Biology subjects

Kroncke, B. M.

Publications and source records attributed to Kroncke, B. M..

9 recordsLinked to original sources

bioRxiv2026

Damaging RBM20 E-rich domain variants are not rescued by gene replacement

The promise of precision therapeutics in genetic cardiomyopathies relies on linking specific therapies to variant mechanisms. Missense variants in the cardiac splice regulator RBM20 cause a highly penetrant and arrhythmogenic dilated cardiomyopathy. Disease-causing variants in RBM20s arginine-serine rich (RS) domain act via formation of toxic gain of function cytoplasmic granules, but this is not true for a small number of clinically adjudicated pathogenic variants in its glutamate(E)-rich domain. To better define the effects of E-rich domain variants, we developed a scalable screen based on induced pluripotent stem cell (iPSC) cardiomyocyte differentiation that identified several additional damaging variants. Several of these reduced RBM20 protein abundance and stability. We therefore hypothesized that, unlike RS domain variants, these E-rich variants might be rescued by RBM20 overexpression. To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with a pathogenic E-rich domain variant (p.E913K), and confirmed reduced RBM20 protein expression in these RBM20+/p.E913K cells after differentiation to iPSC-derived cardiomyocytes (iPSC-CM, vs. engineered isogenic RBM20+/+). These iPSC-CMs also displayed aberrant transcriptional splicing, reduced contractility, increased calcium-induced calcium release, and nuclear localization of RBM20 protein, often with more than the two expected RBM20-centric splice factories. AAV-based overexpression of RBM20 reversed some, but not all of the mis-splicing events identified in RBM20+/p.E913K iPSC-CMs, and did not improve their abnormal contractility, calcium handling or supernumerary RBM20 nuclear granules. In summary, our data indicate that pathogenic E-rich domain variants reduce RBM20 protein abundance, but that their mechanism is unlikely to be explained by haploinsufficiency alone.

genetics↗

bioRxiv2025

Patient-derived Cellular Models of Polygenic Scores: Application to Long QT Syndrome

Polygenic scores (PGS) have emerged as important modifiers of disease risk, drug response, and rare variant penetrance, but the biological mechanisms underlying these associations remain poorly understood. To experimentally investigate these relationships, we derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from individuals at the 1st, 50th, and 99th percentiles of a genome-wide QT interval polygenic score (QT-PGS). We examined baseline repolarization, drug response to the hERG inhibitor E4031, and penetrance of two KCNH2 variants (p.Arg148Trp and p.Arg823Trp) in these cellular models. While baseline field potential durations (FPDs) did not differ across PGS levels, high-PGS iPSC-CMs showed exaggerated prolongation in response to E4031 and increased phenotypic expression of both KCNH2 variants. These findings provide the first experimental demonstration that polygenic background can shape cardiac electrophysiologic phenotypes and modulate the functional impact of both pharmacologic and genetic perturbations. This work establishes a scalable platform for mechanistic studies of polygenic risk.

genetics↗

bioRxiv2024

EFP Analyzer: A fast, accurate, and easy-to-teach program for analyzing Extracellular Field Potentials from iPSC-derived cardiomyocytes

RationaleInduced 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. ObjectiveThe objective of this study was to develop an easy-to-use, easy-to-teach, reproducible software tool to measure EFPs. Methods-ResultsWe 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. ConclusionsThe EFP-Analyzer tool is a useful tool that enables the efficient use of iPSC-CMs as a model to study drug effects and disease.

pharmacology and toxicology↗

bioRxiv2021

A calibrated functional patch clamp assay to enhance clinical variant interpretation in KCNH2-related long QT syndrome

PurposeModern sequencing technologies have revolutionised our detection of gene variants. In most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUS). The aim of this study is to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in the KCNH2 gene. MethodsThe assay was designed according to recommendations of the ClinGen sequence variant interpretation framework. Thirty-one control variants of known clinical significance (17 pathogenic/likely pathogenic, 14 benign/likely benign) were heterozygously expressed in Flp-In HEK293 cells. Variants were analysed for effects on current density and channel gating. A panel of 44 VUS was then assessed for reclassification. ResultsAll 17 pathogenic variant controls had reduced current density and 13/14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying moderate or supporting evidence strength for variant classification. Inclusion of KCNH2 functional assay evidence enabled us to reclassify 6 out of 44 VUS as likely pathogenic. ConclusionThe high-throughput patch clamp assay can provide moderate strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value proposition for developing automated patch clamp assays for other ion channel genes.

genetics↗

bioRxiv2021

A Massively Parallel Trafficking Assay Accurately Predicts Loss of Channel Function in KCNH2 Variants

High throughput genomics has greatly facilitated identification of genetic variants. However, determining which variants contribute to disease causation is challenging with more than half of all missense variants now classified as variants of uncertain significance (VUS). A VUS leaves patients and their clinicians unable to utilize the variant information in clinical decision-making. In long QT syndrome type 2, KCNH2 channel function is directly associated with disease presentation. Therefore, functional phenotyping of KCNH2 variants can provide direct evidence to aid variant classification. Here, we investigated the expression of all codon variants in exon 2 of KCNH2 using a massively parallel trafficking assay and for a subset of 458 single nucleotide variants compared the results with peak tail current density and gating using automated patch clamp electrophysiology. Trafficking could correctly classify loss of peak tail current density variants with an AUC reaching 0.94 compared to AUCs of 0.75 to 0.8 for in silico variant classifiers. We suggest massively parallel trafficking assays can provide prospective and accurate functional assessment for all missense variants in KCNH2 and most likely many other ion channels and membrane proteins.

biophysics↗

bioRxiv2020

High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KCNH2

BackgroundKCHN2 encodes the KV11.1 potassium channel responsible for IKr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that decrease IKr can cause Type 2 Long QT syndrome, usually due to mistrafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would help clinicians identify and treat individuals at risk of a major cardiac event. The volume of reported non-synonymous KCNH2 variants preclude the use of conventional electrophysiologic methods for functional study. ObjectiveTo report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in KCNH2. MethodsWe develop a method to quantitate KCNH2 variant trafficking on a pilot region of 11 residues in the S5 helix, and generate trafficking scores for 220/231 missense variants in this region. ResultsFor 5/5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We additionally compare our results with planar patch electrophysiology and find that loss-of-trafficking variants do not produce IKr, but that some variants which traffic normally may still be functionally compromised. ConclusionsHere, we describe a new method for detecting trafficking-deficient variants in KCNH2 in a multiplexed assay. This new method accurately generates trafficking data for variants in KCNH2 and can be readily extended to all residues in Kv11.1 and to other cell surface proteins. CLINICAL IMPLICATIONSHundreds of KCNH2 variants have been observed to date, and thousands more will be found as clinical and population sequencing efforts become increasingly widespread. The major mechanism of KV11.1 loss of function is misfolding and failure to traffic to the cell surface. Deep mutational scanning of KCNH2 trafficking is a scalable, high-throughput method that can help identify new loss of function variants and decipher the large number of KCNH2 variants being found in the population.

genetics↗

bioRxiv2019

High-throughput reclassification of SCN5A variants

RationalePartial 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 (VUS). The designation of VUS is a barrier to the use of sequence data in clinical care. ObjectiveWe selected 83 variants for study: 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 patients 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. Methods and ResultsTen previously studied variants had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wildtype-like function. 22 suspected BrS variants had loss of channel function (<10% normalized peak current) and 23 variants had partial loss of function (10-50% normalized peak current). The 73 previously unstudied variants were initially classified as likely benign (n=2), likely pathogenic (n=11), or VUS (n=60). After the patch clamp studies, 16 variants were benign/likely benign, 47 were pathogenic/likely pathogenic, and only 10 were still VUS. 8/22 loss of function variants were partially rescuable by incubation at lower temperature or pretreatment with a sodium channel blocker. Structural modeling identified likely mechanisms for loss of function including altered thermostability, and disruptions to alpha helices, disulfide bonds, or the permeation pore. ConclusionsHigh-throughput automated patch clamping enabled the reclassification of the majority of tested VUSs in SCN5A.

genetics↗

bioRxiv2019

Deep Mutational Scan of a cardiac sodium channel voltage sensor

Variants in ion channel genes have classically been studied in low-throughput by patch clamping. Deep Mutational Scanning (DMS) is a complementary approach that can simultaneously assess function of thousands of variants. We have developed and validated a method to perform a DMS 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. In preliminary experiments, challenge with three drugs (veratridine, brevetoxin, and ouabain) could discriminate wildtype channels from gain and loss of function pathogenic variants. High-throughput sequencing of the pre- and post-drug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The DMS scores identified 40 putative gain of function and 33 putative loss of function variants. For 8/9 variants, patch clamping data was consistent with the scores. 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.

genetics↗

bioRxiv2019

A rare variant on a common risk haplotype of HFE causes increased risk of hereditary hemochromatosis

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of excess iron absorption. The most common form, HH1, is caused by loss of function variants in HFE. HFE encodes a cell surface protein that binds to the Transferrin Receptor (TfR1), reducing TfR1s affinity for the transferrin/iron complex and thereby limiting cellular iron uptake. Two common missense alleles for HH1 have been identified, HFE C282Y and HFE H63D; H63D is considered to be a less penetrant allele. When we deployed Phenotype Risk Scores (PheRS), a method that aggregates multiple symptoms together in Electronic Health Records (EHRs), we identified HFE E168Q as a novel variant associated with HH. E168Q is on the same haplotype as H63D, and in a crystal structure HFE E168 lies at the interface of the HFE-TfR1 interaction and makes multiple salt bridge connections with TfR1. In in vitro cell surface abundance experiments, the HFE E168Q+H63D double mutation surprisingly increased cell surface abundance of HFE by 10-fold compared to wildtype. In coimmunoprecipitation experiments, however, HFE C282Y, E168Q, and E168Q+H63D completely abolished the interaction between HFE and TfR1, while H63D alone only partially reduced binding. These findings provide mechanistic insight to validate the PheRS result that HFE E168Q is an HH1-associated allele and lead to the reclassification of E168Q from a variant of uncertain significance to a pathogenic variant, according to ACMG guidelines. HFE E168Q results in loss of HFE function by disrupting the HFE-TfR1 interaction. In addition, some disease manifestations attributed to H63D may reflect the functional effects of E168Q.

genetics↗