Biology subjects

Roden, D. M.

Publications and source records attributed to Roden, D. M..

15 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

Scaled multidimensional assays of variant effect identify sequence-function relationships in hypertrophic cardiomyopathy

BackgroundAn estimated 1 in 500 people live with hypertrophic cardiomyopathy (HCM), a disease for which genetic diagnosis can identify family members at risk, and increasingly guide therapy. Mutations in the myosin binding protein C3 (MYBPC3) gene account for a significant proportion of HCM cases. However, many of these variants are classified as variants of uncertain significance (VUS), complicating clinical decision-making. Scalable methods for variant interpretation in disease-specific cell types are crucial for understanding variant impact and uncovering disease mechanisms. MethodsWe developed a scaled multidimensional mapping strategy to evaluate the functional impact of variants across a critical domain of MYBPC3. We incorporate saturation base editing at the native MYBPC3 locus, a long-read RNA sequencing-enabled assay of variant splice effects, and measurements of HCM-relevant phenotypes, including MYBPC3 abundance, hypertrophic signaling, and ubiquitin-proteasome function in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). ResultsOur multidimensional mapping strategy enabled high-resolution functional analysis of MYBPC3 variants in iPSC-CMs. Targeted transient base editing generated a comprehensive variant library at the native locus, capturing diverse variant effects on cellular HCM-relevant phenotypes. Our massively parallel splicing assay identified novel splice-disrupting variants. Integration of functional assays revealed that decreased MYBPC3 abundance is a key driver of HCM-related phenotypes. In parallel, downregulation of protein degradation was observed as a compensatory response to MYBPC3 loss of function, and novel disease mechanisms were identified for missense variants near a critical binding domain, underscoring their contribution to pathogenesis. Bayesian estimates of variant effects enable the reclassification of clinical variants. ConclusionsThis work provides a platform for extending genome engineering in iPSCs to multiplexed assays of variant effects across diverse disease-relevant cellular phenotypes, enhancing the understanding of variant pathogenicity and uncovering novel biological mechanisms that could inform therapeutic strategies.

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↗

bioRxiv2024

Multifocal Ectopic Purkinje Premature Contractions due to neutralization of an SCN5A negative charge: structural insights into the gating pore hypothesis

BackgroundWe 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 NaV1.5 and generate a gating pore current. Methods and ResultsE171 is a highly conserved negatively-charged residue located in the S2 transmembrane helix of NaV1.5 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 {micro}M). 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. ConclusionPreviously 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.

genetics↗

bioRxiv2023

High-throughput functional mapping of variants in an arrhythmia gene, KCNE1, reveals novel biology

BackgroundKCNE1 encodes a 129-residue cardiac potassium channel (IKs) 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. ResultsHere, we demonstrate 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. We comprehensively assayed KCNE1 variant cell surface expression (2,554/2,709 possible single amino acid variants) and function (2,539 variants). We identified 470 loss-of-surface expression and 588 loss-of-function variants. Out of the 588 loss-of-function variants, only 155 had low cell surface expression. 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 were in predicted close contact with binding partners KCNQ1 or calmodulin. Our data were highly concordant with gold standard electrophysiological data ({rho} = -0.65), population and patient cohorts (32/38 concordant variants), and computational metrics ({rho} = -0.55). Our data provide moderate-strength evidence for the ACMG/AMP functional criteria for benign and pathogenic variants. ConclusionsComprehensive variant effect maps of KCNE1 can both provide insight into IKs channel biology and help reclassify variants of uncertain significance.

genomics↗

bioRxiv2022

Cloud gazing: demonstrating paths for unlocking the value of cloud genomics through cross-cohort analysis

The rapid growth of genomic data has led to a new research paradigm where data are stored centrally in Trusted Research Environments (TREs) such as the All of Us Researcher Workbench (AoU RW) and the UK Biobank Research Analysis Platform (RAP). To characterize the advantages and drawbacks of different TRE attributes in facilitating cross-cohort analysis, we conducted a Genome-Wide Association Study (GWAS) of standard lipid measures on the UKB RAP and AoU RW using two approaches: meta-analysis and pooled analysis. We curated lipid measurements for 37,754 All of Us participants with whole genome sequence (WGS) data and 190,982 UK Biobank participants with whole exome sequence (WES) data. For the meta-analysis, we performed a GWAS of each cohort in their respective platform and meta-analyzed the results. We separately performed a pooled GWAS on both datasets combined. We identified 490 and 464 significant variants in meta-analysis and pooled analysis, respectively. Comparison of full summary data from both meta-analysis and pooled analysis with an external study showed strong correlation of known loci with lipid levels (R2[~]83-97%). Importantly, 90 variants met the significance threshold only in the meta-analysis and 64 variants were significant only in pooled analysis. These method-specific differences may be explained by differences in cohort size, ancestry, and phenotype distributions in All of Us and UK Biobank. We noted approximately 20% of variants significant in only the pooled analysis or significant in only the meta-analysis were most prevalent in non-European, non-Asian ancestry individuals. Pooled analyses included more variants than meta-analyses. Pooled analysis required about half as many computational steps as meta-analysis. These findings have important implications for both platform implementations and researchers undertaking large-scale cross-cohort analyses, as technical and policy choices lead to cross-cohort analyses generating similar, but not identical results, particularly for non-European ancestral populations.

genetics↗

bioRxiv2022

Functional Assays Reclassify Suspected Splice-Altering Variants of Uncertain Significance in Mendelian Channelopathies

BackgroundRare protein-altering variants in SCN5A, KCNQ1, and KCNH2 are major causes of Brugada Syndrome (BrS) and the congenital Long QT Syndrome (LQTS). While splice-altering variants lying outside 2-bp canonical splice sites can cause these diseases, their role remains poorly described. ObjectiveWe implemented two functional assays to assess 12 recently reported putative splice-altering variants of uncertain significance (VUS) and 1 likely pathogenic (LP) variant without functional data observed in BrS and LQTS probands. MethodsWe 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 (iPSCs) by CRISPR genome editing. We differentiated cells into iPSC-derived cardiomyocytes (iPSC-CMs) and studied splicing outcomes by reverse transcription-polymerase chain reaction (RT-PCR). We used the American College of Medical Genetics and Genomics functional assay criteria (PS3/BS3) to reclassify variants. ResultsWe identified aberrant splicing, with presumed disruption of protein sequence, in 8/10 variants studied using the minigene assay and 1/3 studied in iPSC-CMs. We reclassified 9 VUS to LP, 1 VUS to Likely Benign, and 1 LP variant to pathogenic. ConclusionsFunctional assays reclassified splice-altering variants outside canonical splice sites in BrS- and LQTS-associated genes.

genetics↗

bioRxiv2021

Dominant negative effects of SCN5A missense variants

IntroductionUp to 30% of patients with Brugada Syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A. Recent studies have suggested that the SCN5A protein product NaV1.5 can form dimers and exert dominant negative effects. MethodsWe identified 35 LoF variants (<10% peak current compared to wild type (WT)) and 15 partial LoF variants (10-50% peak current compared to WT) that we assessed for dominant negative behavior. SCN5A variants were studied in HEK293T cells alone or in heterozygous co-expression with WT SCN5A using automated patch clamp. To assess clinical risk, we compared the prevalence of dominant negative vs. putative haploinsufficient (frameshift/splice site) variants in a BrS case consortium and the gnomAD population database. ResultsIn heterozygous expression with WT, 32/35 LoF variants and 6/15 partial LoF showed reduction to <75% of WT-alone peak INa, demonstrating a dominant negative effect. Carriers of dominant negative LoF missense variants had an enriched disease burden compared to putative haploinsufficient variant carriers (2.7-fold enrichment in BrS cases, p=0.019). ConclusionsMost SCN5A missense LoF variants exert a dominant negative effect. Cohort analyses reveal that this class of variant confers an especially high burden of BrS.

genetics↗

bioRxiv2021

The 3D spatial constraint on 6.1 million amino acid sites in the human proteome

Quantification of the tolerance of protein-coding sites to genetic variation within human populations has become a cornerstone of the prediction of the function of genomic variants. We hypothesize that the constraint on missense variation at individual amino acid sites is largely shaped by direct 3D interactions with neighboring sites. To quantify the constraint on protein-coding genetic variation in 3D spatial neighborhoods, we introduce a new framework called COntact Set MISsense tolerance (or COSMIS) for estimating constraint. Leveraging recent advances in computational structure prediction, large-scale sequencing data from gnomAD, and a mutation-spectrum-aware statistical model, we comprehensively map the landscape of 3D spatial constraint on 6.1 amino acid sites covering >80% (16,533) of human proteins. We show that the human proteome is broadly under 3D spatial constraint and that the level of spatial constraint is strongly associated with disease relevance both at the individual site level and the protein level. We demonstrate that COSMIS performs significantly better at a range of variant interpretation tasks than other population-based constraint metrics while also providing biophysical insight into the potential functional roles of constrained sites. We make our constraint maps freely available and anticipate that the structural landscape of constrained sites identified by COSMIS will facilitate interpretation of protein-coding variation in human evolution and prioritization of sites for mechanistic or functional investigation.

genomics↗

bioRxiv2020

Robust, flexible, and scalable tests for Hardy-Weinberg Equilibrium across diverse ancestries

Traditional Hardy-Weinberg equilibrium (HWE) tests (the {chi}2 test and the exact test) have long been used as a metric for evaluating genotype quality, as technical artifacts leading to incorrect genotype calls often can be identified as deviations from HWE. However, in datasets comprised of individuals from diverse ancestries, HWE can be violated even without genotyping error, complicating the use of HWE testing to assess genotype data quality. In this manuscript, we present the Robust Unified Test for HWE (RUTH) to test for HWE while accounting for population structure and genotype uncertainty, and evaluate the impact of population heterogeneity and genotype uncertainty on the standard HWE tests and alternative methods using simulated and real sequence datasets. Our results demonstrate that ignoring population structure or genotype uncertainty in HWE tests can inflate false positive rates by many orders of magnitude. Our evaluations demonstrate different tradeoffs between false positives and statistical power across the methods, with RUTH consistently amongst the best across all evaluations. RUTH is implemented as a practical and scalable software tool to rapidly perform HWE tests across millions of markers and hundreds of thousands of individuals while supporting standard VCF/BCF formats. RUTH is publicly available at https://www.github.com/statgen/ruth.

bioinformatics↗

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

A Bayesian method using sparse data to estimate penetrance of disease-associated genetic variants

PurposeA major challenge in genomic medicine is how to best predict risk of disease from rare variants discovered in Mendelian disease genes but with limited phenotypic data. We have recently used Bayesian methods to show that in vitro functional measurements and computational pathogenicity classification of variants in the cardiac gene SCN5A correlate with rare arrhythmia penetrance. We hypothesized that similar predictors could be used to impute variant-specific penetrance prior probabilities.\n\nMethodsFrom a review of 756 publications, we developed a pattern mixture algorithm, based on a Bayesian Beta-Binomial model, to generate SCN5A variant-specific penetrance priors for the heart arrhythmia Brugada syndrome (BrS).\n\nResultsThe resulting priors correlate with mean BrS penetrance posteriors (cross validated R2= 0.41). SCN5A variant function and structural context provide the most information predictive of BrS penetrance. The resulting priors are interpretable as equivalent to the observation of affected and unaffected carriers.\n\nConclusionsBayesian estimates of penetrance can efficiently integrate variant-specific data (e.g. functional, structural, and sequence) to accurately estimate disease risk attributable to individual variants. We suggest this formulation of penetrance is quantitative, probabilistic, and more precise than, but consistent with, discrete pathogenicity classification approaches.

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↗

bioRxiv2017

Mutations in RPL3L and MYZAP increase risk of atrial fibrillation

We performed a meta-analysis of genome-wide association studies on atrial fibrillation (AF) among 14,710 cases and 373,897 controls from Iceland and 14,792 cases and 393,863 controls from the UK Biobank, focusing on low frequency coding and splice mutations, with follow-up in samples from Norway and the US. We observed associations with two missense (OR=1.19 for both) and one splice-donor mutation (OR=1.52) in RPL3L, encoding a ribosomal protein primarily expressed in skeletal muscle and heart. Analysis of 167 RNA samples from the right atrium revealed that the splice donor mutation in RPL3L results in exon skipping. AF is the first disease associated with RPL3L and RPL3L is the first ribosomal gene implicated in AF. This finding is consistent with tissue specialization of ribosomal function. We also found an association with a missense variant in MYZAP (OR=1.37), encoding a component of the intercalated discs of cardiomyocytes, the organelle harbouring most of the mutated proteins involved in arrhythmogenic right ventricular cardiomyopathy. Both discoveries emphasize the close relationship between the mechanical and electrical function of the heart.

genetics↗