µL using: 10 pmol each of forward primer 5’-CT- GGAGACCACTCCCATCCTTTCT-3’ and reverse primer 5’-GATGTGGCCATCTTCGTCAGAT-3’; 200 ng of genomic DNA; 3 mM of magnesium chloride; 50 mM of potassium chloride; 10 mM of trisamino- methane hydrochloride at a pH of 8.4; 0.5 mM each of deoxyribonucleotide triphosphate; and 2 U of Thermus aquaticus (Taq) polymerase (Bioline Reagents Ltd., London, UK). The PCR amplification was performed using an initial denaturation at 94 °C for five minutes, followed by 35 cycles of denaturation at 94 °C for 45 seconds, annealing at 60 °C for one minute and 15 seconds, extension at 72 °C for two minutes and 30 seconds and final extension at 72 °C for five minutes. The amplified products were separated on a 2.0% agarose gel stained with 0.5 ug/mL of ethidium bromide and visualised using a bioimaging and gel documentation system (UVP LLC, Upland, California, USA). The GSTM1 and GSTT1 genetic polymorphisms were analysed by genomic DNA amplification using the multiplex-PCR technique (MJ Mini™ Thermal Cycler, Bio Rad Laboratories Inc., Hercules, California, USA).15 Primers for GSTM1 were 5’-AACTCCCTGAAAAGCTAAAGC-3’ and 5’-GTT- GGGCTCAAATATACGGTGG-3’, while primers for GSTT1 were 5’-TTCCTTACTGGTCCTCACATCT C-3’ and 5’-TCACCGGACATGGCCAGCA-3’. To avoid false-negative readings, β-globin was used as an internal control. Primers for β-globin were 5’-CAACTTCATCCACGTTCACC-3’ and 5’-GAA GAGCCAAGGACAGGTAC-3’. PCR amplification was conducted in a total volume of 25 μL using: 10 pmol of each primer; 100 ng of genomic DNA; 2.5 mmol/L of magnesium chloride; 0.2 mmol/L each of deoxyribonucleotide triphosphate; and 1 U of Taq polymerase (Bioline Reagents Ltd). The PCR amplification was performed using an initial denaturation at 94 °C for five minutes, followed by 30 cycles at 94 °C for one minute, 64 °C for one minute, 72 °C for one minute and a final extension of 72 °C for seven minutes. Amplified products were separated on a 1.5% agarose gel stained with 0.5 μg/mL of ethidium bromide and visualised using a bioimaging and gel documentation system (UVP LLC). Direct sequencing was carried out for approximately 10% of all samples for both polymorphisms studied and genotyping for 20% of the samples was further duplicated. The results were verified by two individual laboratory technicians to confirm genotyping results. Statistical analysis was performed using the Statis- tical Package for the Social Sciences (SPSS), Version 12 (IBM Corp., Chicago, Illinois, USA). Variables were tested for normality using the Kolmogorov- Smirnov test. Variables with a normal distribution were expressed as means ± standard deviation while variables with a non-normal distribution were expressed as medians and interquartile ranges and were log transformed before statistical analysis. A Chi-squared test was used to compare genotyping data between cases and controls. A P value of ≤0.050 was considered significant. The odds ratio (OR) and 95% confidence interval (CI) was calculated to test the relative risk for association. Other variables were compared using the Student’s t-test for normally- distributed variables. Ethical approval for this study was granted by the Institutional Ethical Committee of the Department of Medicine at Era’s Lucknow Medical College & Hospital (#ELMC/E-1/3942). Written informed consent was obtained from each subject prior to data collection. Results A total of 378 patients were included in this study, Association of Angiotensin-Converting Enzyme and Glutathione S-Transferase Gene Polymorphisms with Body Mass Index among Hypertensive North Indians e480 | SQU Medical Journal, November 2015, Volume 15, Issue 4 ranging in age from 26‒60 years old. There were 103 subjects in the hypertensive normal weight group, 150 subjects in the hypertensive overweight/obese group and 125 subjects in the control group. Obesity-related parameters were higher in the hypertensive groups compared to the control group [Table 1]. For ACE polymorphisms, a single 490 bp band represented an insertion/insertion genotype, two bands of 490 bp and 190 bp represented an insertion/deletion genotype, whereas the deletion/deletion genotype was confirmed by a single band of 190 bp [Figure 1]. For GST genetic polymorphisms, the band sizes observed were 215 bp, 480 bp and 268 bp for GSTM1, GSTT1 and β-globin, respectively [Figure 2]. Negligible discrepancy in genotyping was observed. The genotype frequencies of ACE insertion/ deletion, deletion/deletion and insertion/insertion genotypes among the hypertensive overweight/obese group and the hypertensive normal weight group were 53.34%, 19.33% and 27.33% and 54.37%, 16.50% and 29.13%, respectively (OR = 0.96, 1.22 and 0.91; 95% CI = 0.56–1.66, 0.59–2.49 and 0.50–1.67; P = Figure 1: Agarose gel image showing polymerase chain reaction products for an angiotensin-converting enzyme polymorphism. Lanes 1, 2 and 8 show the insertion/ insertion genotype at 490 base pairs (bp), lanes 6 and 7 show the insertion/deletion genotype at 490 bp and lanes 3 and 5 show the deletion/deletion genotype in a single band of 190 bp. Lane 4 is a 100 bp ladder. Figure 2: Agarose gel image showing polymerase chain reaction products of glutathione S-transferase (GST) polymorphisms. The presence or absence of GST mu 1 (GSTM1) and GST theta 1 (GSTT1) was detected by 480 base pairs (bp) and 210 bp bands, respectively. β-globin was considered an internal control at 260 bp. Lanes 1 and 7 show GSTM1- and GSTT1-null genotypes, respectively, and lanes 2–5 show GSTM1 and GSTT1 wild-type genotypes. Lane 4 is a 100 bp ladder. Table 1: Characteristics of overweight/obese* hypertensive subjects and hypertensive subjects of a normal weight† in comparison to an age-, gender- and ethnically-matched control group† in North India (N = 378) Characteristic Mean ± SD P value‡ Mean ± SD P value‡ HH group (n = 150) Control group (n = 125) HN group (n = 103) Control group (n = 125) Male-to-female ratio 83:67 72:53 0.706 58:45 72:53 0.845 Age in years 43.42 ± 13.29 40.03 ± 10.28 0.017 42.02 ± 16.65 40.03 ± 10.28 0.290 BMI in kg/m2 28.87 ± 3.78 21.92 ± 2.60 <0.001 21.68 ± 1.93 21.92 ± 2.60 0.424 SBP in mm Hg 147.63 ± 21.16 110.00 ± 10.61 <0.001 146.96 ± 17.87 110.00 ± 10.61 <0.001 DBP in mm Hg 97.85 ± 4.02 75.00 ± 9.20 <0.001 93.50 ± 5.67 75.00 ± 9.20 <0.001 SC in mg/dL 194.92 ± 43.27 166.73 ± 24.17 <0.001 181.11 ± 56.88 166.73 ± 24.17 0.017 Median TG in mmol/L-1 (IQR) 2.12 (1.50–2.62) 1.32 (0.95–2.10) <0.001 1.94 (0.96–1.98) 1.32 (0.95–2.10) <0.001 HDL in mg/dL 41.24 ± 8.53 55.23 ± 9.59 <0.001 45.77 ± 8.79 55.23 ± 9.59 <0.001 VLDL in mg/dL 34.41 ± 20.55 30.83 ± 11.64 0.070 33.53 ± 16.7 30.83 ± 11.64 0.166 LDL in mg/dL 102.82 ± 47.44 87.80 ± 19.83 <0.001 103.78 ± 31.93 87.80 ± 19.83 <0.001 WHR 0.93 ± 0.02 0.86 ± 0.08 <0.001 0.86 ± 0.06 0.86 ± 0.08 0.667 SD = standard deviation; HH = hypertensive overweight/obese group; HN =hypertensive normal weight group; BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; SC = serum cholesterol; TG = triglycerides; IQR = interquartile range; HDL = high-density lipoproteins; VLDL = very low-density lipoproteins; LDL = low-density lipoproteins; WHR = waist-to-hip ratio. *BMI ≥25 kg/m2. †BMI <25 kg/m2. ‡Calculated using the Student’s t-test. A P value of ≤0.050 was considered significant. Saliha Rizvi, Syed T. Raza, Zeba Siddiqi, Shania Abbas and Farzana Mahdi Clinical and Basic Research | e481 0.889, 0.586 and 0.757). In the control group, the same ACE genotype frequencies were 59.20%, 27.20% and 13.60%, respectively (OR = 0.86, 0.68 and 2.37; 95% CI = 0.49–1.48, 0.35–1.31 and 1.16–4.83; P = 0.575, 0.245 and 0.015). Frequencies of insertion and deletion alleles were 54.00% and 46.00% for the hypertensive overweight/obese group compared to 56.31% and 43.69% for the hypertensive normal weight group (OR = 0.91 and 1.10; 95% CI = 0.61–1.34 and 0.75–1.63; P = 0.620 each) and 43.20% and 56.80% for the control subjects, respectively (OR = 1.60 and 0.63; 95% CI = 1.08–2.36 and 0.42–0.92; P = 0.018 each) [Tables 2 and 3]. The GSTM1-null genotype frequency was 33.33% for the hypertensive overweight/obese group, while it was 36.89% and 45.60% for the hypertensive normal weight and control groups, respectively (OR = 0.84 and 0.61; 95% CI = 0.47–1.50 and 0.35–1.07; P = 0.559 Table 2: Frequencies of angiotensin-converting enzyme and glutathione S-transferase genetic polymorphisms among overweight/obese* hypertensive subjects and hypertensive subjects of a normal weight† in North India (N = 253) Polymorphism n (%) OR 95% CI χ2 P value‡ Power HN group (n = 103) HH group (n = 150) I/D 56 (54.37) 80 (53.34) 0.96 0.56–1.66 0.02 0.889 0.610 D/D 17 (16.50) 29 (19.33) 1.22 0.59–2.49 0.30 0.586 0.813 I/I 30 (29.13) 41 (27.33) 0.91 0.50–1.67 0.10 0.757 0.622 I 116 (56.31) 162 (54.00) 0.91 0.61–1.34 0.25 0.620 0.735 D 90 (43.69) 138 (46.00) 1.10 0.75–1.63 0.25 0.620 0.735 GSTT1/GSTM1-positive 49 (47.57) 80 (53.34) 1.26 0.73–2.18 0.70 0.403 0.900 GSTT1-null 16 (15.53) 20 (13.33) 0.86 0.39–1.86 0.16 0.693 0.654 GSTM1-positive 65 (63.12) 100 (66.67) 1.19 0.67–2.11 0.34 0.559 0.822 GSTT1 -positive 87 (84.47) 130 (86.67) 1.17 0.54–2.54 0.16 0.693 0.753 GSTM1-null 38 (36.89) 50 (33.33) 0.84 0.47–1.50 0.34 0.559 0.763 OR = odds ratio; CI = confidence interval; HN = hypertensive normal weight group; HH = hypertensive overweight/obese group; ACE = angiotensin- converting enzyme; I = insertion; D = deletion; GST = glutathione S-transferase; GSTT1 = GST theta 1; GSTM1 = GST mu 1. *BMI ≥25 kg/m2. †BMI <25 kg/m2. ‡Calculated using the Student’s t-test. A P value of ≤0.050 was considered significant. A C E G ST G en ot yp e G en ot yp e A lle le A lle le Table 3: Frequencies of angiotensin-converting enzyme and glutathione S-transferase genetic polymorphisms among overweight/obese* hypertensive subjects in comparison to an age-, gender- and ethnically-matched control group† in North India (N = 275) Polymorphism n (%) OR 95% CI χ2 P value Power Control group (n = 125) HH group (n = 150) I/D 74 (59.20) 80 (53.34) 0.86 0.49–1.48 0.31 0.575 0.812 D/D 34 (27.20) 29 (19.33) 0.68 0.35–1.31 1.35 0.245 0.922 I/I 17 (13.60) 41 (27.33) 2.37 1.16–4.83 5.86 0.015 0.908 I 108 (43.20) 162 (54.00) 1.60 1.08–2.36 5.60 0.018 0.882 D 142 (56.80) 138 (46.00) 0.63 0.42–0.92 5.60 0.018 0.882 GSTT1/GSTM1-positive 63 (50.40) 80 (53.34) 1.22 0.70–2.10 0.49 0.486 0.811 GSTT1-null 5 (4.00) 20 (13.33) 3.89 1.23–12.27 6.09 0.014 0.900 GSTM1-positive 68 (54.40) 100 (66.67) 1.84 1.05–3.24 4.54 0.033 0.900 GSTT1 -positive 120 (96.00) 130 (86.67) 0.46 0.18–1.20 2.60 0.107 0.885 GSTM1-null 57 (45.60) 50 (33.33) 0.61 0.35–1.07 2.94 0.086 0.879 OR = odds ratio; CI = confidence interval; HH = hypertensive overweight/obese group; ACE = angiotensin-converting enzyme; I = insertion; D = deletion; GST = glutathione S-transferase; GSTT1 = GST theta 1; GSTM1 = GST mu 1. *BMI ≥25 kg/m2. †BMI <25 kg/m2. ‡Calculated using the Student’s t-test. A P value of ≤0.050 was considered significant. A C E G ST G en ot yp e G en ot yp e A lle le A lle le Association of Angiotensin-Converting Enzyme and Glutathione S-Transferase Gene Polymorphisms with Body Mass Index among Hypertensive North Indians e482 | SQU Medical Journal, November 2015, Volume 15, Issue 4 and 0.086). The frequency of the GSTT1-null genotype was 13.33% among the hypertensive overweight/obese group as compared to 15.53% and 4.00% among the hypertensive normal weight and control groups, respectively (OR = 0.86 and 3.89; 95% CI = 0.39–1.86 and 1.23–12.27; P = 0.693 and 0.014). The frequencies of GSTM1- and GSTT1-positive genotypes were 66.67% and 86.67% in the hypertensive overweight/ obese group, while they were 63.12% and 84.47% for the hypertensive normal weight group (OR = 1.19 and 1.17; 95% CI = 0.67–2.11 and 0.54–2.54; P = 0.559 and 0.693) and 54.40% and 96.00% in the control group, respectively (OR = 1.84 and 0.46; 95% CI = 1.05–3.24 and 0.18–1.20; P = 0.033 and 0.107). The frequency of the GSTT1-/GSTM1-positive genotype was 53.34% in the hypertensive overweight/obese group, 47.57% for the hypertensive normal weight group and 50.40% in the control group (OR = 1.26 and 1.22; 95% CI = 0.73–2.18 and 0.70–2.10; P = 0.403 and 0.486) [Tables 2 and 3]. The ACE insertion/insertion genotype frequency among the combined overweight/obese and normal weight hypertensive groups was 28.07% compared to 13.60% in the control group (OR = 2.41; 95% CI = 1.27–4.57; P = 0.006). The deletion/deletion genotype frequency was 17.18% among hypertensives compared to 27.20% among healthy controls (OR = 0.59; 95% CI = 0.34–1.04; P = 0.068). Among the hypertensives, frequencies of the insertion and deletion alleles were 55.14% and 44.86%, respectively, compared with 43.20% and 56.80% in the control group, respectively (OR = 1.59 and 0.63; 95% CI = 1.13–2.24 and 0.45– 0.88; P = 0.007 each). The frequencies of the GSTT1- and GSTM1-null genotypes in the hypertensive groups were 14.69% and 34.78%, respectively (OR = 4.09 and 0.63; 95% CI = 1.40–11.96 and 0.39–1.03; P = 0.006 and 0.062). A significant difference was found between the GSTT1-null (14.69% versus 4.00%) and -positive (85.37% versus 96.00%; OR = 0.24; 95% CI = 0.08–0.71; P = 0.006) genotypes among the hypertensive groups in comparison to the control group. However, there was a non-significant difference between frequencies of GSTM1-positive genotypes (65.22% versus 54.40%; OR = 1.59; 95% CI = 0.97–2.58; P = 0.062) [Table 4]. Discussion Hypertension and obesity may have a mutual or reciprocal relationship, where the presence of one potentially leads to the other.4 Studies have shown that the prevalence of hypertension is directly related to a higher BMI.2,3 In the current study, lipid profile variables (including serum cholesterol, triglycerides, low- and very low-density lipoproteins and WHR) were higher among the hypertensive individuals than the healthy controls, regardless of BMI category. Various studies have demonstrated increased plasma renin activity and plasma angiotensinogen, angiotensin II and aldosterone levels in obese individuals.16–18 Cooper et al. pointed out that the ACE insertion/ deletion polymorphism was linked to changes in ACE levels, but was inconsistently associated with BMI.19 Table 4: Frequencies of angiotensin-converting enzyme and glutathione S-transferase genetic polymorphisms among hypertensive subjects in comparison to an age-, gender- and ethnically-matched control group in North India (N = 378) Polymorphism n (%) OR 95% CI χ2 P value* Power Hypertensive subjects (n = 253) Control group (n = 125) I/D 137 (54.15) 74 (59.20) 0.81 0.50–1.32 0.71 0.398 0.913 D/D 45 (17.18) 34 (27.20) 0.59 0.34–1.04 3.33 0.068 0.935 I/I 71 (28.07) 17 (13.60) 2.42 1.27–4.57 7.50 0.006 0.969 I 279 (55.14) 108 (43.20) 1.59 1.13–2.24 7.25 0.007 0.940 D 227 (44.86) 142 (56.80) 0.63 0.45–0.88 7.25 0.007 0.940 GSTT1/GSTM1-positive 128 (50.59) 63 (50.40) 1.02 0.63–1.64 0.01 0.936 N/A GSTT1-null 37 (14.69) 5 (4.00) 4.09 1.40–11.96 7.61 0.006 0.969 GSTM1-positive 165 (65.22) 68 (54.40) 1.59 0.97–2.58 3.47 0.062 0.880 GSTT1 -positive 216 (85.37) 120 (96.00) 0.24 0.08–0.71 7.61 0.006 0.984 GSTM1-null 88 (34.78) 57 (45.60) 0.63 0.39–1.03 3.47 0.062 0.910 OR = odds ratio; CI = confidence interval; ACE = angiotensin-converting enzyme; I = insertion; D = deletion; GST = glutathione S-transferase; GSTT1 = GST theta 1; GSTM1 = GST mu 1. *Calculated using the Student’s t-test. A P value of ≤0.050 was considered significant. A C E G ST G en ot yp e G en ot yp e A lle le A lle le Saliha Rizvi, Syed T. Raza, Zeba Siddiqi, Shania Abbas and Farzana Mahdi Clinical and Basic Research | e483 A study of a Saudi population noted a higher frequency of the mutant deletion allele in hypertensive cases with a BMI of ≥30 kg/m2 in comparison to controls, although the difference was not significant.20 However, this result was reversed in the current study, as the frequency of the deletion allele among the hypertensive overweight/obese group was significantly lower than the control group. A similar study performed on Korean women also confirmed this finding (P = 0.063).21 Similarly, Passaro et al. showed that subjects with different polymorphisms in the ACE gene did not show significant variability in BMI.22 The frequency of the ACE deletion/deletion genotype among hypertensive overweight/obese individuals in the current study (19.33%) was similar to that found among an overweight Pakistani population (18.00%).8 The frequency of the ACE insertion/ insertion genotype in the hypertensive overweight/ obese group of the current study (27.33%) was similar to that found among obese Pakistani individuals (26.00%); however, it was lower than the frequency found among overweight Pakistanis (32.00%).8 Eisenmann et al. reported that children with the ACE deletion/deletion genotype had a higher BMI compared to insertion/insertion carriers;23 this is consistent with the results obtained in the current study when comparing the hypertensive groups according to BMI, although this was not found to be significant. These results are contradictory to another report which demonstrated that the mutant deletion allele was associated with a low BMI.24 On further comparison of the hypertensive overweight/obese and control groups in the current study, a significant difference was found between the frequency of the insertion/insertion genotype and between the allele frequencies of insertion and deletion alleles. While no significant association was found between the ACE insertion/deletion polymorphism and BMI in the hypertensives, a significant association was observed with hypertension. This suggests that the ACE insertion/deletion polymorphism affects hypertension as opposed to BMI, with carriers of the insertion/ insertion genotype being more prone to hypertension. The insertion/insertion genotype frequency in the combined North Indian hypertensive population of the current study (28.07%) was similar to that found in hypertensive Pakistanis (29.30%) and another North Indian population (28.62%).15,25 There was a significant association between the ACE insertion/insertion genotype and hypertension; this contrasts with the results of a study conducted among South Indians, although it is similar to that of the other study on North Indians.15,26 The current study found an association between the ACE deletion/deletion genotype and hypertension, although this was not significant. The frequency of this genotype among hypertensives in the current study (17.18%) was higher than that found for Malaysian and Bangladeshi individuals (10.76% and 11.40%, respectively); however, a Chinese population had a lower frequency.27–29 The insertion and deletion alleles were significantly related to hypertension in the current study, which correlates with the findings of a similar North Indian study.15 A positive correlation has been reported between various obesity variables like BMI and WHR, including oxidative stress markers such as reduced erythrocyte glutathione.30 In the current study, significant variation was found between the GSTT1-null genotype frequency among the hypertensive overweight/ obese and control groups. Similarly, the frequency of the GSTM1-positive genotype was significantly higher in the hypertensive overweight/obese subjects. However, this difference was not significant when compared with the hypertensive normal weight group. The frequencies of the GSTM1- and GSTT1- null genotypes among hypertensive overweight/obese subjects in the present study (33.33% and 13.33%, respectively) were lower than those of non-Hispanics (45.30% and 81.60%, respectively), African Americans (72.20% and 76.20%, respectively) and Hispanics (46.70% and 81.70%, respectively) with higher BMIs.31 In contrast, the frequencies of GSTM1- and GSTT1- positive genotypes were higher when compared to overweight/obese non-Hispanics (54.70% and 18.40%, respectively), African Americans (27.80% and 23.80%, respectively) and Hispanics (53.30% and 18.30%, respectively).31 The GSTM1- and GSTT1-positive genotype frequencies were somewhat higher among the overweight/obese hypertensives in the current study than for hypertensives of a normal weight, while the null genotype frequencies were lower among the former than the latter. However, these differences were not significant. The GSTM1- and GSTT1-positive genotype findings were similar across all three groups; nevertheless, there was a slightly higher although non- significant frequency in the hypertensive overweight/ obese group than the hypertensive normal weight group. No association was found between GST polymorphisms and BMI in Indian diabetic patients with null and positive GST genotypes.11,32 Similarly, in Korean schizophrenic patients, no association was observed between GST polymorphisms and drug- induced weight gain.12 In view of these results, it can be concluded that GST polymorphisms are not associated with a higher BMI among hypertensive patients. Association of Angiotensin-Converting Enzyme and Glutathione S-Transferase Gene Polymorphisms with Body Mass Index among Hypertensive North Indians e484 | SQU Medical Journal, November 2015, Volume 15, Issue 4 In the present study, a significant difference was found between the GSTT1-null and -positive genotypes among hypertensives in comparison to control subjects. However, frequencies of GSTM1- null and-positive genotypes were not significant. The frequencies of the GSTT1- and GSTM1-null genotypes among hypertensive patients in the current study (14.69% and 34.78%, respectively) were lower in comparison to findings from Arab (30.00% and 60.00%, respectively), Chinese (58.10% and 60.00%, respectively) and Korean (55.40% and 51.80%, respectively, and 61.20% and 62.40%, respectively) populations, but similar to findings from another North Indian study (11.59% and 34.05%, respectively).15,33–36 In comparison, the GSTM1-positive genotype was found in 65.22% of the hypertensives in the current study; this was higher than the results from Arab (40.00%), Chinese (40.00%) and Korean (48.20% and 37.60%) populations, but again similar to results from a North Indian study.15,33–36 Conclusion Among the studied North Indian population, ACE and GST genetic polymorphisms (particularly ACE insertion/insertion and GSTT1-null and -positive genotypes) were significantly associated with hypertension but not BMI. 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