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Matching in observational studies faces complications when units enroll in treatment on a rolling basis. While each treated unit has a specific time of entry into the study, control units each have many possible comparison, or “pseudo-treatment,” times. Valid inference must account for correlations between repeated measures for a single unit, and researchers must decide how flexibly to match across time and units. We provide three important innovations. First, we introduce a new matched design, GroupMatch with instance replacement, allowing maximum flexibility in control selection. This new design searches over all possible comparison times for each treated-control pairing and is more amenable to analysis than past methods. Second, we propose a block bootstrap approach for inference in matched designs with rolling enrollment and demonstrate that it accounts properly for complex correlations across matched sets in our new design and several other contexts. Third, we develop a falsification test to detect violations of the timepoint agnosticism assumption, which is needed to permit flexible matching across time. We demonstrate the practical value of these tools via simulations and a case study of the impact of short-term injuries on batting performance in major league baseball.

Rationale: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are an emerging model for determining drug effects and modeling disease. Specialized devices can generate Extracellular Field Potential (EFP) measurements from these cells, analogous to the ventricular complex of the electrocardiogram., Objective: The objective of this study was to develop an easy-to-use, easy-to-teach, reproducible software tool to measure EFPs., Methods-Results: We present the EFP-Analyzer (EFPA), a semi-automized analyzer for EFP traces, which identifies and averages beats, identifies landmarks, and calculates intervals. We evaluated the tool in an analysis of 358 EFP traces from 22 patient-derived lines. We analyzed spontaneously beating iPSC-CMs, as well as optically paced iPSC-CMs through channelrhodopsin. We developed stringent quality criteria and measured EFP intervals, including Field Potential Duration (FPD). FPD from optically paced iPSC-CMs were shorter than those of spontaneously beating iPSC-CMs (283.7.0±54.2 vs. 293.0±47.5, p: 0.32, respectively). We further analyzed the usability and data replicability of EFPA through an inter-intra observer analysis. Correlation coefficient for inter-reader tangent and threshold measurements for these FPD ranged between r: 0.93-1.00. Bland-Altman plots comparing inter observer results for spontaneously beating and paced iPSC-CMs showed 95% limits of agreement (-13.6 to 19.4ms and -13.2 to 15.3ms, respectively). The EFP-analyzer could accurately detect FPD prolongation due to drug (moxifloxacin) or pathogenic loss of function mutations ( CACNA1C N639T). This program is available for download at https://github.com/kroncke-lab/EFPA . The instructions will be available at the same listed website under the README section of the Github main page., Conclusions: The EFP-Analyzer tool is a useful tool that enables the efficient use of iPSC-CMs as a model to study drug effects and disease.

It is pointed out that many authors are unaware that the particular choice of unit-cell origin determines the irreducible representations to which octahedral tilts in perovskites belong. Furthermore, a recommendation is made that the preferred option is with the origin at the B-cation site rather than that of the A site., (open access.)

Interpreting the clinical significance of putative splice-altering variants outside canonical splice sites remains difficult without time-intensive experimental studies. To address this, we introduce Parallel Splice Effect Sequencing (ParSE-seq), a multiplexed assay to quantify variant effects on RNA splicing. We first apply this technique to study hundreds of variants in the arrhythmia-associated gene SCN5A. Variants are studied in 'minigene' plasmids with molecular barcodes to allow pooled variant effect quantification. We perform experiments in two cell types, including disease-relevant induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The assay strongly separates known control variants from ClinVar, enabling quantitative calibration of the ParSE-seq assay. Using these evidence strengths and experimental data, we reclassify 29 of 34 variants with conflicting interpretations and 11 of 42 variants of uncertain significance. In addition to intronic variants, we show that many synonymous and missense variants disrupted RNA splicing. Two splice-altering variants in the assay also disrupt splicing and sodium current when introduced into iPSC-CMs by CRISPR-Cas9 editing. ParSE-seq provides high-throughput experimental data for RNA-splicing to support precision medicine efforts and can be readily adopted to study other loss-of-function genotype-phenotype relationships., (© 2024. The Author(s).)

Background: Long QT syndrome is a lethal arrhythmia syndrome, frequently caused by rare loss-of-function variants in the potassium channel encoded by KCNH2 . Variant classification is difficult, often because of lack of functional data. Moreover, variant-based risk stratification is also complicated by heterogenous clinical data and incomplete penetrance. Here we sought to test whether variant-specific information, primarily from high-throughput functional assays, could improve both classification and cardiac event risk stratification in a large, harmonized cohort of KCNH2 missense variant heterozygotes., Methods: We quantified cell-surface trafficking of 18 796 variants in KCNH2 using a multiplexed assay of variant effect (MAVE). We recorded KCNH2 current density for 533 variants by automated patch clamping. We calibrated the strength of evidence of MAVE data according to ClinGen guidelines. We deeply phenotyped 1458 patients with KCNH2 missense variants, including QTc, cardiac event history, and mortality. We correlated variant functional data and Bayesian long QT syndrome penetrance estimates with cohort phenotypes and assessed hazard ratios for cardiac events., Results: Variant MAVE trafficking scores and automated patch clamping peak tail currents were highly correlated (Spearman rank-order ρ=0.69; n=433). The MAVE data were found to provide up to pathogenic very strong evidence for severe loss-of-function variants. In the cohort, both functional assays and Bayesian long QT syndrome penetrance estimates were significantly predictive of cardiac events when independently modeled with patient sex and adjusted QT interval (QTc); however, MAVE data became nonsignificant when peak tail current and penetrance estimates were also available. The area under the receiver operator characteristic curve for 20-year event outcomes based on patient-specific sex and QTc (area under the curve, 0.80 [0.76-0.83]) was improved with prospectively available penetrance scores conditioned on MAVE (area under the curve, 0.86 [0.83-0.89]) or attainable automated patch clamping peak tail current data (area under the curve, 0.84 [0.81-0.88])., Conclusions: High-throughput KCNH2 variant MAVE data meaningfully contribute to variant classification at scale, whereas long QT syndrome penetrance estimates and automated patch clamping peak tail current measurements meaningfully contribute to risk stratification of cardiac events in patients with heterozygous KCNH2 missense variants.

Background: Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A . Interpreting the pathogenicity of SCN5A missense variants is challenging, and ≈79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification., Methods: An in vitro SCN5A -Brugada syndrome automated patch clamp assay was independently performed at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function., Results: Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak I Na density ( R 2 =0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values were 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic., Conclusions: This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A -Brugada syndrome variants of uncertain significance., Competing Interests: Dr Glazer is a consultant for BioMarin Inc Victoria Parikh is a scientific advisory board member (Lexeo Therapeutics), clinical advisor (Constantiam Biosciences), and consultant (BioMarin Inc, viz.ai). Dr Parikh also receives research support from BioMarin, Inc. The other authors report no conflicts.

Background: Long QT syndrome (LQTS) is a lethal arrhythmia syndrome, frequently caused by rare loss-of-function variants in the potassium channel encoded by KCNH2 . Variant classification is difficult, often owing to lack of functional data. Moreover, variant-based risk stratification is also complicated by heterogenous clinical data and incomplete penetrance. Here, we sought to test whether variant-specific information, primarily from high-throughput functional assays, could improve both classification and cardiac event risk stratification in a large, harmonized cohort of KCNH2 missense variant heterozygotes., Methods: We quantified cell-surface trafficking of 18,796 variants in KCNH2 using a Multiplexed Assay of Variant Effect (MAVE). We recorded KCNH2 current density for 533 variants by automated patch clamping (APC). We calibrated the strength of evidence of MAVE data according to ClinGen guidelines. We deeply phenotyped 1,458 patients with KCNH2 missense variants, including QTc, cardiac event history, and mortality. We correlated variant functional data and Bayesian LQTS penetrance estimates with cohort phenotypes and assessed hazard ratios for cardiac events., Results: Variant MAVE trafficking scores and APC peak tail currents were highly correlated (Spearman Rank-order ρ = 0.69). The MAVE data were found to provide up to pathogenic very strong evidence for severe loss-of-function variants. In the cohort, both functional assays and Bayesian LQTS penetrance estimates were significantly predictive of cardiac events when independently modeled with patient sex and adjusted QT interval (QTc); however, MAVE data became non-significant when peak-tail current and penetrance estimates were also available. The area under the ROC for 20-year event outcomes based on patient-specific sex and QTc (AUC 0.80 [0.76-0.83]) was improved with prospectively available penetrance scores conditioned on MAVE (AUC 0.86 [0.83-0.89]) or attainable APC peak tail current data (AUC 0.84 [0.81-0.88])., Conclusion: High throughput KCNH2 variant MAVE data meaningfully contribute to variant classification at scale while LQTS penetrance estimates and APC peak tail current measurements meaningfully contribute to risk stratification of cardiac events in patients with heterozygous KCNH2 missense variants.

Background: KCNE1 encodes a 129-residue cardiac potassium channel (I Ks ) subunit. KCNE1 variants are associated with long QT syndrome and atrial fibrillation. However, most variants have insufficient evidence of clinical consequences and thus limited clinical utility., Methods: In this study, we leveraged the power of variant effect mapping, which couples saturation mutagenesis with high-throughput sequencing, to ascertain the function of thousands of protein-coding KCNE1 variants., Results: We comprehensively assayed KCNE1 variant cell surface expression (2554/2709 possible single-amino-acid variants) and function (2534 variants). Our study identified 470 loss- or partial loss-of-surface expression and 574 loss- or partial loss-of-function variants. Of the 574 loss- or partial loss-of-function variants, 152 (26.5%) had reduced cell surface expression, indicating that most functionally deleterious variants affect channel gating. Nonsense variants at residues 56-104 generally had WT-like trafficking scores but decreased functional scores, indicating that the latter half of the protein is dispensable for protein trafficking but essential for channel function. 22 of the 30 KCNE1 residues (73%) highly intolerant of variation (with > 70% loss-of-function variants) were in predicted close contact with binding partners KCNQ1 or calmodulin. Our functional assay data were consistent with gold standard electrophysiological data (ρ =  - 0.64), population and patient cohorts (32/38 presumed benign or pathogenic variants with consistent scores), and computational predictors (ρ =  - 0.62). Our data provide moderate-strength evidence for the American College of Medical Genetics/Association of Molecular Pathology functional criteria for benign and pathogenic variants., Conclusions: Comprehensive variant effect maps of KCNE1 can both provide insight into I Ks channel biology and help reclassify variants of uncertain significance., (© 2024. The Author(s).)

Aims: While variants in KCNQ1 are the commonest cause of the congenital long QT syndrome, we and others find only a small IKs in cardiomyocytes from human-induced pluripotent stem cells (iPSC-CMs) or human ventricular myocytes., Methods and Results: We studied population control iPSC-CMs and iPSC-CMs from a patient with Jervell and Lange-Nielsen (JLN) syndrome due to compound heterozygous loss-of-function (LOF) KCNQ1 variants. We compared the effects of pharmacologic IKs block to those of genetic KCNQ1 ablation, using JLN cells, cells homozygous for the KCNQ1 LOF allele G643S, or siRNAs reducing KCNQ1 expression. We also studied the effects of two blockers of IKr, the other major cardiac repolarizing current, in the setting of pharmacologic or genetic ablation of KCNQ1: moxifloxacin, associated with a very low risk of drug-induced long QT, and dofetilide, a high-risk drug. In control cells, a small IKs was readily recorded but the pharmacologic IKs block produced no change in action potential duration at 90% repolarization (APD90). In contrast, in cells with genetic ablation of KCNQ1 (JLN), baseline APD90 was markedly prolonged compared with control cells (469 ± 20 vs. 310 ± 16 ms). JLN cells displayed increased sensitivity to acute IKr block: the concentration (μM) of moxifloxacin required to prolong APD90 100 msec was 237.4 [median, interquartile range (IQR) 100.6-391.6, n = 7] in population cells vs. 23.7 (17.3-28.7, n = 11) in JLN cells. In control cells, chronic moxifloxacin exposure (300 μM) mildly prolonged APD90 (10%) and increased IKs, while chronic exposure to dofetilide (5 nM) produced greater prolongation (67%) and no increase in IKs. However, in the siRNA-treated cells, moxifloxacin did not increase IKs and markedly prolonged APD90., Conclusion: Our data strongly suggest that KCNQ1 expression modulates baseline cardiac repolarization, and the response to IKr block, through mechanisms beyond simply generating IKs., Competing Interests: Conflict of interest: All authors report no conflict of interest., (Published by Oxford University Press on behalf of the European Society of Cardiology 2024.)

Multiplexed assays of variant effect (MAVEs) have emerged as a powerful approach for interrogating thousands of genetic variants in a single experiment. The flexibility and widespread adoption of these techniques across diverse disciplines have led to a heterogeneous mix of data formats and descriptions, which complicates the downstream use of the resulting datasets. To address these issues and promote reproducibility and reuse of MAVE data, we define a set of minimum information standards for MAVE data and metadata and outline a controlled vocabulary aligned with established biomedical ontologies for describing these experimental designs., (© 2024. The Author(s).)

Background/aim: Sentinel lymph node biopsy (SLNB) for patients with cutaneous melanoma is primarily a prognostic procedure that broadly identifies patients who may have disease progression and may warrant additional intervention. However, 88% of patients undergoing SLNB receive a negative result and of those, some will succumb to their disease. One clinical utility of the integrated 31-GEP test, which combines gene expression data with clinicopathologic factors to provide a personalized, precise risk of SLN positivity, is SLNB guidance. This study compared the i31-GEP for SLNB to a nomogram that predicts SLN positivity using only clinicopathologic factors., Patients and Methods: Patients with T1-T2 tumors and known SLN status (N=465) were analyzed by the i31-GEP for SLNB and a nomogram developed at Memorial Sloan Kettering Cancer Center (MSKCC). A 5% risk threshold was used to conform with national guidelines., Results: In patients with <5% predicted risk, SLN positivity was 2.7% (3/111) for i31-GEP versus 10.0% (11/110, p=0.026) for MSKCC. In each T-category, the i31-GEP maintained a false-negative rate below the 5% risk threshold in those predicted to have a <5% risk, while the MSKCC nomogram did not., Conclusion: Integrating the 31-GEP with traditional factors outperformed a nomogram that uses clinicopathologic factors alone to predict SLN status. Incorporating the i31-GEP into clinical practice could improve identification of patients for SLNB, resulting in better risk-aligned management., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Truncating variants in filamin C (FLNC) can cause arrhythmogenic cardiomyopathy (ACM) through haploinsufficiency. Noncanonical splice-altering variants may contribute to this phenotype., Objective: The purpose of this study was to investigate the clinical and functional consequences of a recurrent FLNC intronic variant of uncertain significance (VUS), c.970-4A>G., Methods: Clinical data in 9 variant heterozygotes from 4 kindreds were obtained from 5 tertiary health care centers. We used in silico predictors and functional studies with peripheral blood and patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Isolated RNA was studied by reverse transcription polymerase chain reaction. iPSC-CMs were further characterized at baseline and after nonsense-mediated decay (NMD) inhibition, using quantitative polymerase chain reaction (qPCR), RNA-sequencing, and cellular electrophysiology. American College of Medical Genetics and Genomics (ACMG) criteria were used to adjudicate variant pathogenicity., Results: Variant heterozygotes displayed a spectrum of disease phenotypes, spanning from mild ventricular dysfunction with palpitations to severe ventricular arrhythmias requiring device shocks or progressive cardiomyopathy requiring heart transplantation. Consistent with in silico predictors, the c.970-4A>G FLNC variant activated a cryptic splice acceptor site, introducing a 3-bp insertion containing a premature termination codon. NMD inhibition upregulated aberrantly spliced transcripts by qPCR and RNA-sequencing. Patch clamp studies revealed irregular spontaneous action potentials, increased action potential duration, and increased sodium late current in proband-derived iPSC-CMs. These findings fulfilled multiple ACMG criteria for pathogenicity., Conclusion: Clinical, in silico, and functional evidence support the prediction that the intronic c.970-4A>G VUS disrupts splicing and drives ACM, enabling reclassification from VUS to pathogenic., (Copyright © 2023. Published by Elsevier Inc.)

Background: Brugada syndrome (BrS) is an inherited arrhythmia syndrome caused by loss-of-function variants in the cardiac sodium channel gene SCN5A (sodium voltage-gated channel alpha subunit 5) in ≈20% of subjects. We identified a family with 4 individuals diagnosed with BrS harboring the rare G145R missense variant in the cardiac transcription factor TBX5 (T-box transcription factor 5) and no SCN5A variant., Methods: We generated induced pluripotent stem cells (iPSCs) from 2 members of a family carrying TBX5-G145R and diagnosed with Brugada syndrome. After differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), electrophysiologic characteristics were assessed by voltage- and current-clamp experiments (n=9 to 21 cells per group) and transcriptional differences by RNA sequencing (n=3 samples per group), and compared with iPSC-CMs in which G145R was corrected by CRISPR/Cas9 approaches. The role of platelet-derived growth factor (PDGF)/phosphoinositide 3-kinase (PI3K) pathway was elucidated by small molecule perturbation. The rate-corrected QT (QTc) interval association with serum PDGF was tested in the Framingham Heart Study cohort (n=1893 individuals)., Results: TBX5-G145R reduced transcriptional activity and caused multiple electrophysiologic abnormalities, including decreased peak and enhanced "late" cardiac sodium current (I Na ), which were entirely corrected by editing G145R to wild-type. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A down-regulation caused decreased peak I Na , and that reduced PDGF receptor ( PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late I Na . Tbx5 regulation of the PDGF axis increased arrhythmia risk due to disruption of PDGF signaling and was conserved in murine model systems. PDGF receptor blockade markedly prolonged normal iPSC-CM action potentials and plasma levels of PDGF in the Framingham Heart Study were inversely correlated with the QTc interval ( P <0.001)., Conclusions: These results not only establish decreased SCN5A transcription by the TBX5 variant as a cause of BrS, but also reveal a new general transcriptional mechanism of arrhythmogenesis of enhanced late sodium current caused by reduced PDGF receptor-mediated PI3K signaling.

Domain boundaries in ferroic materials are found to have various physical properties not observed in the surrounding domains. Such differences can be enhanced and bring promising functionalities when centrosymmetric nonpolar materials encounter polar domain boundaries. In this work, a tunable polar domain boundary is discovered in an antiferroelectric single crystal. Under a small stress or electric field, the density, volume, and polarity of the boundaries are successfully controlled., (© 2023 Wiley-VCH GmbH.)

Background: Rare protein-altering variants in SCN5A , KCNQ1 , and KCNH2 are major causes of Brugada syndrome and the congenital long QT syndrome. While splice-altering variants lying outside 2-bp canonical splice sites can cause these diseases, their role remains poorly described. We implemented 2 functional assays to assess 12 recently reported putative splice-altering variants of uncertain significance and 1 likely pathogenic variant without functional data observed in Brugada syndrome and long QT syndrome probands., Methods: We deployed minigene assays to assess the splicing consequences of 10 variants. Three variants incompatible with the minigene approach were introduced into control induced pluripotent stem cells by CRISPR genome editing. We differentiated cells into induced pluripotent stem cell-derived cardiomyocytes and studied splicing outcomes by reverse transcription-polymerase chain reaction. We used the American College of Medical Genetics and Genomics functional assay criteria (PS3/BS3) to reclassify variants., Results: We identified aberrant splicing, with presumed disruption of protein sequence, in 8/10 variants studied using the minigene assay and 1/3 studied in induced pluripotent stem cell-derived cardiomyocytes. We reclassified 8 variants of uncertain significance to likely pathogenic, 1 variant of uncertain significance to likely benign, and 1 likely pathogenic variant to pathogenic., Conclusions: Functional assays reclassified splice-altering variants outside canonical splice sites in Brugada Syndrome- and long QT syndrome-associated genes.

Congenital arrhythmia syndromes are rare genetic disorders that can cause a high risk of sudden cardiac death. Expert panels have affirmed 15 genes that are linked to congenital arrhythmias. These genes mostly encode cardiac ion channel proteins or associated regulatory proteins that generate the cardiac action potential. Common genetic variation modulates the risk of rare variants and partially explains the incomplete penetrance of these disorders. As genetic testing becomes more prevalent, a major challenge is that most detected variants are annotated as variants of uncertain significance. This review will highlight emerging methods that are refining our understanding of arrhythmia genetics, including phenotype risk scores, large cohorts, in vitro functional assays, structural models, and computational predictions., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Intensive investigations of PbZr 1-x Ti x O 3 (PZT) materials with the ABO 3 perovskite structure are connected with their extraordinary piezoelectric properties. Especially well known are PZT ceramics at the Morphotropic Phase Boundary (MPB), with x~0.48, whose applications are the most numerous among ferroelectrics. These piezoelectric properties are often obtained by doping with various ions at the B sites. Interestingly, we have found similar properties for undoped PZT single crystals with low Ti content, for which we have confirmed the existence of the tricritical point near x~0.06. For a PbZr 0.95 ± 0.01 Ti 0.05 ∓ 0.01 O 3 crystal, we describe the ultrahigh strain, dielectric, optical and piezoelectric properties. We interpret the ultrahigh strain observed in the region of the antiferroelectric-ferroelectric transition as an inverse piezoelectric effect generated by the coexistence of domains of different symmetries. The complex domain coexistence was confirmed by determining optical indicatrix orientations in domains. The piezoelectric coefficient in this region reached an extremely high value of 5000 pm/V. We also verified that the properties of the PZT single crystals from the region near the tricritical point are incredibly susceptible to a slight deviation in the Ti content.

Importance: Patients with early-onset atrial fibrillation (AF) are enriched for rare variants in cardiomyopathy and arrhythmia genes. The clinical significance of these rare variants in patients with early-onset AF is unknown., Objective: To assess the association between rare variants in cardiomyopathy and arrhythmia genes detected in patients with early-onset AF and time to death., Design, Setting, and Participants: This prospective cohort study included participants with AF diagnosed before 66 years of age who underwent whole-genome sequencing through the National Heart, Lung and Blood Institute's Trans-Omics for Precision Medicine program. Participants were enrolled from November 23, 1999, to June 2, 2015. Data were analyzed from February 26 to September 19, 2021., Exposures: Rare variants identified in a panel of 145 genes that are included in cardiomyopathy and arrhythmia panels used by commercial clinical genetic testing laboratories., Main Outcomes and Measures: The primary study outcome was time to death and was adjudicated from medical records and the National Death Index. Multivariable Cox proportional hazards regression was used to evaluate the association of disease-associated variants with risk of death after adjustment for age at AF diagnosis, sex, race, body mass index, left ventricular ejection fraction, and an interaction term of age at AF diagnosis and disease-associated variant status., Results: Among 1293 participants (934 [72%] male; median age at enrollment, 56.0 years; IQR, 48.0-61.0 years), disease-associated (pathogenic or likely pathogenic) rare variants were found in 131 (10%). During a median follow-up of 9.9 years (IQR, 6.9-13.2 years), 219 participants (17%) died. In univariable analysis, disease-associated variants were associated with an increased risk of mortality (hazard ratio, [HR], 1.5; 95% CI, 1.0-2.1; P = .05); the association remained significant in multivariable modeling when adjusted for age at AF diagnosis, sex, race, body mass index, left ventricular ejection fraction, and an interaction term between disease-associated variant status and age at AF diagnosis. The interaction demonstrated that disease-associated variants were associated with a significantly higher risk of mortality compared with no disease-associated variant when AF was diagnosed at a younger age (P = .008 for interaction). Higher body mass index (per IQR: HR, 1.4; 95% CI, 1.2-1.6; P < .001) and lower left ventricular ejection fraction (per IQR: HR, 0.8; 95% CI, 0.7-0.8; P < .001) were associated with higher mortality risk. There were 73 cardiomyopathy-related deaths, 40 sudden deaths, and 10 stroke-related deaths. Mortality among patients with the most prevalent genes with disease-associated variants was 26% (10 of 38 patients) for TTN, 33% (6 of 18) for MYH7, 22% (2 of 9) for LMNA, 0% (0 of 10) for MYH6, and 0% (0 of 8) for KCNQ1., Conclusions and Relevance: The findings suggest that rare variants in cardiomyopathy and arrhythmia genes may be associated with increased risk of mortality among patients with early-onset AF, especially those diagnosed at a younger age. Genetic testing may provide important prognostic information for patients with early-onset AF.

Purpose: Up to 30% of patients with Brugada syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A encoding for the protein Na V 1.5. Recent studies suggested that Na V 1.5 can dimerize, and some variants exert dominant negative effects. In this study, we sought to explore the generality of missense variant Na V 1.5 dominant negative effects and their clinical severity., Methods: We identified 35 LoF variants (<10% of wild type [WT] peak current) and 15 partial LoF variants (10%-50% of WT peak current) that we assessed for dominant negative effects. SCN5A variants were studied in HEK293T cells, alone or in heterozygous coexpression with WT SCN5A using automated patch clamp. To assess the clinical risk, we compared the prevalence of dominant negative vs putative haploinsufficient (frameshift, splice, or nonsense) variants in a BrS consortium and the Genome Aggregation Database population database., Results: In heterozygous expression with WT, 32 of 35 LoF and 6 of 15 partial LoF variants showed reduction to <75% of WT-alone peak current, showing a dominant negative effect. Individuals with dominant negative LoF variants had an elevated disease burden compared with the individuals with putative haploinsufficient variants (2.7-fold enrichment in BrS cases, P = .019)., Conclusion: Most SCN5A missense LoF variants exert a dominant negative effect. This class of variant confers an especially high burden of BrS., Competing Interests: Conflict of Interest The authors, M.J.O., A.M., B.L., Y.W., L.H., J.F.S., L.S., D.M.R., and A.M.G., have all read the paper and reviewed potential conflicts of interest. The authors declare no conflicts of interest., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Background: Sequencing Mendelian arrhythmia genes in individuals without an indication for arrhythmia genetic testing can identify carriers of pathogenic or likely pathogenic (P/LP) variants. However, the extent to which these variants are associated with clinically meaningful phenotypes before or after return of variant results is unclear. In addition, the majority of discovered variants are currently classified as variants of uncertain significance, limiting clinical actionability., Methods: The eMERGE-III study (Electronic Medical Records and Genomics Phase III) is a multicenter prospective cohort that included 21 846 participants without previous indication for cardiac genetic testing. Participants were sequenced for 109 Mendelian disease genes, including 10 linked to arrhythmia syndromes. Variant carriers were assessed with electronic health record-derived phenotypes and follow-up clinical examination. Selected variants of uncertain significance (n=50) were characterized in vitro with automated electrophysiology experiments in HEK293 cells., Results: As previously reported, 3.0% of participants had P/LP variants in the 109 genes. Herein, we report 120 participants (0.6%) with P/LP arrhythmia variants. Compared with noncarriers, arrhythmia P/LP carriers had a significantly higher burden of arrhythmia phenotypes in their electronic health records. Fifty-four participants had variant results returned. Nineteen of these 54 participants had inherited arrhythmia syndrome diagnoses (primarily long-QT syndrome), and 12 of these 19 diagnoses were made only after variant results were returned (0.05%). After in vitro functional evaluation of 50 variants of uncertain significance, we reclassified 11 variants: 3 to likely benign and 8 to P/LP., Conclusions: Genome sequencing in a large population without indication for arrhythmia genetic testing identified phenotype-positive carriers of variants in congenital arrhythmia syndrome disease genes. As the genomes of large numbers of people are sequenced, the disease risk from rare variants in arrhythmia genes can be assessed by integrating genomic screening, electronic health record phenotypes, and in vitro functional studies., Registration: URL: https://www., Clinicaltrials: gov; Unique identifier; NCT03394859.

The cardiac sodium ion channel (Na V 1.5) is a protein with four domains (DI-DIV), each with six transmembrane segments. Its opening and subsequent inactivation results in the brief rapid influx of Na + ions resulting in the depolarization of cardiomyocytes. The neurotoxin veratridine (VTD) inhibits Na V 1.5 inactivation resulting in longer channel opening times, and potentially fatal action potential prolongation. VTD is predicted to bind at the channel pore, but alternative binding sites have not been ruled out. To determine the binding site of VTD on Na V 1.5, we perform docking calculations and high-throughput electrophysiology experiments in the present study. The docking calculations identified two distinct binding regions. The first site was in the pore, close to the binding site of Na V 1.4 and Na V 1.5 blocking drugs in experimental structures. The second site was at the "mouth" of the pore at the cytosolic side, partly solvent-exposed. Mutations at this site (L409, E417, and I1466) had large effects on VTD binding, while residues deeper in the pore had no effect, consistent with VTD binding at the mouth site. Overall, our results suggest a VTD binding site close to the cytoplasmic mouth of the channel pore. Binding at this alternative site might indicate an allosteric inactivation mechanism for VTD at Na V 1.5.

Background: Multiple reports associate the cardiac sodium channel gene ( SCN5A ) variants S1103Y and R1193Q with type 3 congenital long QT syndrome and drug-induced long QT syndrome. These variants are too common in ancestral populations to be highly arrhythmogenic at baseline, however: S1103Y allele frequency is 8.1% in African Americans and R1193Q 6.1% in East Asians. R1193Q is known to increase late sodium current ( I Na-L ) in cardiomyocytes derived from induced pluripotent stem cells but the role of these variants in modulating repolarization remains poorly understood., Methods: We determined the effect of S1103Y on QT intervals among African-American participants in a large electronic health record. Using cardiomyocytes derived from induced pluripotent stem cells carrying naturally occurring or genome-edited variants, we studied action potential durations (APDs) at baseline and after challenge with the repolarizing potassium current ( I Kr ) blocker dofetilide and I Na-L and I Kr at baseline., Results: In 1479 African-American participants with no confounding medications or diagnoses of heart disease, QT intervals in S1103Y carriers was no different from that in noncarriers. Baseline APD was no different in cells expressing the Y allele (SY, YY cells) compared with isogenic cells with the reference allele (SS cells). However, I Na-L was increased in SY and YY cells and the I Na-L blocker GS967 shortened APD in SY/YY but not SS cells ( P <0.001). I Kr was increased almost 2-fold in SY/YY cells compared with SS cells (tail current: 0.66±0.1 versus 1.2±0.1 pA/pF; P <0.001). Dofetilide challenge prolonged APD at much lower concentrations in SY (4.1 nmol/L [interquartile range, 1.5-9.3]; n=11) and YY (4.2 nmol/L [1.7-5.0]; n=5) than in SS cells (249 nmol/L [22.3-2905]; n=14; P <0.001 and P <0.01, respectively) and elicited afterdepolarizations in 8/16 SY/YY cells but only in 1/14 SS cells. R1193Q cells similarly displayed no difference in baseline APD but increased I Kr and increased dofetilide sensitivity., Conclusions: These common ancestry-specific variants do not affect baseline repolarization, despite generating increased I Na-L . We propose that increased I Kr serves to maintain normal repolarization but increases the risk of manifest QT prolongation with I Kr block in variant carriers. Our findings emphasize the need for inclusion of diverse populations in the study of adverse drug reactions.

Importance: Early-onset atrial fibrillation (AF) can be the initial manifestation of a more serious underlying inherited cardiomyopathy or arrhythmia syndrome., Objective: To examine the results of genetic testing for early-onset AF., Design, Setting, and Participants: This prospective, observational cohort study enrolled participants from an academic medical center who had AF diagnosed before 66 years of age and underwent whole genome sequencing through the National Heart, Lung, and Blood Institute's Trans-Omics for Precision Medicine program. Participants were enrolled from November 23, 1999, to June 2, 2015. Data analysis was performed from October 24, 2020, to March 11, 2021., Exposures: Rare variants identified in a panel of 145 genes that are included on cardiomyopathy and arrhythmia panels used by commercial clinical genetic testing laboratories., Main Outcomes and Measures: Sequencing data were analyzed using an automated process followed by manual review by a panel of independent, blinded reviewers. The primary outcome was classification of rare variants using American College of Medical Genetics and Genomics criteria: benign, likely benign, variant of undetermined significance, likely pathogenic, or pathogenic. Disease-associated variants were defined as pathogenic/likely pathogenic variants in genes associated with autosomal dominant or X-linked dominant disorders., Results: Among 1293 participants (934 [72.2%] male; median [interquartile range] age at enrollment, 56 [48-61] years; median [interquartile range] age at AF diagnosis, 50 [41-56] years), genetic testing identified 131 participants (10.1%) with a disease-associated variant, 812 (62.8%) with a variant of undetermined significance, 92 (7.1%) as heterozygous carriers for an autosomal recessive disorder, and 258 (20.0%) with no suspicious variant. The likelihood of a disease-associated variant was highest in participants with AF diagnosed before the age of 30 years (20 of 119 [16.8%; 95% CI, 10.0%-23.6%]) and lowest after the age of 60 years (8 of 112 [7.1%; 95% CI, 2.4%-11.9%]). Disease-associated variants were more often associated with inherited cardiomyopathy syndromes compared with inherited arrhythmias. The most common genes were TTN (n = 38), MYH7 (n = 18), MYH6 (n = 10), LMNA (n = 9), and KCNQ1 (n = 8)., Conclusions and Relevance: In this cohort study, genetic testing identified a disease-associated variant in 10% of patients with early-onset AF (the percentage was higher if diagnosed before the age of 30 years and lower if diagnosed after the age of 60 years). Most pathogenic/likely pathogenic variants are in genes associated with cardiomyopathy. These results support the use of genetic testing in early-onset AF.

Background: The proliferation of genetic profiling has revealed many associations between genetic variations and disease. However, large-scale phenotyping efforts in largely healthy populations, coupled with DNA sequencing, suggest variants currently annotated as pathogenic are more common in healthy populations than previously thought. In addition, novel and rare variants are frequently observed in genes associated with disease both in healthy individuals and those under suspicion of disease. This raises the question of whether these variants can be useful predictors of disease. To answer this question, we assessed the degree to which the presence of a variant in the cardiac potassium channel gene KCNH2 was diagnostically predictive for the autosomal dominant long QT syndrome., Methods: We estimated the probability of a long QT diagnosis given the presence of each KCNH2 variant using Bayesian methods that incorporated variant features such as changes in variant function, protein structure, and in silico predictions. We call this estimate the posttest probability of disease. Our method was applied to over 4000 individuals heterozygous for 871 missense or in-frame insertion/deletion variants in KCNH2 and validated against a separate international cohort of 933 individuals heterozygous for 266 missense or in-frame insertion/deletion variants., Results: Our method was well-calibrated for the observed fraction of heterozygotes diagnosed with long QT syndrome. Heuristically, we found that the innate diagnostic information one learns about a variant from 3-dimensional variant location, in vitro functional data, and in silico predictors is equivalent to the diagnostic information one learns about that same variant by clinically phenotyping 10 heterozygotes. Most importantly, these data can be obtained in the absence of any clinical observations., Conclusions: We show how variant-specific features can inform a prior probability of disease for rare variants even in the absence of clinically phenotyped heterozygotes.

Background: Male hypogonadism, arising from a range of etiologies including androgen-deprivation therapies (ADTs), has been reported as a risk factor for acquired long-QT syndrome (aLQTS) and torsades de pointes (TdP). A full description of the clinical features of aLQTS associated with ADT and of underlying mechanisms is lacking., Methods: We searched the international pharmacovigilance database VigiBase for men (n=6 560 565 individual case safety reports) presenting with aLQTS, TdP, or sudden death associated with ADT. In cardiomyocytes derived from induced pluripotent stem cells from men, we studied electrophysiological effects of ADT and dihydrotestosterone., Results: Among subjects receiving ADT in VigiBase, we identified 184 cases of aLQTS (n=168) and/or TdP (n=68; 11% fatal), and 99 with sudden death. Of the 10 ADT drugs examined, 7 had a disproportional association (reporting odds ratio=1.4-4.7; P<0.05) with aLQTS, TdP, or sudden death. The minimum and median times to sudden death were 0.25 and 92 days, respectively. The androgen receptor antagonist enzalutamide was associated with more deaths (5430/31 896 [17%]; P<0.0001) than other ADT used for prostate cancer (4208/52 089 [8.1%]). In induced pluripotent stem cells, acute and chronic enzalutamide (25μM) significantly prolonged action potential durations (action potential duration at 90% when paced at 0.5Hz; 429.7±27.1 (control) versus 982.4±33.2 (acute, P<0.001) and 1062.3±28.9ms (chronic; P<0.001), and generated afterdepolarizations and/or triggered activity in drug-treated cells (11/20 acutely and 8/15 chronically). Enzalutamide acutely and chronically inhibited delayed rectifier potassium current, and chronically enhanced late sodium current. Dihydrotestosterone (30nM) reversed enzalutamide electrophysiological effects on induced pluripotent stem cells., Conclusion: QT prolongation and TdP are a risk in men receiving enzalutamide and other ADTs., Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT03193138., (Copyright © 2020. Published by Elsevier Masson SAS.)

Background: KCHN2 encodes the K V 11.1 potassium channel responsible for I Kr , a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that lead to decreased I Kr have been associated with long QT syndrome type 2 (LQT2). The mechanism of LQT2 is most often induced loss of K V 11.1 trafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would aid in understanding the deleterious nature of these variants; however, the volume of reported nonsynonymous KCNH2 variants precludes the use of conventional methods for functional study., Objective: The purpose of this study was to report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in the resulting K V 11.1 channel., Methods: We developed a method to quantitate K V 11.1 variant trafficking on a pilot region of 11 residues in the S5 helix., Results: We generated trafficking scores for 220 of 231 missense variants in the pilot region. For 5 of 5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We further explored these results with planar patch electrophysiology and found that loss-of-trafficking variants do not produce I Kr . Conversely, but expectedly, some variants that traffic normally were still functionally compromised., Conclusion: We describe a new method for detecting K V 11.1 trafficking-deficient variants in a multiplexed assay. This new method accurately generated trafficking data for variants in K V 11.1 and is extendable both to all residues in K V 11.1 and to other cell surface proteins., (Copyright © 2020 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: In population-based research exome sequencing, the path from variant discovery to return of results is not well established. Variants discovered by research exome sequencing have the potential to improve population health., Methods: Population-based exome sequencing and agnostic ExWAS were performed 5521 Amish individuals. Additional phenotyping and in vitro studies enabled reclassification of a KCNQ1 variant from variant of unknown significance to pathogenic. Results were returned to participants in a community setting., Results: A missense variant was identified in KCNQ1 (c.671C>T, p.T224M), a gene associated with long QT syndrome type 1, which can cause syncope and sudden cardiac death. The p.T224M variant, present in 1/45 Amish individuals is rare in the general population (1/248 566 in gnomAD) and was highly associated with QTc on electro-cardiogram ( P =5.53E-24, β=20.2 ms/allele). Because of the potential importance of this variant to the health of the population, additional phenotyping was performed in 88 p.T224M carriers and 54 noncarriers. There was stronger clinical evidence of long QT syndrome in carriers (38.6% versus 5.5%, P =0.0006), greater history of syncope (32% versus 17%, P =0.020), and higher rate of sudden cardiac death in first degree relatives

Partial or complete loss-of-function variants in SCN5A are the most common genetic cause of the arrhythmia disorder Brugada syndrome (BrS1). However, the pathogenicity of SCN5A variants is often unknown or disputed; 80% of the 1,390 SCN5A missense variants observed in at least one individual to date are variants of uncertain significance (VUSs). The designation of VUS is a barrier to the use of sequence data in clinical care. We selected 83 variants: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada syndrome-associated variants, selected on the basis of their frequency in the general population and in individuals with Brugada syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data. The ten previously studied controls had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wild-type-like function. 22 suspected BrS variants had loss of channel function (<10% normalized peak current) and 22 variants had partial loss of function (10%-50% normalized peak current). The previously unstudied variants were initially classified as likely benign (n = 2), likely pathogenic (n = 10), or VUSs (n = 61). After the patch clamp studies, 16 variants were benign/likely benign, 45 were pathogenic/likely pathogenic, and only 12 were still VUSs. Structural modeling identified likely mechanisms for loss of function including altered thermostability and disruptions to alpha helices, disulfide bonds, or the permeation pore. High-throughput patch clamping enabled reclassification of the majority of tested VUSs in SCN5A., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

A major challenge emerging in genomic medicine is how to assess best disease risk from rare or novel variants found in disease-related genes. The expanding volume of data generated by very large phenotyping efforts coupled to DNA sequence data presents an opportunity to reinterpret genetic liability of disease risk. Here we propose a framework to estimate the probability of disease given the presence of a genetic variant conditioned on features of that variant. We refer to this as the penetrance, the fraction of all variant heterozygotes that will present with disease. We demonstrate this methodology using a well-established disease-gene pair, the cardiac sodium channel gene SCN5A and the heart arrhythmia Brugada syndrome. From a review of 756 publications, we developed a pattern mixture algorithm, based on a Bayesian Beta-Binomial model, to generate SCN5A penetrance probabilities for the Brugada syndrome conditioned on variant-specific attributes. These probabilities are determined from variant-specific features (e.g. function, structural context, and sequence conservation) and from observations of affected and unaffected heterozygotes. Variant functional perturbation and structural context prove most predictive of Brugada syndrome penetrance., Competing Interests: The authors have declared that no competing interests exist.

Background: Variants in ion channel genes have classically been studied in low throughput by patch clamping. Deep mutational scanning is a complementary approach that can simultaneously assess function of thousands of variants., Methods: We have developed and validated a method to perform a deep mutational scan of variants in SCN5A , which encodes the major voltage-gated sodium channel in the heart. We created a library of nearly all possible variants in a 36 base region of SCN5A in the S4 voltage sensor of domain IV and stably integrated the library into HEK293T cells., Results: In preliminary experiments, challenge with 3 drugs (veratridine, brevetoxin, and ouabain) could discriminate wild-type channels from gain- and loss-of-function pathogenic variants. High-throughput sequencing of the pre- and postdrug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The deep mutational scan scores identified 40 putative gain-of-function and 33 putative loss-of-function variants. For 8 of 9 variants, patch clamping data were consistent with the scores., Conclusions: These experiments demonstrate the accuracy of a high-throughput in vitro scan of SCN5A variant function, which can be used to identify deleterious variants in SCN5A and other ion channel genes.

Background: Commercial genetic testing for long QT syndrome (LQTS) has rapidly expanded, but the inability to accurately predict whether a rare variant is pathogenic has limited its clinical benefit. Novel missense variants are routinely reported as variant of unknown significance (VUS) and cannot be used to screen family members at risk for sudden cardiac death. Better approaches to determine the pathogenicity of VUS are needed., Objective: The purpose of this study was to rapidly determine the pathogenicity of a CACNA1C variant reported by commercial genetic testing as a VUS using a patient-independent human induced pluripotent stem cell (hiPSC) model., Methods: Using CRISPR/Cas9 genome editing, CACNA1C-p.N639T was introduced into a previously established hiPSC from an unrelated healthy volunteer, thereby generating a patient-independent hiPSC model. Three independent heterozygous N639T hiPSC lines were generated and differentiated into cardiomyocytes (CM). Electrophysiological properties of N639T hiPSC-CM were compared to those of isogenic and population control hiPSC-CM by measuring the extracellular field potential (EFP) of 96-well hiPSC-CM monolayers and by patch clamp., Results: Significant EFP prolongation was observed only in optically stimulated but not in spontaneously beating N639T hiPSC-CM. Patch-clamp studies revealed that N639T prolonged the ventricular action potential by slowing voltage-dependent inactivation of Ca V 1.2 currents. Heterologous expression studies confirmed the effect of N639T on Ca V 1.2 inactivation., Conclusion: The patient-independent hiPSC model enabled rapid generation of functional data to support reclassification of a CACNA1C VUS to likely pathogenic, thereby establishing a novel LQTS type 8 mutation. Furthermore, our results indicate the importance of controlling beating rates to evaluate the functional significance of LQTS VUS in high-throughput hiPSC-CM assays., (Copyright © 2019 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Male hypogonadism, arising from a range of etiologies including androgen-deprivation therapies (ADTs), has been reported as a risk factor for acquired long-QT syndrome (aLQTS) and torsades de pointes (TdP). A full description of the clinical features of aLQTS associated with ADT and of underlying mechanisms is lacking., Methods: We searched the international pharmacovigilance database VigiBase for men (n=6 560 565 individual case safety reports) presenting with aLQTS, TdP, or sudden death associated with ADT. In cardiomyocytes derived from induced pluripotent stem cells from men, we studied electrophysiological effects of ADT and dihydrotestosterone., Results: Among subjects receiving ADT in VigiBase, we identified 184 cases of aLQTS (n=168) and/or TdP (n=68; 11% fatal), and 99 with sudden death. Of the 10 ADT drugs examined, 7 had a disproportional association (reporting odds ratio=1.4-4.7; P <0.05) with aLQTS, TdP, or sudden death. The minimum and median times to sudden death were 0.25 and 92 days, respectively. The androgen receptor antagonist enzalutamide was associated with more deaths (5430/31 896 [17%]; P <0.0001) than other ADT used for prostate cancer (4208/52 089 [8.1%]). In induced pluripotent stem cells, acute and chronic enzalutamide (25 µM) significantly prolonged action potential durations (action potential duration at 90% when paced at 0.5 Hz; 429.7±27.1 (control) versus 982.4±33.2 (acute, P <0.001) and 1062.3±28.9 ms (chronic; P <0.001), and generated afterdepolarizations and/or triggered activity in drug-treated cells (11/20 acutely and 8/15 chronically). Enzalutamide acutely and chronically inhibited delayed rectifier potassium current, and chronically enhanced late sodium current. Dihydrotestosterone (30 nM) reversed enzalutamide electrophysiological effects on induced pluripotent stem cells., Conclusions: QT prolongation and TdP are a risk in men receiving enzalutamide and other ADTs., Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT03193138.

Importance: Thyroid hormone levels are tightly regulated through feedback inhibition by thyrotropin, produced by the pituitary gland. Hyperthyroidism is overwhelmingly due to thyroid disorders and is well recognized to contribute to a wide spectrum of cardiovascular morbidity, particularly the increasingly common arrhythmia atrial fibrillation (AF)., Objective: To determine the association between genetically determined thyrotropin levels and AF., Design, Setting, and Participants: This phenome-wide association study scanned 1318 phenotypes associated with a polygenic predictor of thyrotropin levels identified by a previously published genome-wide association study that included participants of European ancestry. North American individuals of European ancestry with longitudinal electronic health records were analyzed from May 2008 to November 2016. Analysis began March 2018., Main Outcomes and Measures: Clinical diagnoses associated with a polygenic predictor of thyrotropin levels., Exposures: Genetically determined thyrotropin levels., Results: Of 37 154 individuals, 19 330 (52%) were men. The thyrotropin polygenic predictor was positively associated with hypothyroidism (odds ratio [OR], 1.10; 95% CI, 1.07-1.14; P = 5 × 10-11) and inversely associated with diagnoses related to hyperthyroidism (OR, 0.64; 95% CI, 0.54-0.74; P = 2 × 10-8 for toxic multinodular goiter). Among nonthyroid associations, the top association was AF/flutter (OR, 0.93; 95% CI, 0.9-0.95; P = 9 × 10-7). When the analyses were repeated excluding 9801 individuals with any diagnoses of a thyroid-related disease, the AF association persisted (OR, 0.91; 95% CI, 0.88-0.95; P = 2.9 × 10-6). To replicate this association, we conducted an inverse-variance weighted average meta-analysis using AF single-nucleotide variant weights from a genome-wide association study of 17 931 AF cases and 115 142 controls. As in the discovery analyses, each SD increase in predicted thyrotropin was associated with a decreased risk of AF (OR, 0.86; 95% CI, 0.79-0.93; P = 4.7 × 10-4). In a set of AF cases (n = 745) and controls (n = 1680) older than 55 years, directly measured thyrotropin levels that fell within the normal range were inversely associated with AF risk (OR, 0.91; 95% CI, 0.83-0.99; P = .04)., Conclusions and Relevance: This study suggests a role for genetically determined variation in thyroid function within a physiologically accepted normal range as a risk factor for AF. The clinical decision to treat subclinical thyroid disease should incorporate the risk for AF as antithyroid medications to treat hyperthyroidism may reduce AF risk and thyroid hormone replacement for hypothyroidism may increase AF risk.