Nathan R. Tucker

Integrating Genetic, Transcriptional, and Functional Analyses to Identify 5 Novel Genes for Atrial Fibrillation

Background— Atrial fibrillation (AF) affects >30 million individuals worldwide and is associated with an increased risk of stroke, heart failure, and death. AF is highly heritable, yet the genetic basis for the arrhythmia remains incompletely understood. Methods and Results— To identify new AF-related genes, we used a multifaceted approach, combining large-scale genotyping in 2 ethnically distinct populations, cis-eQTL (expression quantitative trait loci) mapping, and functional validation. Four novel loci were identified in individuals of European descent near the genes NEURL (rs12415501; relative risk [RR]=1.18; 95% confidence interval [CI], 1.13–1.23; P=6.5×10−16), GJA1 (rs13216675; RR=1.10; 95% CI, 1.06–1.14; P=2.2×10−8), TBX5 (rs10507248; RR=1.12; 95% CI, 1.08–1.16; P=5.7×10−11), and CAND2 (rs4642101; RR=1.10; 95% CI, 1.06–1.14; P=9.8×10−9). In Japanese, novel loci were identified near NEURL (rs6584555; RR=1.32; 95% CI, 1.26–1.39; P=2.0×10−25) and CUX2 (rs6490029; RR=1.12; 95% CI, 1.08–1.16; P=3.9×10−9). The top single-nucleotide polymorphisms or their proxies were identified as cis-eQTLs for the genes CAND2 (P=2.6×10−19), GJA1 (P=2.66×10−6), and TBX5 (P=1.36×10−5). Knockdown of the zebrafish orthologs of NEURL and CAND2 resulted in prolongation of the atrial action potential duration (17% and 45%, respectively). Conclusions— We have identified 5 novel loci for AF. Our results expand the diversity of genetic pathways implicated in AF and provide novel molecular targets for future biological and pharmacological investigation.

Emerging Directions in the Genetics of Atrial Fibrillation

Atrial fibrillation (AF) is the most common arrhythmia and is associated with increased morbidity. As the population ages and the prevalence of AF continues to rise, the socioeconomic consequences of AF will become increasingly burdensome. Although there are well-defined clinical risk factors for AF, a significant heritable component is also recognized. To identify the molecular basis for the heritability of AF, investigators have used a combination of classical Mendelian genetics, candidate gene screening, and genome-wide association studies. However, these avenues have, as yet, failed to define the majority of the heritability of AF. The goal of this review is to describe the results from both candidate gene and genome-wide studies, as well as to outline potential future avenues for creating a more complete understanding of AF genetics. Ultimately, a more comprehensive view of the genetic underpinnings for AF will lead to the identification of novel molecular pathways and improved risk prediction of this complex arrhythmia.

Overexpression of KCNN3 results in sudden cardiac death

BACKGROUND A recent genome-wide association study identified a susceptibility locus for atrial fibrillation at the KCNN3 gene. Since the KCNN3 gene encodes for a small conductance calcium-activated potassium channel, we hypothesized that overexpression of the SK3 channel increases susceptibility to cardiac arrhythmias. METHODS AND RESULTS We characterized the cardiac electrophysiological phenotype of a mouse line with overexpression of the SK3 channel. We generated homozygote (SK3(T/T)) and heterozygote (SK3(+/T)) mice with overexpression of the channel and compared them with wild-type (WT) controls. We observed a high incidence of sudden death among SK3(T/T) mice (7 of 19 SK3(T/T) mice). Ambulatory monitoring demonstrated that sudden death was due to heart block and bradyarrhythmias. SK3(T/T) mice displayed normal body weight, temperature, and cardiac function on echocardiography; however, histological analysis demonstrated that these mice have abnormal atrioventricular node morphology. Optical mapping demonstrated that SK3(T/T) mice have slower ventricular conduction compared with WT controls (SK3(T/T) vs. WT; 0.45 ± 0.04 vs. 0.60 ± 0.09 mm/ms, P = 0.001). Programmed stimulation in 1-month-old SK3(T/T) mice demonstrated inducible atrial arrhythmias (50% of SK3(T/T) vs. 0% of WT mice) and also a shorter atrioventricular nodal refractory period (SK3(T/T) vs. WT; 43 ± 6 vs. 52 ± 9 ms, P = 0.02). Three-month-old SK3(T/T) mice on the other hand displayed a trend towards a more prolonged atrioventricular nodal refractory period (SK3(T/T) vs. WT; 61 ± 1 vs. 52 ± 6 ms, P = 0.06). CONCLUSION Overexpression of the SK3 channel causes an increased risk of sudden death associated with bradyarrhythmias and heart block, possibly due to atrioventricular nodal dysfunction.

PHACTR1 Is a Genetic Susceptibility Locus for Fibromuscular Dysplasia Supporting Its Complex Genetic Pattern of Inheritance

Fibromuscular dysplasia (FMD) is a nonatherosclerotic vascular disease leading to stenosis, dissection and aneurysm affecting mainly the renal and cerebrovascular arteries. FMD is often an underdiagnosed cause of hypertension and stroke, has higher prevalence in females (~80%) but its pathophysiology is unclear. We analyzed ~26K common variants (MAF>0.05) generated by exome-chip arrays in 249 FMD patients and 689 controls. We replicated 13 loci (P<10−4) in 402 cases and 2,537 controls and confirmed an association between FMD and a variant in the phosphatase and actin regulator 1 gene (PHACTR1). Three additional case control cohorts including 512 cases and 669 replicated this result and overall reached the genomic level of significance (OR = 1.39, P = 7.4×10−10, 1,154 cases and 3,895 controls). The top variant, rs9349379, is intronic to PHACTR1, a risk locus for coronary artery disease, migraine, and cervical artery dissection. The analyses of geometrical parameters of carotids from ~2,500 healthy volunteers indicate higher intima media thickness (P = 1.97×10−4) and wall to lumen ratio (P = 0.002) in rs9349379-A carriers, suggesting indices of carotid hypertrophy previously described in carotids of FMD patients. Immunohistochemistry detected PHACTR1 in endothelium and smooth muscle cells of FMD and normal human carotids. The expression of PHACTR1 by genotypes in primary human fibroblasts showed higher expression in rs9349379-A carriers (N = 86, P = 0.003). Phactr1 knockdown in zebrafish resulted in dilated vessels indicating subtle impaired vascular development. We report the first susceptibility locus for FMD and provide evidence for a complex genetic pattern of inheritance and indices of shared pathophysiology between FMD and other cardiovascular and neurovascular diseases.

Genetic association analyses highlight biological pathways underlying mitral valve prolapse

Nonsyndromic mitral valve prolapse (MVP) is a common degenerative cardiac valvulopathy of unknown etiology that predisposes to mitral regurgitation, heart failure and sudden death. Previous family and pathophysiological studies suggest a complex pattern of inheritance. We performed a meta-analysis of 2 genome-wide association studies in 1,412 MVP cases and 2,439 controls. We identified 6 loci, which we replicated in 1,422 cases and 6,779 controls, and provide functional evidence for candidate genes. We highlight LMCD1 (LIM and cysteine-rich domains 1), which encodes a transcription factor and for which morpholino knockdown of the ortholog in zebrafish resulted in atrioventricular valve regurgitation. A similar zebrafish phenotype was obtained with knockdown of the ortholog of TNS1, which encodes tensin 1, a focal adhesion protein involved in cytoskeleton organization. We also showed expression of tensin 1 during valve morphogenesis and describe enlarged posterior mitral leaflets in Tns1−/− mice. This study identifies the first risk loci for MVP and suggests new mechanisms involved in mitral valve regurgitation, the most common indication for mitral valve repair.

A novel trafficking-defective HCN4 mutation is associated with early-onset atrial fibrillation

BACKGROUND Atrial fibrillation (AF) is the most common arrhythmia, and a recent genome-wide association study identified the hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) as a novel AF susceptibility locus. HCN4 encodes for the cardiac pacemaker channel, and HCN4 mutations are associated with familial sinus bradycardia and AF. OBJECTIVE The purpose of this study was to determine whether novel variants in the coding region of HCN4 contribute to the susceptibility for AF. METHODS We sequenced the coding region of HCN4 for novel variants from 527 cases with early-onset AF from the Massachusetts General Hospital AF Study and 443 referents from the Framingham Heart Study. We used site-directed mutagenesis, cellular electrophysiology, immunocytochemistry, and confocal microscopy to functionally characterize novel variants. RESULTS We found the frequency of novel coding HCN4 variants was 2-fold greater for individuals with AF (7 variants) compared to the referents (3 variants). We determined that of the 7 novel HCN4 variants in our AF cases, 1 (p.Pro257Ser, located in the amino-terminus adjacent to the first transmembrane spanning domain) did not traffic to cell membrane, whereas the remaining 6 were not functionally different from wild type. In addition, the 3 novel variants in our referents did not alter function compared to wild-type. Coexpression studies showed that the p.Pro257Ser mutant channel failed to colocalize with the wild-type HCN4 channel on the cell membrane. CONCLUSION Our findings are consistent with HCN4 haploinsufficiency as the likely mechanism for early-onset AF in the p.Pro257Ser carrier.

Common variation in atrial fibrillation: navigating the path from genetic association to mechanism

Atrial fibrillation (AF) is the most common cardiac arrhythmia with well-established clinical and genetic risk components. Genome-wide association studies (GWAS) have identified 17 independent susceptibility signals for AF at 14 genomic regions, but the mechanisms through which these loci confer risk to AF remain largely undefined. This problem is not unique to AF, as the field of functional genomics, which attempts to bridge this gap from genotype to phenotype, has only uncovered the mechanisms for a handful of GWAS loci. Recent functional genomic studies have made great strides towards translating genetic discoveries to an underlying mechanism, but the large-scale application of these techniques to AF has remain limited. These advances, as well as the continued unresolved challenges for both common variation in AF and the functional genomics field in general, will be the subject of the following review.

Novel Mutation in FLNC (Filamin C) Causes Familial Restrictive CardiomyopathyCLINICAL PERSPECTIVE

Background— Restrictive cardiomyopathy (RCM) is a rare cardiomyopathy characterized by impaired diastolic ventricular function resulting in a poor clinical prognosis. Rarely, heritable forms of RCM have been reported, and mutations underlying RCM have been identified in genes that govern the contractile function of the cardiomyocytes. Methods and Results— We evaluated 8 family members across 4 generations by history, physical examination, electrocardiography, and echocardiography. Affected individuals presented with a pleitropic syndrome of progressive RCM, atrioventricular septal defects, and a high prevalence of atrial fibrillation. Exome sequencing of 5 affected members identified a single novel missense variant in a highly conserved residue of FLNC (filamin C; p.V2297M). FLNC encodes filamin C—a protein that acts as both a scaffold for the assembly and organization of the central contractile unit of striated muscle and also as a mechanosensitive signaling molecule during cell migration and shear stress. Immunohistochemical analysis of FLNC localization in cardiac tissue from an affected family member revealed a diminished localization at the z disk, whereas traditional localization at the intercalated disk was preserved. Stem cell-derived cardiomyocytes mutated to carry the effect allele had diminished contractile activity when compared with controls. Conclusion— We have identified a novel variant in FLNC as pathogenic variant for familial RCM—a finding that further expands on the genetic basis of this rare and morbid cardiomyopathy.

CO-43: Genetic study identifies common variation in phactr1 to associate with fibromuscular dysplasia

BACKGROUND Fibromuscular dysplasia (FMD) is a nonatherosclerotic vascular disease leading to arterial stenosis, aneurysm and dissection, mainly in renal and carotid artery. FMD has higher prevalence in females (80-90%) and is associated with hypertension and stroke. The pathophysiology of FMD is unclear and a genetic origin is suspected. METHODS We performed a genetic association study in European ancestry individuals. The discovery included 249 cases and 689 controls, in which we analyzed 25,606 common variants (MAF>0.05) using an exome-chip array. RESULTS We followed up 13 loci (p<10(-4)) in 393 cases and 2537 controls and replicated a signal on Chr6. Three additional studies (combined n cases = 512, n controls = 669) confirmed this association, with an overall OR of 1.39, (p = 7.4×10(-10), n all cases = 1154, n all controls = 3895). The FMD risk variant is intronic to the phosphatase and actin regulator 1 gene (PHACTR1), involved in angiogenesis and cell migration. PHACTR1 is a risk locus for coronary artery disease, migraine, and cervical artery dissection, which may occur in FMD. We found a significant association between the risk allele and higher central pulse pressure (p=0.0009), increased intima media thickness (p=0.001) and wall cross-sectional area (p=0.003) of carotids assessed by echotracking in 3800 population-based individuals. RNA expression of PHACTR1 in primary cultured human fibroblasts is 1.7 fold higher in FMD patients (n=20, matched to 20 controls) and we showed that FMD risk allele is an eQTL for PHACTR1 in fibroblast of 57 FMD patients (p=0.02). Finally, Phactr1 knockdown of zebrafish showed significantly dilated vessels (p=0.003) indicating impaired development of vasculature. CONCLUSIONS Here we report the first risk locus for FMD with the largest genetic association study conducted so far. Our data reveal a common genetic variant at PHACTR1 providing indices of shared pathophysiology between FMD and other cardiovascular and neurovascular diseases.

Gain-of-function mutations in GATA6 lead to atrial fibrillation.

BACKGROUND The genetic basis of atrial fibrillation (AF) and congenital heart disease remains incompletely understood. OBJECTIVE We sought to determine the causative mutation in a family with AF, atrial septal defects, and ventricular septal defects. METHODS We evaluated a pedigree with 16 family members, 1 with an atrial septal defect, 1 with a ventricular septal defect, and 3 with AF; we performed whole exome sequencing in 3 affected family members. Given that early-onset AF was prominent in the family, we then screened individuals with early-onset AF, defined as an age of onset <66 years, for mutations in GATA6. Variants were functionally characterized using reporter assays in a mammalian cell line. RESULTS Exome sequencing in 3 affected individuals identified a conserved mutation, R585L, in the transcription factor gene GATA6. In the Massachusetts General Hospital Atrial Fibrillation (MGH AF) Study, the mean age of AF onset was 47.1 ± 10.9 years; 79% of the participants were men; and there was no evidence of structural heart disease. We identified 3 GATA6 variants (P91S, A177T, and A543G). Using wild-type and mutant GATA6 constructs driving atrial natriuretic peptide promoter reporter, we found that 3 of the 4 variants had a marked upregulation of luciferase activity (R585L: 4.1-fold, P < .0001; P91S: 2.5-fold, P = .0002; A177T; 1.7-fold, P = .03). In addition, when co-overexpressed with GATA4 and MEF2C, GATA6 variants exhibited upregulation of the alpha myosin heavy chain and atrial natriuretic peptide reporter activity. CONCLUSION Overall, we found gain-of-function mutations in GATA6 in both a family with early-onset AF and atrioventricular septal defects as well as in a family with sporadic, early-onset AF.