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The study was approved by the Institutional Review Board at the University of Illinois at Chicago. Written, informed consent was obtained from all patients prior to study enrollment.
African Americans (by self-report) at least18 years of age, with a diagnosis of heart failure (either with reduced or preserved ejection fraction) for at least 3 months were included. Additional inclusion criteria were treatment with an ACE inhibitor or angiotensin receptor blocker (or if contraindicated, the combination of hydralazine and nitrates) for at least 6 months with no change in doses of these medications for at least 2 months. Patients with a history of liver disease were excluded.
After obtaining written, informed consent, a buccal cell or venous blood sample was collected for determination of genotype and, in a subset of patients, additional blood was collected for determination of serum aldosterone concentration. Since serum aldosterone exhibits diurnal variation and may be influenced by body position, all blood samples were drawn between 8 am and 1 pm while patients were seated after being upright for at least 2 hours. [33] Samples were stored at −80°C until further analysis. Demographic, clinical, and social data were collected at the time of enrollment. AF was defined by a University of Illinois Hospital and Health Science System (UI-Health) cardiologist at study enrollment and diagnosed by documentation in either the electronic medical record or evidence on 12-lead electrocardiography (ECG) and/or Holter monitoring, as previously published [34]. Atrial fibrillation cases associated with a recent surgery or hyperthyroidism were excluded.
Transthoracic echocardiographic studies were performed within 12 months of enrollment using an Acuson SC2000TM ultrasound system. Echocardiographic measurements of left ventricular end diastolic diameter (LVEDD) were performed using standard 2D and M-Mode methods. Left ventricular ejection fraction was assessed using 2D methods and the Simpson method of discs, and left atrial size was determined using linear measurements as outlined by the American Society of Echocardiography [35]. Severity of mitral regurgitation was determined using color Doppler and the PISA method (when appropriate) as outlined by the American Society of Echocardiography [36].
Samples for aldosterone were assayed using a commercially available kit containing I-125-labeled aldosterone (Beckman Coulter, Brea, CA), as previously described [37]. All samples were assayed in duplicate. Intra-assay and inter-assay coefficients of variation for this assay were 1.5% and 1.9% respectively [37].
Genomic DNA was isolated from buccal cells or whole blood using a Puregene® kit (Qiagen, Valencia, CA). Genotyping for the CYP11B2 -344T>C (rs1799998) polymorphism was done via PCR and capillary sequencing, with primers and annealing temperatures shown in Table S1. Genotype results were verified using a different primer set. Each genotype was scored by two independent investigators blinded to AF status. Individual genetic ancestry was determined for each person using 105 autosomal DNA ancestry informative markers for West African, Native American, and European genetic ancestry using published methods [38], [39]. Each participant was then scored from 0% to 100% for individual estimates of West African, Native American and European ancestry.
Creatinine clearance was calculated using the equation of Cockcroft and Gault and ideal body weight [40]. Hardy-Weinberg equilibrium was tested by χ2 analysis. Normally distributed continuous data are presented as mean ± SD and were compared by unpaired t-tests and analysis of variance. Continuous data that were non-normally distributed are presented as median (IQR) and were compared with Mann Whitney U and Kruskal Wallis tests. The χ2 or Fischer's exact test was used to compare categorical data, and the Cochran-Armitage trend test was used to compare allele frequencies between groups. Multiple logistic regression permitted tests of association (odds ratio) between CYP11B2 -344T>C and presence of AF while holding clinical factors, echocardiographic measurements, and genetic ancestry constant. Dominant, additive, recessive and genotypic effects models were all used to test the association of CYP11B2 −344T>C genotype and presence of AF. Based on previous data, risk factors for AF included as covariates in the multiple logistic regression models were age, sex, body size, systemic hypertension, diabetes, coronary artery disease, creatinine clearance, left atrial size, mitral regurgitation and genetic ancestry [41], [42], [43], [44], [45], [46]. Marginal standardization was used for the final logistic regression model to estimate adjusted prevalence differences between genotype groups [47]. Bootstrapping was used to quantify the confidence intervals of the prevalence difference generated from the marginal standardization [48]. Given the low prevalence of the CC homozygous genotype, we used permutation to generate a distribution genotypic effects under the assumption of a true null hypothesis, which creates an empirical p value for the association of the CYP11B2 −344T>C recessive effects model and AF [49]. For the exploratory analysis of serum aldosterone, linear regression (ordinary least squares) was used to examine the association between genetic ancestry and aldosterone levels. Serum aldosterone was natural–log transformed to produce a more normal distribution of regression residuals, as done previously [50]. Mean log serum aldosterone was compared between genotype and AF groups by the unpaired t-test. We also examined the association between genotype and extreme elevation of log aldosterone despite standard heart failure therapy, which was defined as a serum log aldosterone level at or above the 90th percentile for the study population, using Fisher's exact test. A two-sided p value of less than 0.05 was considered as statistical significance. Statistical analyses were performed with the SAS software package, version 9.2 (SAS Institute, Cary, NC, USA), and Stata/SE software, Version 12.1 (StataCorp, College Station, TX, USA).
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