Megan C. Lancaster

BACKGROUND: Variants in the ion channel gene KCNQ1 are the leading cause of congenital long QT syndrome (LQTS), a disorder characterized by QT prolongation and risk of fatal arrhythmias. The expanding use of genetic testing has led to the identification of numerous variants of uncertain significance, complicating clinical interpretation and risk stratification. Functional characterization of KCNQ1 variants is essential to define their role in long QT syndrome and advance precision medicine in cardiovascular care. METHODS: We generated a nearly comprehensive variant library of 13 403 protein-coding variants in KCNQ1 . Using 4 pooled assays followed by high-throughput sequencing, we characterized the variants’ effects on cell surface abundance and channel function in both the variant-only and heterozygous states. Case–control analyses and data from large-scale biobanks were used to assess the clinical impact of loss-of-function variants on QT prolongation and long QT syndrome. Heterozygous assay results were used to determine dominant-negative effects, which were also evaluated for association with arrhythmia risk. Protein structural clustering of distinct classes of deleterious variants was analyzed. RESULTS: Across the 4 assays, we generated 49 305 functional measurements of KCNQ1 variants. Of the 13 403 KCNQ1 studied variants, 1757 demonstrated strong pathogenic evidence, and 6660 showed moderate benign evidence. Loss-of-function variants were significantly associated with QT prolongation and long QT syndrome in case–control and biobank analyses. Deleterious variants clustered within distinct structural domains based on their functional classification. A majority of pathogenic variants exhibited dominant-negative effects; these variants were linked to a higher risk of arrhythmia diagnoses than were haploinsufficient variants. CONCLUSIONS: This comprehensive KCNQ1 functional atlas provides novel functional evidence for most KCNQ1 variants of uncertain significance. It also reveals mechanistic principles linking genetic variation to ion channel biology and arrhythmia susceptibility, advancing cardiovascular precision medicine.

BACKGROUND: Individuals of African ancestry are underrepresented in genetic studies, contributing to disproportionately higher rates of variants of uncertain significance (VUS) and fewer actionable results in genetic testing for cardiomyopathies and arrhythmias. We aimed to determine whether ancestry-enriched VUS confer measurable cardiovascular risk among individuals of African ancestry. METHODS: We identified VUS enriched in individuals of African ancestry in 18 cardiomyopathy and arrhythmia genes. We defined enriched as allele frequency in gnomAD ≥2-fold higher than in European (non-Finnish) individuals, and we confined the analysis to VUS with allele frequency >0.05% in individuals of African ancestry. Associations with cardiovascular phenotypes were assessed in 96 897 individuals of African ancestry from the All of Us (n=65 481) and BioVU (n=31 416) biobanks using fixed-effects meta-analysis. Analyses were stratified by heart failure (HF) status and conventional cardiovascular risk factors. RESULTS: We identified 82 ancestry-enriched VUS. Exploratory analysis in All of Us identified 10 variants associated with composite cardiovascular outcome, 4 of which were associated with individual cardiovascular phenotypes in pooled meta-analysis across both cohorts. PKP2 p.Val558Ile was associated with a 4-fold increased risk of ventricular arrhythmias or sudden cardiac death (adjusted odds ratio [aOR], 4.02 [95% CI, 1.85–8.71]; P =0.004). ELAC2 p.Ile396Val was associated with HF (aOR, 1.67 [95% CI, 1.17–2.39]; P =0.02) and atrial arrhythmias (aOR, 1.88 [95% CI, 1.27–2.78]; P =0.02). FLNC p.Gly11Ser and PKP2 p.Val842Ile were associated with HF (aOR, 1.96 [95% CI, 1.24–3.10]; P =0.02 and aOR, 1.99 [95% CI, 1.16–3.41]; P =0.039). The presence of cardiovascular risk factors was associated with earlier onset of HF (adjusted hazard ratio, 1.71 [95% CI, 1.25–2.33]; P =0.0006) and atrial arrhythmias (adjusted hazard ratio, 1.17 [95% CI, 1.06–1.29]; P =0.0019) in pooled variant carriers. PKP2 p.Val558Ile, SCN5A p.Gln1832Glu, and FLNC p.Gly11Ser demonstrated increased arrhythmia burden specifically in participants with HF. Using American College of Medical Genetics and Genomics guidelines, PKP2 p.Val558Ile met criteria for likely pathogenic classification, potentially affecting an estimated 24 000 Black adults in the United States. CONCLUSIONS: Large-scale biobank analysis identified variants classified as VUS that conferred increased risk of cardiomyopathy and arrhythmia in individuals with African ancestry. The risk associated with these variants was increased in the presence of cardiovascular risk factors and HF.

Variants in the familial hypercholesterolemia gene LDLR —the most important genetic driver of cardiovascular disease—can raise circulating low-density lipoprotein (LDL) cholesterol concentrations and increase the risk of premature atherosclerosis. Definitive classifications are lacking for nearly half of clinically encountered LDLR missense variants, limiting interventions that reduce disease burden. We tested the impact of ~17,000 (nearly all possible) LDLR coding variants on both LDLR cell-surface abundance and LDL uptake, yielding sequence–function maps that recapitulate known biochemistry, offer functional insights, and provide evidence for interpreting clinical variants. Functional scores correlated with hyperlipidemia phenotypes in prospective human cohorts and augmented polygenic scores to improve risk inference, highlighting the potential of this resource to accelerate familial hypercholesterolemia diagnosis and improve patient outcomes.

Cardiovascular diseases are leading global causes of death and disability, often presenting as interrelated phenotypes of atherosclerotic vascular disease, heart failure and arrhythmias. Cardiovascular diseases arise from interactions between environmental factors and predisposing genotypes and include common Mendelian lipid disorders, cardiomyopathies and arrhythmia syndromes. The identification of a pathogenic variant through genetic testing can inform disease diagnosis, risk prediction, treatment and family screening. However, a major roadblock in genomic medicine is that for many variants, especially missense variants, we lack sufficient evidence to enable a definitive classification, and therefore these variants are deemed as ‘variants of uncertain significance’. In this Review, we describe how multiplexed assays of variant effects can enable the functional assessment of nearly all coding variants in a target sequence, potentially offering a proactive approach to identifying the functional significance of gene variants that are observed later in a patient. We discuss validation, including the role of in silico variant effect predictors, and how multiplexed experimental methods are informing cardiovascular disease biology and ultimately resolving the problem of variants of uncertain significance at scale.

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 corrected 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.

Rare genetic diseases are typically studied in referral populations, resulting in underdiagnosis and biased assessment of penetrance and phenotype. To address this, we develop a generalizable method of genotype inference based on distant relatedness and deploy this to identify undiagnosed Type 5 Long QT Syndrome (LQT5) rare variant carriers in a non-referral population. We identify 9 LQT5 families referred to a single specialty clinic, each carrying p.Asp76Asn, the most common LQT5 variant. We uncover recent common ancestry and a single shared haplotype among probands. Application to a non-referral population of 69,819 BioVU biobank subjects identifies 22 additional subjects sharing this haplotype, which we confirm to carry p.Asp76Asn. Referral and non-referral carriers have prolonged QT interval corrected for heart rate (QTc) compared to controls, and, among carriers, the QTc polygenic score is independently associated with QTc prolongation. Thus, our innovative analysis of shared chromosomal segments identifies undiagnosed cases of genetic disease and refines the understanding of LQT5 penetrance and phenotype.

Background: Drug-induced QT interval prolongation raises the risk of fatal arrhythmia and is a central issue in safety pharmacology. Yet individual QT drug response is highly variable, and risk stratification remains a challenge. We hypothesized that genetic factors underlying the baseline QT influence this variation in QT drug response. Methods: A cohort of 990 healthy subjects was prospectively challenged with a single 80 mg oral sotalol dose. Exclusion criteria included abnormal ECG, cardiac disease, and QT-prolonging drug use. ECGs were obtained at baseline and 3 hours post-dose. QT response was defined as absolute change in Fridericia heart-rate corrected QT (ΔQTc). Plasma sotalol was measured 3 hours post-dose. Subjects were genotyped on the MEGA array. Principal component analysis of genotypes was used to define genetic ancestry. A polygenic risk score (PRS) for the baseline QTc comprised of 465,399 common variants was calculated from assayed and imputed genotypes. The difference in PRS between subjects with high QT response (ΔQTc≥60 ms) and those with ΔQTcResults: Of the 990 subjects, 978 (99%) passed genomic quality control and were included in analysis. 62% were female (n=607), with median age 23 (interquartile range (IQR) 21-33). The median ΔQTc 3 hours post-sotalol was 21 ms (IQR 12-30). Ten subjects (1%) had ΔQTc≥60 ms, and their PRS was significantly higher compared to those with ΔQTc<60 ms (P=0.0082). In the regression model, PRS was positively associated with ΔQTc (P=0.0002). Conclusion: Common genetic variants associated with the baseline QT also capture part of the repolarization response to sotalol. This adds to the emerging concept that the genetic architecture of a trait can predict drug response.

Background: We identified a novel SCN5A variant, E171Q, in a neonate with very frequent ectopy and reduced ejection fraction which normalized after arrhythmia suppression by flecainide. This clinical picture is consistent with multifocal ectopic Purkinje-related premature contractions (MEPPC). Most previous reports of MEPPC have implicated SCN5A variants such as R222Q that neutralize positive charges in the S4 voltage sensor helix of the channel protein Na _V 1.5 and generate a gating pore current. Methods and Results: E171 is a highly conserved negatively-charged residue located in the S2 transmembrane helix of Na _V 1.5 domain I. E171 is a key component of the Gating Charge Transfer Center, a region thought to be critical for normal movement of the S4 voltage sensor helix. We used heterologous expression, CRISPR-edited induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and molecular dynamics simulations to demonstrate that E171Q generates a gating pore current, which was suppressed by a low concentration of flecainide (IC50 = 0.71±0.07 µM). R222Q shifts voltage dependence of activation and inactivation in a negative direction but we observed positive shifts with E171Q. E171Q iPSC-CMs demonstrated abnormal spontaneous activity and prolonged action potentials. Molecular dynamics simulations revealed that both R222Q and E171Q proteins generate a water-filled permeation pathway that underlies generation of the gating pore current. Conclusion: Previously identified MEPPC-associated variants that create gating pore currents are located in positively-charged residues in the S4 voltage sensor and generate negative shifts in the voltage dependence of activation and inactivation. We demonstrate that neutralizing a negatively charged S2 helix residue in the Gating Charge Transfer Center generates positive shifts but also create a gating pore pathway. These findings implicate the gating pore pathway as the primary functional and structural determinant of MEPPC and widen the spectrum of variants that are associated with gating pore-related disease in voltage-gated ion channels.