Arrhythmia Variant Associations and Reclassifications in the eMERGE-III Sequencing Study

Abstract

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.

Keywords: arrhythmias; cardiac; electronic health records; electrophysiology; genetic testing; long QT syndrome.

Fig. 1:. Penetrance of arrhythmia variants by gene

Abbreviations: SCD: sudden cardiac death, LQTS—long QT syndrome, VT—ventricular tachycardia, VF—ventricular fibrillation, AF/AFL—atrial fibrillation/atrial flutter, PVC—premature ventricular contraction, Ad.AVB—advanced atrioventricular block, SSS—sick sinus syndrome, CIEDs—cardiovascular implantable electronic devices, LBBB—left bundle branch block, RBBB—right bundle branch block, P/LP—pathogenic/likely pathogenic, VUS—variant of uncertain significance. * indicates false discovery rate < 0.1 (adjusted for the p-values from comparisons made in this figure). P values were derived from a logistic regression analysis which modeled severe or extreme phenotype status as a function of variant carrier status (P/LP vs. no variant or VUS vs. no variant), sex, site, age, and the first 10 principal components of ancestry.

Fig. 2:. ECG interval associations and frequency of LQTS diagnoses

A-D) Comparison between individuals with P/LP variants in all genes or in specific genes and individuals with no P/LP or ultra-rare VUS (“no variant”). Only groups with at least 5 carriers are shown. A-C) Electrocardiographic intervals (mean+/− SE) derived from the EHR from 4 of the 10 sites in the study. Red indicates significant associations (FDR<0.1), when compared to the no variant group. FDRs were calculated from all the p-values in panels A-C. D) Percentage of variant carriers with a long QT syndrome (LQTS) code in their EHR. OR=odds ratio, compared to individuals with no P/LP or ultra-rare VUS (“no variant”). *FDR<0.1. FDRs for panel D were calculated from all the p-values in Table S3 (including the other EHR-based phenotypes analyzed in this study). An additional summary of the data in panel D is presented in Table S10, including numbers of individuals in each group, and a similar analysis for VUS is presented in Table S11.

Fig. 3:. Return of variant results

Abbreviations: DCM—Dilated cardiomyopathy; Atrial Fib—Atrial Fibrillation; PVC—Premature Ventricular Contractions; V. Tach—Ventricular Tachycardia.

Fig. 4:. Percentage of variant carriers with arrhythmia phenotypes

Text columns indicate gene, variant name, variant ACMG classification, and number of individuals who were heterozygous for that variant in this study. Selected variants in KCNE1, KCNH2, KCNQ1, LMNA, and SCN5A are shown. A similar plot for the remaining genes in this study is presented in Figure S2. Shown variants have a gnomAD minor allele frequency below 2.5e-5 and are either 1) present in at least 3 eMERGE-III heterozygous participants or 2) present in at least 1 heterozygous participant with an extreme or severe phenotype. This figure focuses on a partial set of variants with 1 or more carrier with an extreme/severe phenotype or 3 or more carriers with a milder phenotype. These cutoffs were used only to limit the number of displayed variants for the figure; a full list of variants is presented in File S2. *Clinical genetic testing indicated that this participant also has a pathogenic variant in DSP p.Arg1269Ter and a VUS in JUP p.Ser323Gly. Variants in bold were subjected to in vitro functional studies (Table 2). Variant reclassifications are indicated with red or green text and are based on a combination of in vitro functional data and revised hotspot criteria (Table 2). Abbreviations: SCD—sudden cardiac death, LQTS—long QT syndrome, VT—ventricular tachycardia, VF—ventricular fibrillation, AF/AFL—atrial fibrillation/atrial flutter, PVC—premature ventricular contraction, Ad.AVB—advanced atrioventricular block, SSS—sick sinus syndrome, CIEDs—cardiovascular implantable electronic devices, LBBB—left bundle branch block, RBBB—right bundle branch block, P/LP—pathogenic/likely pathogenic, VUS—variant of uncertain significance. Cardiomyopathy and echocardiographic phenotypes for LMNA carriers were not assessed in this analysis.

Fig. 5:. In vitro functional characterization of KCNQ1 and SCN5A variants

A. Representative homotetramer current-voltage plots for selected KCNQ1 variants. B. Representative heterotetramer current-voltage plots for selected KCNQ1 variants. p.Gly314Ser is a control variant with dominant negative properties. For A-B wildtype KCNE1 was coexpressed and for B a 1:1 ratio of WT:variant KCNQ1 was expressed. C. KCNQ1 current density for all variants studied in homotetrameric form. D. KCNQ1 current density for all variants studied in heterotetrameric form. KCNQ1 current densities were measured during the depolarizing 2 second activation pulses. E. Representative current-voltage plots for selected SCN5A variants. F. SCN5A peak current density for all variants studied. Peak current density was measured as pA/pF and normalized to wildtype. G. Representative and quantification (inset) of late-current tracings for wildtype and p.Gly1605Asp variant. Late current measurements were calculated as a percentage of peak current from the same cell. *p=0.0028. Data in A-F were generated using the SyncroPatch instrument. Data in G were generated with manual patch-clamping. Chinese Hamster Ovary cells were used for A-D and Human Embryonic Kidney 293 cells were used for E-G. At least 20 replicate cells were analyzed per variant. The full patch clamp dataset, including numbers of replicate cells for each variant are listed in File S4. LOF: Loss of function, GOF: gain of function, WT: Wildtype. Dark Blue: severe GOF; light blue: mild GOF; Light Green: near normal; Dark green: normal; Yellow: Mild LOF; Red: Severe LOF; Pink: LOF.