The study was approved by Ethical Review Committee of Kursk State Medical University. A total of 2 995 Russian unrelated subjects from Kursk (discovery cohort) and Belgorod (replication population) regions of Central Russia were included in the study. The discovery cohort comprising 2 216 subjects (1 362 EH patients and 843 healthy subjects with normal blood pressure) was recruited at Cardiology Clinics of Kursk Regional Clinical Hospital and Neurology Clinics of Kursk Emergency Medicine Hospital over two periods: between 2003 and 2006 [7 (link)] and between 2010 and 2013. The replication population included DNA samples from 779 individuals (357 EH patients and 422 population controls) which have been obtained from the biobank of Belgorod State National Research University, as part of a large population-based study of Belgorod region [8 (link)]. The baseline characteristics of the study patients are listed in Table 1 . As can be seen from Table 1 , hypertensive patients were matched to controls on sex and age (P > 0.05). Diagnosis of essential hypertension in both populations was verified by qualified cardiologists. Individuals were defined as hypertensive according to World Health Organization criteria or if they had a history of receiving any antihypertensive drug. Diagnosis of EH in untreated patients was defined by a seated systolic and/or diastolic blood pressure greater than 140 and/or 90 mmHg, respectively, on at least 2 separate measurements. All EH patients had no clinical signs, symptoms, and laboratory findings suggestive of secondary hypertension.
Genomic DNA was isolated from peripheral blood samples using a standard phenol/chloroform procedure. Genotyping of polymorphism E158K of the FMO3 gene was done using PCR followed by RFLP analysis as described elsewhere [9 ]. The genotyping results were scored by two independent investigators blindly to the patient's case/control status and regenotyping of about 5% of randomly selected samples yielded 100% reproducibility.
The association between the polymorphism and hypertension risk was estimated by odds ratio (OR) with 95% confidence interval (CI) using multiple logistic regression analysis with adjustment for confounding variables such as age and gender. Each FMO3 genotype was assessed according to dominant, recessive, and additive genetic models, and the chi-squire (Wald's statistic) odds ratio with 95% confidence interval was calculated. Odds ratios were calculated as a measure of the association of the FMO3 genotype with hypertension risk, with the effects of the allele 158K assumed to be additive (with scores of 0, 1, and 2 assigned for EE, EK, and KK genotypes, resp.), dominant (with scores of 0 for EE genotype and 1 for EK and KK genotypes combined), or recessive (with scores of 0 for EE and EK genotypes combined and 1 for KK genotype). The statistical significance was established at P ≤ 0.05. Bonferroni correction for P values (Padj) was applied in cases when multiple tests were performed. Statistical calculations were performed with Statistica for Windows 8.0 (StatSoft Inc., Tulsa, OK, USA).
Genomic DNA was isolated from peripheral blood samples using a standard phenol/chloroform procedure. Genotyping of polymorphism E158K of the FMO3 gene was done using PCR followed by RFLP analysis as described elsewhere [9 ]. The genotyping results were scored by two independent investigators blindly to the patient's case/control status and regenotyping of about 5% of randomly selected samples yielded 100% reproducibility.
The association between the polymorphism and hypertension risk was estimated by odds ratio (OR) with 95% confidence interval (CI) using multiple logistic regression analysis with adjustment for confounding variables such as age and gender. Each FMO3 genotype was assessed according to dominant, recessive, and additive genetic models, and the chi-squire (Wald's statistic) odds ratio with 95% confidence interval was calculated. Odds ratios were calculated as a measure of the association of the FMO3 genotype with hypertension risk, with the effects of the allele 158K assumed to be additive (with scores of 0, 1, and 2 assigned for EE, EK, and KK genotypes, resp.), dominant (with scores of 0 for EE genotype and 1 for EK and KK genotypes combined), or recessive (with scores of 0 for EE and EK genotypes combined and 1 for KK genotype). The statistical significance was established at P ≤ 0.05. Bonferroni correction for P values (Padj) was applied in cases when multiple tests were performed. Statistical calculations were performed with Statistica for Windows 8.0 (StatSoft Inc., Tulsa, OK, USA).
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