Although multiple lines of evidence suggest variable expression of the cardiac sodium channel gene SCN5A plays a role in susceptibility to arrhythmia, little is known about its transcriptional regulation.
We used in silico and in vitro experiments to identify possible non-coding sequences important for transcriptional regulation of SCN5A. The results were extended to mice in which a putative regulatory region was deleted.
Methods and Results
We identified 92 non-coding regions highly conserved (>70%) between human and mouse SCN5A orthologs. Three conserved non-coding sequences (CNS) showed significant (>5-fold) activity in luciferase assays. Further in vitro studies indicated one, CNS28 in intron 1, as potential regulatory region. Using Recombinase-Mediated Cassette Exchange (RMCE), we generated mice in which a 435 bp region encompassing CNS28 was removed. Animals homozygous for the deletion showed significant increases in SCN5A transcripts, NaV1.5 protein abundance, and sodium current measured in isolated ventricular myocytes. ECGs revealed a significantly shorter QRS (10.7±0.2ms in controls vs. 9.7±0.2ms in knockouts) indicating more rapid ventricular conduction. In vitro analysis of CNS28 identified a short 3′ segment within this region required for regulatory activity and including an E-box motif. Deletion of this segment reduced reporter activity to 3.6±0.3% of baseline in CHO cells and 16±3% in myocytes (both P<0.05), and mutation of individual sites in the E-box restored activity to 62±4% and 57±2% of baseline in CHO cells and myocytes, respectively (both P<0.05).
These findings establish that regulation of cardiac sodium channel expression modulates channel function in vivo, and identify a non-coding region underlying this regulation.
Gene Expression Regulation; Sodium Channels; Mice; Transgenic
Randomized studies have shown that ventricular rate-control is an acceptable treatment strategy in patients with atrial fibrillation (AF). However, identification of patients who will adequately respond to rate control therapy remains a challenge.
In this study, we evaluated the impact of two common β1-adrenergic receptor (β1-ADR) polymorphisms (G389R and S49G) on response to ventricular rate control therapy in patients with AF.
We studied 543 subjects (63% men; age 61.8 ± 14) prospectively enrolled in the Vanderbilt AF registry and managed with rate control strategy. A ‘responder’ displayed adequate ventricular rate control based on the AFFIRM criteria: 1. Average heart rate (HR) at rest ≤80 bpm and 2. Maximum HR during 6-min walk test ≤110 bpm or average HR during 24-hr Holter ≤100 bpm.
295 (54.3%) met the AFFIRM criteria. Baseline clinical characteristics were similar in responders and non-responders except for mean resting heart rate (76 ± 20 vs. 70 ± 15; P <0.01) and smoking (6% vs. 1%; P <0.01). Multiple clinical variables (age, gender, HTN) failed to predict response to rate-control therapy. By contrast, carriers of Gly variant at 389 were more likely to respond favorably to rate control therapy; 60% vs. 51% in Arg389Arg genotype, P = 0.04. This association persisted after correction for multiple clinical factors (OR 1.42, 95% CI 1.00–2.03, P<0.05). Among responders, subjects carrying the Gly389 variant required the lowest doses of rate-control meds; atenolol 92 mg vs. 68 mg; carvedilol 44 mg vs. 20 mg; metoprolol 80 mg vs. 72mg; diltiazem 212 mg vs. 180 mg and verapamil 276 mg vs. 200 mg respectively (P < 0.01 for all comparisons).
We have identified a common β1-ADR polymorphism, G389R that is associated with adequate response to rate control therapy in AF patients. Gly389 is a ‘loss of function’ variant, consequently, for the same adrenergic stimulation it produces reduced levels of adenyl cyclase and hence attenuates the β-adrenergic cascade. Mechanistically, the effect of rate-control drugs will be synergistic with that of the Gly389 variant, which could possibly explain our findings. These findings represent a step forward in development of a long-term strategy of selecting treatment options in AF based on genotype.
atrial fibrillation; genomics; beta-adrenergic receptor; polymorphisms; rate control
The degree of QT prolongation by drug is highly variable and related to risk for polymorphic ventricular tachycardia due to drugs.
to determine factors that affect the degree of QT prolongation by drugs.
QT and QTc were measured before and after administration of the QT-prolonging drug ibutilide in 253 normal volunteers aged 18-40 years. Drug effect on QTc prolongation was defined as ΔQTc = QTc after drug minus QTc before drug.
Ibutilide prolonged QT from 396 ± 31 ms to 418 ± 39 ms (P<0.001) and QTc from 406 ± 15 ms to 446 ± 33 ms (P<0.001). The ΔQTc did not correlate with baseline QTc (Pearson correlation 0.016, P = 0.8). Post-drug QTc was correlated weakly with pre-drug QTc (Pearson correlation 0.484, p<0.001), and strongly with ΔQTc (Pearson correlation 0.882, P < 0.001). ΔQTc was identical for men and women (39 ± 29 ms vs. 39 ± 27 ms, P = 0.9), but displayed significant differences among body mass index categories (P<0.001). Overweight (48 ± 27 ms) and obese (61 ± 31 ms) subjects had significantly more QT prolongation by drug than normal (31 ± 25 ms) or underweight (24 ± 12 ms) subjects.
QT prolongation by ibutilide does not correlate to baseline QTc, and does not differ between men and women. Overweight and obese subjects have greater drug effect on QTc than subjects with normal or low body mass index. These findings have implications for drug-induced long QT syndrome.
Electrocardiography; Obesity; Ion channels; Pharmacology; QT interval
This study tested the hypothesis that two common polymorphisms in the chromosome 4q25 region that have been associated with atrial fibrillation (AF) contribute to the variable penetrance of familial AF.
Although mutations in ion channels, gap junction proteins, and signaling molecules have been described for Mendelian forms of AF, penetrance is highly variable. Recent studies have consistently identified 2 common single nucleotide polymorphisms (SNPs) in the chromosome 4q25 region as independent AF susceptibility alleles.
We studied 11 families in which AF was present in ≥2 individuals who also shared a candidate gene mutation. These mutations were identified in all subjects with familial lone AF (n=33) as well as apparently unaffected family members (age >50 yrs with no AF; n=17).
Mutations were identified in SCN5A (n=6); NPPA (n=2); KCNQ1 (n=1); KCNA5 (n=1) and NKX2.5 (n=1). In genetic association analyses, un-stratified and stratified according to age of onset of AF and unaffected age > 50 yrs, there was a highly statistically significant association between the presence of both common (rs2200733, rs10033464) as well as rare variants and AF (un-stratified P=1×10−8; stratified [age of onset <50 yrs and unaffected age >50 yrs], P=7.6×10−5) (un-stratified P<0.0001; stratified [age of onset <50 yrs and unaffected age >50 yrs], P<0.0001). Genetic association analyses showed that the presence of common 4q25 risk alleles predicted whether carriers of rare mutations developed AF (P = 2.2×10−4).
Common AF-associated 4q25 polymorphisms modify the clinical expression of latent cardiac ion channel and signaling molecule gene mutations associated with familial AF. These findings support the idea that the genetic architecture of AF is complex and includes both rare and common genetic variants.
Patients vary in their responses to drug therapy, and some of that variability is genetically-determined. This review outlines general approaches used to identify genetic variation that influences drug response. Examples from specific therapeutic areas are presented: cholesterol management, arrhythmias, heart failure, hypertension, warfarin anticoagulation, and anti-platelet agents. A brief view of potential pathways to implementation is presented.
genetics; pharmacogenetics; pharmacogenomics; drug therapy
Atrial fibrillation (AF) is the most common sustained arrhythmia affecting over 700,000 individuals in Japan and 2.2 million in the USA. The proper management of patients with AF is critical due to the well-documented association with heart failure and stroke. A strategy to better define the emergency department (ED) management, admission decisions, and spectrum of risk from low to high is needed.
Methods and subjects
The Atrial Fibrillation and Flutter Outcomes & Risk Determination investigation is a prospective, observational cohort study to develop a multivariable clinical prediction rule that accurately estimates risk for adverse outcomes in patients presenting to the ED with symptomatic AF. We will enroll 430 patients at 2 sites who present to the ED with symptomatic AF defined as a new or established diagnosis of AF or atrial flutter that require ED evaluation for a complaint thought related to their rhythm disturbance. The study’s endpoint is to develop an accurate, objective, internally validated, reliable clinical prediction rule to risk-stratify ED patients presenting with AF exacerbations. The rule will incorporate patient history and examination findings and laboratory studies obtained upon ED presentation, as well as trends over the first 2 hours of care. This investigation’s primary outcome is the incidence of any AF-related adverse event at 5 days and 30 days. We expect to complete the study by the end of 2014. The study was registered at Clinicaltrials.gov NCT01138644.
Atrial fibrillation; Atrial flutter; Prognosis; Emergency care
Atrial fibrillation (AF) is more common in those with obstructive sleep apnea (OSA) than in unaffected individuals and recurs more frequently in the presence of severe OSA after electrical cardioversion and AF ablation. However, it is unknown whether severity of OSA influences the efficacy of anti-arrhythmic drug (AAD) therapy in patients with OSA and AF. This study examined the impact of OSA severity on treatment of symptomatic AF with AADs. We studied 61 patients (62 ± 15 years; 21 women) treated with AADs for symptomatic AF who had overnight polysomnography. Rhythm control was prospectively defined as successful if a patient remained on the same AAD therapy for a minimum of 6 months with ≥75% reduction in symptomatic AF burden. Twenty-four patients (40%) had severe OSA. Thirty patients (49%) were rhythm controlled with AADs. Non-responders to AADs were more likely to have severe OSA than milder disease (52% vs 23%; p < 0.05); those with severe OSA were less likely to respond to AADs than participants with non-severe OSA (39% vs 70%; p = 0.02). Non-responders had higher apnea-hypopnea indices than responders (34 ± 25 vs 22 ± 18 events/hour; p = 0.05), but there were no differences between these groups in minimum oxygen saturation or % time spent in REM sleep. In conclusion, patients with severe OSA are less likely to respond to AAD therapy for AF than those with milder forms of OSA.
obstructive sleep apnea; atrial fibrillation; anti-arrhythmic drugs
Important human phenotypes like height or facial appearance run in families—that has been known for millennia. Systematic studies of the way in which crossing pea plants resulted in changes in important pea plant phenotypes such as flower color or leaf number were defined in the mid-nineteenth century by Mendel  and the chemical basis for “inherited inborn errors of metabolism” by Garrod  at the turn of the twentieth century. Thus, some of the fundamental and familiar rules that we accept in a contemporary understanding of human genetics were laid down decades ago. However, an understanding of the mechanisms whereby genetic information is transmitted from generation to generation and how this information modulates important physiologic or disease susceptibility traits has been more recent. The fundamental discovery was the double-helix structure of DNA, which immediately led to the inference that DNA replication might replicate itself . The last 50 years has seen the development of increasingly robust techniques for sequencing DNA and for using DNA as a laboratory reagent.
Phenotypes; Genotypes; DNA
Understanding variation in the normal electrical activity of the heart, assessed by the electrocardiogram (ECG), may provide a starting point for studies of susceptibility to serious arrhythmias, such as sudden cardiac death during myocardial infarction or drug therapy. Recent genetic association studies of one ECG trait, the QT interval, have identified common variation in European-descent populations, but little is known about the genetic determinants of ECG traits in populations of African-descent.
Methods and Results
To identify genetic risk factors, we have undertaken a candidate gene study of ECG traits in collaboration with the Jackson Heart Study (JHS), a longitudinal study of 5,301 African Americans ascertained from the Jackson, Mississippi area. Nine quantitative ECG traits were evaluated: P, PR, QRS, QT, and QTc durations, heart rate and P, QRS and T axes. We genotyped 72 variations in the predominant sodium channel gene expressed in heart, SCN5A, encoding the Nav1.5 voltage-gated sodium channel in 4,558 subjects. Both rare and common variants in this gene have previously been associated with inherited arrhythmia syndromes and variable conduction. Adjusting for age, sex, and European ancestry, we performed tests of association in 3,054 unrelated participants and identified 14 significant associations (p<1.0×10−4), of which 13 are independent based upon linkage disequilibrium. These variants explain up to 2% of the variation in ECG traits in the JHS.
These results suggest that SCN5A variation contributes to ECG trait distributions in African Americans and these same variations may be risk or protective factors associated with susceptibility to arrhythmias.
electrocardiography; arrhythmia; genetics; ion channels; cardiovascular diseases
genetic polymorphisms; genetics; pharmacogenetics; pharmacogenetics anticoagulants; pharmacogenetics cholesterol
Despite a greater burden of risk factors, atrial fibrillation (AF) is less common among African Americans than European-descent populations. Genome-wide association studies (GWAS) for AF in European-descent populations have identified three predominant genomic regions associated with increased risk (1q21, 4q25, and 16q22). The contribution of these loci to AF risk in African American is unknown.
We studied 73 African Americans with AF from the Vanderbilt-Meharry AF registry and 71 African American controls, with no history of AF including after cardiac surgery. Tests of association were performed for 148 SNPs across the three regions associated with AF, and 22 SNPs were significantly associated with AF (P<0.05). The SNPs with the strongest associations in African Americans were both different from the index SNPs identified in European-descent populations and independent from the index European-descent population SNPs (r2<0.40 in HapMap CEU): 1q21 rs4845396 (odds ratio [OR] 0.30, 95% confidence interval [CI] 0.13–0.67, P = 0.003), 4q25 rs4631108 (OR 3.43, 95% CI 1.59–7.42, P = 0.002), and 16q22 rs16971547 (OR 8.1, 95% CI 1.46–45.4, P = 0.016). Estimates of European ancestry were similar among cases (23.6%) and controls (23.8%). Accordingly, the probability of having two copies of the European derived chromosomes at each region did not differ between cases and controls.
Variable European admixture at known AF loci does not explain decreased AF susceptibility in African Americans. These data support the role of 1q21, 4q25, and 16q22 variants in AF risk for African Americans, although the index SNPs differ from those identified in European-descent populations.
Traditional electrocardiographic reference ranges were derived from studies in communities or clinical trial populations. The distribution of ECG parameters in a large population presenting to a healthcare system has not been studied.
The objective of this study is to define the contribution of age, race, gender, height, body mass index (BMI), and type 2 diabetes mellitus (T2D) to normal electrocardiographic parameters in a population presenting to a healthcare system.
Study subjects were obtained from the Vanderbilt Synthetic Derivative, a de-identified image of the electronic medical record (EMR), containing more than 20 years of records on 1.7 million subjects. We identified 63,177 unique subjects with an ECG read as ‘normal’ by the reviewing cardiologist. Using combinations of natural language processing, laboratory and billing code queries, we identified a subset of 32,949 subjects without cardiovascular disease, interfering medications, or abnormal electrolytes. The ethnic makeup was 77% Caucasian, 13% African American, 1% Hispanic, 1% Asian, and 8% unknown.
The range that included 95% of normal PR intervals was 125–196 msec; QRS 69–103 msec; QTcB 365–458 msec; and HR 54–96 bpm. Linear regression modeling of patient characteristic effects reproduced known age and gender effects and identified novel associations with race, BMI, and T2D. A web-based application for patient-specific normal ranges has been made available online at http://biostat.mc.vanderbilt.edu/ECGPredictionInterval.
Analysis of a large set of EMR-derived normal ECGs reproduced known associations, found new relationships, and established patient-specific normal ranges. Such knowledge informs clinical and genetic research and may improve understanding of normal cardiac physiology.
electrocardiogram; cardiac conduction; electronic medical record; natural language processing
The QT interval is the electrocardiographic manifestation of ventricular repolarization, is variable under physiologic conditions, and is measurably prolonged by many drugs. Rarely, however, individuals with normal base-line intervals may display exaggerated QT interval prolongation, and the potentially fatal polymorphic ventricular tachycardia torsade de pointes, with drugs or other environmental stressors such as heart block or heart failure. This review summarizes the molecular and cellular mechanisms underlying this acquired or drug-induced form of long QT syndrome, describes approaches to the analysis of a role for DNA variants in the mediation of individual susceptibility, and proposes that these concepts may be generalizable to common acquired arrhythmias.
Expression of voltage-gated K+ channel, shaker-related subfamily, member 5 (KCNA5) underlies the human atrial ultra-rapid delayed rectifier K+ current (IKur). The KCNA5 polymorphism resulting in P532L in the C terminus generates IKur that is indistinguishable from wild type at baseline but strikingly resistant to drug block. In the present study, truncating the C terminus of KCNA5 generated a channel with wild-type drug sensitivity, which indicated that P532 is not a drug-binding site. Secondary structure prediction algorithms identified a probable α-helix in P532L that is absent in wild-type channels. We therefore assessed drug sensitivity of IKur generated in vitro in CHO and HEK cells by channels predicted to exhibit or lack this C-terminal α-helix. All constructs displayed near-identical IKur in the absence of drug challenge. However, those predicted to lack the C-terminal α-helix generated quinidine-sensitive currents (43–51% block by 10 μM quinidine), while the currents generated by those constructs predicted to generate a C-terminal α-helix were inhibited less than 12%. Circular dichroism spectroscopy revealed an α-helical signature with peptides derived from drug-resistant channels and no organized structure in those associated with wild-type drug sensitivity. In conclusion, we found that this secondary structure in the KCNA5 C terminus, absent in wild-type channels but generated by a naturally occurring DNA polymorphism, does not alter baseline currents but renders the channel drug resistant. Our data support a model in which this structure impairs access of the drug to a pore-binding site.
As genotyping technology has progressed, genome-wide association studies (GWAS) have matured into efficient and effective tools for mapping genes underlying human phenotypes. Recent studies have demonstrated the utility of the GWAS approach for examining pharmacogenomic traits, including drug metabolism, efficacy, and toxicity. Application of GWAS to pharmacogenomic outcomes presents unique challenges and opportunities. In the current review, we discuss the potential promises and potential caveats of this approach specifically as it relates to pharmacogenomic studies. Concerns with study design, power and sample size, and analysis are reviewed. We further examine the features of successful pharmacogenomic GWAS, and describe consortia efforts that are likely to expand the reach of pharmacogenomic GWAS in the future.
Genome-wide association; GWAS; pharmacogenetic; pharmacogenomic; drug response; drug metabolism; toxicity
Atrial fibrillation (AF) affects over 2 million people in the United States and accounts for nearly 1% of emergency department (ED) visits. Physicians have little information to guide risk stratification of patients with symptomatic AF and admit more than 65%. Our aim was to assess whether data available in the ED management of symptomatic AF can estimate a patient's risk of experiencing a 30-day adverse event.
We systematically reviewed the electronic medical records of all ED patients presenting with symptomatic AF between August 2005 and July 2008. Predefined adverse outcomes included 30-day ED return visit, unscheduled hospitalization, cardiovascular complication or death. We performed multivariable logistic regression to identify predictors of 30-day adverse events. The model was validated using 300 bootstrap replications.
During the 3-year study period, 914 patients accounted for 1228 ED visits. Eighty patients were excluded for non-AF related complaints and 2 patients had no follow-up recorded. Of 832 eligible patients, 216 (25.9%) experienced at least one of the 30-day adverse events. Increasing age (odds ratio [OR] and [95% CI]: 1.20 per decade [1.06, 1.36]), complaint of dyspnea (OR: 1.57 [1.12, 2.20]), smokers (OR: 2.35 [1.47, 3.76]), inadequate ventricular rate control (OR: 1.58 [1.13, 2.21]), and patients taking beta-blockers (OR: 1.44 [1.02, 2.04]) were independently associated with higher risk for adverse events. C-index was 0.67.
In ED patients with symptomatic AF, increased age, inadequate ED ventricular rate control, dyspnea, smoking, and beta-blocker treatment were associated with an increased risk of a 30-day adverse event.
In this paper, we describe using Synthesis-View, a new method of presenting complex genetic data, to revisit results of a study from the BioVU Vanderbilt DNA databank. BioVU is a biorepository of DNA samples coupled with de-identified electronic medical records (EMR). In the Ritchie et al. study 1 ~10,000 BioVU samples were genotyped for 21 SNPs that were previously associated with 5 diseases: atrial fibrillation, Crohn Disease, multiple sclerosis, rheumatoid arthritis, and type 2 diabetes. In the proof-of-concept study, the 21 tests of association replicated previous findings where sample size provided adequate power. The majority of the BioVU results were originally presented in tabular form. Herein we have revisited the results of this study using Synthesis-View. The Synthesis-View software tool visually synthesizes the results of complex, multi-layered studies that aim to characterize associations between small numbers of single-nucleotide polymorphisms (SNPs) and diseases and/or phenotypes, such as the results of replication and meta-analysis studies. Using Synthesis-View with the data of the Ritchie et al. study and presenting these data in this integrated visual format demonstrates new ways to investigate and interpret these kinds of data. Synthesis-View is freely available for non-commercial research institutions, for full details see https://chgr.mc.vanderbilt.edu/synthesisview.
We describe a two-stage analytical approach for characterizing morbidity profile dissimilarity among patient cohorts using electronic medical records. We capture morbidities using the International Statistical Classification of Diseases and Related Health Problems (ICD-9) codes. In the first stage of the approach separate logistic regression analyses for ICD-9 sections (e.g., “hypertensive disease” or “appendicitis”) are conducted, and the odds ratios that describe adjusted differences in prevalence between two cohorts are displayed graphically. In the second stage, the results from ICD-9 section analyses are combined into a general morbidity dissimilarity index (MDI). For illustration, we examine nine cohorts of patients representing six phenotypes (or controls) derived from five institutions, each a participant in the electronic MEdical REcords and GEnomics (eMERGE) network. The phenotypes studied include type II diabetes and type II diabetes controls, peripheral arterial disease and peripheral arterial disease controls, normal cardiac conduction as measures by electrocardiography, and senile cataracts.
Electronic medical records; ICD-9; dissimilarity index; comorbidity index; population comparison; morbidity dissimilarity index
hERG protein, MeSH; C500089, MeSH entry term; HERG protein, human, KCNH2 MeSH; C500089, MeSH entry term; KCNH2 potassium channel, human, long QT syndrome MeSH; D008133, LQTS, pharmacogenomics, QT-prolonging drugs
QT interval for a given heart rate differs between exercise and recovery (QT hysteresis) due to slow QT adaptation to changes in heart rate. We hypothesized that QT hysteresis is evident within stages of exercise and investigated which component of the QT contributes to hysteresis.
Methods and Results
Nineteen healthy volunteers performed a staged exercise test (four stages, 3 min each). Continuous telemetry was analyzed with software to compare QT intervals in a rate-independent fashion. QRST complexes during each minute were sorted by RR interval, and complexes in bins of 20 ms width were signal-averaged. QT and QTp (onset of QRS to peak T wave) were measured, and terminal QT calculated (peak to end of T wave, Tpe = QT − QTp). QT, QTp, and Tpe at the same heart rate were compared between the first and last minute of each stage. QT shortened from the first to last minute of exercise in each stage (Stage I: 358 ± 30 to 346 ± 25 ms, P < 0.001; Stage II: 342 ± 27 to 331 ± 24 ms, P = 0.003; Stage III: 329 ± 21 to 322 ± 18 ms, P = 0.03; Stage IV: 313 ± 22 to 303 ± 23 ms, P = 0.005). QTp also shortened in each stage, while Tpe was unchanged.
QT hysteresis occurs during exercise in normals, and the major determinant is shortening of the first component of the T wave. Terminal repolarization (peak to end of T wave), a surrogate for transmural dispersion of repolarization, does not shorten significantly with exercise.
exercise; QT interval; QT hysteresis