Association Between Titin Loss-of-Function Variants and Early-Onset Atrial Fibrillation.

Importance: Atrial fibrillation (AF) is the most common arrhythmia affecting 1% of the population. Young individuals with AF have a strong genetic association with the disease, but the mechanisms remain incompletely understood. Objective: To perform large-scale whole-genome sequencing to identify genetic variants related to AF. Design, Setting, and Participants: The National Heart, Lung, and Blood Institute's Trans-Omics for Precision Medicine Program includes longitudinal and cohort studies that underwent high-depth whole-genome sequencing between 2014 and 2017 in 18 526 individuals from the United States, Mexico, Puerto Rico, Costa Rica, Barbados, and Samoa. This case-control study included 2781 patients with early-onset AF from 9 studies and identified 4959 controls of European ancestry from the remaining participants. Results were replicated in the UK Biobank (346 546 participants) and the MyCode Study (42 782 participants). Exposures: Loss-of-function (LOF) variants in genes at AF loci and common genetic variation across the whole genome. Main Outcomes and Measures: Early-onset AF (defined as AF onset in persons <66 years of age). Due to multiple testing, the significance threshold for the rare variant analysis was P = 4.55 × 10-3. Results: Among 2781 participants with early-onset AF (the case group), 72.1% were men, and the mean (SD) age of AF onset was 48.7 (10.2) years. Participants underwent whole-genome sequencing at a mean depth of 37.8 fold and mean genome coverage of 99.1%. At least 1 LOF variant in TTN, the gene encoding the sarcomeric protein titin, was present in 2.1% of case participants compared with 1.1% in control participants (odds ratio [OR], 1.76 [95% CI, 1.04-2.97]). The proportion of individuals with early-onset AF who carried a LOF variant in TTN increased with an earlier age of AF onset (P value for trend, 4.92 × 10-4), and 6.5% of individuals with AF onset prior to age 30 carried a TTN LOF variant (OR, 5.94 [95% CI, 2.64-13.35]; P = 1.65 × 10-5). The association between TTN LOF variants and AF was replicated in an independent study of 1582 patients with early-onset AF (cases) and 41 200 control participants (OR, 2.16 [95% CI, 1.19-3.92]; P = .01). Conclusions and Relevance: In a case-control study, there was a statistically significant association between an LOF variant in the TTN gene and early-onset AF, with the variant present in a small percentage of participants with early-onset AF (the case group). Further research is necessary to understand whether this is a causal relationship.

Novel Mutation in FLNC (Filamin C) Causes Familial Restrictive Cardiomyopathy.

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.

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.

Mutation of a common amino acid in NKX2.5 results in dilated cardiomyopathy in two large families.

BACKGROUND: The genetic basis for dilated cardiomyopathy (DCM) can be difficult to determine, particularly in familial cases with complex phenotypes. Next generation sequencing may be useful in the management of such cases. METHODS: We report two large families with pleiotropic inherited cardiomyopathy. In addition to DCM, the phenotypes included atrial and ventricular septal defects, cardiac arrhythmia and sudden death. Probands underwent whole exome sequencing to identify potentially causative variants. RESULTS: Each whole exome sequence yielded over 18,000 variants. We identified distinct mutations affecting a common amino acid in NKX2.5. Segregation analysis of the families support the pathogenic role of these variants. CONCLUSION: Our study emphasizes the utility of next generation sequencing in identifying causative mutations in complex inherited cardiac disease. We also report a novel pathogenic NKX2.5 mutation.

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.

Independent susceptibility markers for atrial fibrillation on chromosome 4q25.

BACKGROUND: Genetic variants on chromosome 4q25 are associated with atrial fibrillation (AF). We sought to determine whether there is more than 1 susceptibility signal at this locus. METHODS AND RESULTS: Thirty-four haplotype-tagging single-nucleotide polymorphisms (SNPs) at the 4q25 locus were genotyped in 790 case and 1177 control subjects from Massachusetts General Hospital and tested for association with AF. We replicated SNPs associated with AF after adjustment for the most significantly associated SNP in 5066 case and 30 661 referent subjects from the German Competence Network for Atrial Fibrillation, Atherosclerosis Risk In Communities Study, Cleveland Clinic Lone AF Study, Cardiovascular Health Study, and Rotterdam Study. All subjects were of European ancestry. A multimarker risk score composed of SNPs that tagged distinct AF susceptibility signals was constructed and tested for association with AF, and all results were subjected to meta-analysis. The previously reported SNP, rs2200733, was most significantly associated with AF (minor allele odds ratio 1.80, 95% confidence interval 1.50 to 2.15, P=1.2 x 10(-20)) in the discovery sample. Adjustment for rs2200733 genotype revealed 2 additional susceptibility signals marked by rs17570669 and rs3853445. A graded risk of AF was observed with an increasing number of AF risk alleles at SNPs that tagged these 3 susceptibility signals. CONCLUSIONS: We identified 2 novel AF susceptibility signals on chromosome 4q25. Consideration of multiple susceptibility signals at chromosome 4q25 identifies individuals with an increased risk of AF and may localize regulatory elements at the locus with biological relevance in the pathogenesis of AF.

Mutation in the S3 segment of KCNQ1 results in familial lone atrial fibrillation.

BACKGROUND: Mutations in several ion channel genes have been reported to cause rare cases of familial atrial fibrillation (AF). OBJECTIVE: The purpose of this study was to determine the genetic basis for AF in a family with autosomal dominant AF. METHODS: Family members were evaluated by 12-lead ECG, echocardiogram, signal-averaged P-wave analysis, and laboratory studies. Fourteen family members in AF-324 were studied. Six individuals had AF, with a mean age at onset of 32 years (range 16-59 years). RESULTS: Compared with unaffected family members, those with AF had a longer mean QRS duration (100 vs 86 ms, P = .015) but no difference in the corrected QT interval (423 +/- 15 ms vs 421 +/- 21 ms). The known loci for AF and other cardiovascular diseases were evaluated. Evidence of linkage was obtained with marker D11S4088 located within KCNQ1, and a highly conserved serine in the third transmembrane region was found to be mutated to a proline (S209P). Compared to the wild-type channel, the S209P mutant activates more rapidly, deactivates more slowly, and has a hyperpolarizing shift in the voltage activation curve. A fraction of the mutant channels are constitutively open at all voltages, resulting in a net increase in I(Ks) current. CONCLUSION: We identified a family with lone AF due to a mutation in the highly conserved S3 domain of KCNQ1, a region of the channel not previously implicated in the pathogenesis of AF.

Cardiac sodium channel mutation in atrial fibrillation.

BACKGROUND: Mutations in the sodium channel SCN5A have been implicated in many cardiac disorders, including the long QT syndrome, Brugada syndrome, conduction system disease, and dilated cardiomyopathy with atrial arrhythmias. OBJECTIVE: In view of the pleiotropic effects of SCN5A mutations, the purpose of this study was to examine a cohort of patients with familial atrial fibrillation (AF) for mutations in the SCN5A gene. METHODS: Probands with AF were enrolled in the study between June 1, 2001 and February 10, 2004. Each patient underwent a standardized evaluation, which included an interview, physical examination, ECG, echocardiogram, and blood sample for genetic analysis. Direct sequencing of the coding region of SCN5A was used to screen for mutations in genomic DNA. RESULTS: One hundred eighty-nine patients with AF were enrolled during the study period. From this cohort, a subset of 57 probands with a family history of AF in at least one first-degree relative was studied. Forty-seven subjects were men (82%); 45 had paroxysmal AF (79%). Echocardiography revealed ejection fraction 62% +/- 6.4 % and left atrial dimension 40 +/- 6.9 mm. A single mutation (N1986K) was observed in one family but was not present in more than 600 control chromosomes. Expression of the N1986K mutant in Xenopus oocytes revealed a hyperpolarizing shift in channel steady-state inactivation. CONCLUSION: In a cohort with familial AF, a single SCN5A mutation causing the arrhythmia in one kindred was identified. These data extend the range of phenotypes observed with SCN5A mutations and suggest that variation in the SCN5A gene is not a major cause of familial AF.

C-Reactive protein in lone atrial fibrillation.

An inflammatory cause of atrial fibrillation (AF) has been proposed on the basis of the presence of lymphocytic infiltrates in the biopsy results of patients with lone AF, alterations of C-reactive protein (CRP) and interleukin-6 levels in subjects with AF, and the time course of postoperative AF. Many previous studies exploring inflammatory factors in AF have been confounded by concomitant medical illnesses. Subjects with lone AF provide a unique opportunity to eliminate the effects of associated conditions. We therefore sought to determine CRP levels in homogenous cohorts of patients with lone AF or AF and hypertension. One hundred twenty-one subjects with lone AF, 52 subjects with AF and hypertension, and 75 control subjects were enrolled and studied. Plasma CRP levels were determined using a commercially available immunoassay. There was no significant difference in CRP levels between subjects with lone AF and controls (1.34 vs 1.21 mg/L, p = 0.18). CRP levels in subjects with AF and hypertension were elevated compared with those of controls and those of subjects with lone AF, although this difference was attributable to increased body mass indexes. CRP levels were not elevated in subjects with lone AF compared with controls. In conclusion, these findings clarify previous observations of elevations in CRP levels in subjects with AF and suggest that this marker of systemic inflammation is associated not with the arrhythmia per se, but rather with underlying cardiovascular disease.

Clinical subtypes of lone atrial fibrillation.

AIMS: In the face of increasing evidence of underlying genetic heterogeneity for lone atrial fibrillation (LAF), we undertook a clinical analysis of subjects to identify the phenotypic subsets of this arrhythmia. METHODS AND RESULTS: We evaluated serial patients who presented with LAF between July 5, 2001 and December 19, 2003. Subjects underwent a standardized interview to elicit a detailed medical history, prior therapies, and precipitants of atrial fibrillation. The results of a physical exam, electrocardiogram and echocardiogram were reviewed. One hundred and eighty subjects with a mean age of 45 years (15-67 years) at the time of diagnosis were enrolled. The majority of patients originally presented with paroxysmal fibrillation (94%), and 7.8% progressed to permanent AF. Reported triggers for AF included sleeping (44%), exercise (36%), alcohol use (36%), and eating (34%). Women with LAF had distinct symptoms, triggers for episodic AF, and over one-fourth had an underlying rheumatologic condition. Several subsets of AF including familial AF (39%), exercise-induced AF (32%), and conduction system disease requiring pacemaker implantation (7%), were identified. CONCLUSIONS: Family history, exercise as a trigger of AF, and a history of a pacemaker identified subtypes of LAF.

Discordant atrial natriuretic peptide and brain natriuretic peptide levels in lone atrial fibrillation.

OBJECTIVES: We sought to characterize natriuretic peptide levels in a cohort of rigorously characterized subjects with lone atrial fibrillation (AF). BACKGROUND: Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are sensitive biomarkers of cardiac contractile dysfunction. Both peptides have been reported to be elevated in cohorts with AF, but previous studies have included subjects with underlying structural heart disease. We studied these hormones in 150 subjects with lone AF. METHODS: Study subjects had electrocardiographic evidence of at least one episode of AF and a structurally normal heart on echocardiography. Subjects were excluded if they had a history of a myocardial infarction, rheumatic heart disease, cardiomyopathy, significant valvular disease, hyperthyroidism, or hypertension that preceded the onset of AF. Control subjects were obtained from a healthy outpatient primary care population. Plasma pro-ANP and N-terminal pro-BNP (nt-pro-BNP) levels were determined using commercially available immunoassays. RESULTS: A total of 150 serial subjects with lone AF were enrolled and studied, the majority during normal sinus rhythm. Median levels of nt-pro-BNP were significantly elevated in subjects with lone AF as compared with control subjects (166 vs. 133 fmol/ml, p=0.0003). There was no significant difference in pro-ANP levels between subjects with lone AF and control subjects (1,730 vs. 1,625 fmol/ml, p=0.90). CONCLUSIONS: Discordant natriuretic peptide levels were observed in this homogeneous population of subjects with lone AF. This biomarker pattern, which is present even in sinus rhythm, may represent an underlying subclinical predisposition to this common arrhythmia.