# Deep Mutational Scanning and Functional Analysis of Repolarization Determinants

> **NIH NIH R01** · UNIVERSITY OF WISCONSIN-MADISON · 2022 · $406,669

## Abstract

PROJECT SUMMARY/ABSTRACT
The underpinnings of sudden cardiac death are related to genetic and acquired ion channel abnormalities and
many are related to potassium channel variants. Gains in phenotype-genotype correlative studies have
revolutionized our understanding of a range of sudden arrhythmic death syndromes, yet currently, identification
of coding variants has far outpaced our ability to correctly classify the variant, and for most genes there are more
unclassified variants (variants of unknown significance, VUS) than classified. This creates barriers for clinical
care, familial cascade screening and, moreover, a functional link to disease. The importance of physiologic and
functional analysis for variant classification has been emphasized, yet contemporary methods are cumbersome
(time and resources) decreasing efficiency in unraveling the arrhythmic risk associated with genetic variants.
Our lab’s work focuses on functional genomics of abnormal cardiac repolarization and cardiac arrhythmic sudden
death syndromes, and we have developed high volume assays to understand variant pathogenicity. Yet most
variant characterization proceeds in a reactive manner (clinical variant identification followed by functional study)
and clinical association is often lacking (siloed research); this creates gaps in optimal and efficient variant
classification. We aim to address these major gaps in knowledge by creating a pro-active, data driven and
mechanistic variant classification scheme cross-validated with clinical data. In Aim 1, Deep Mutational Scanning
(DMS) of Kir2.1, a K+ channel essential for repolarization, and MAVE (multiplexed assay of variant effects)
creation will unveil functional annotation of all possible variants simultaneously to create a comprehensive fitness
landscape. In Aim 2 MAVE will be applied to all K+ channel variants identified from TOPMed and the UK Biobank
that have effects on repolarization to triangularly validate phenomic-genomic-functional data for genetic variant
classification. Lastly, in Aim 3 we integrate genetic variant and MAVE results with traditional cellular markers of
abnormal repolarization using an iPS-cardiomyocyte model and molecular computational modeling. Our central
hypothesis is that DMS will uncover loss of function variants in regulatory regions of Kir2.1, MAVE of low
frequency K+ channel coding variants from the TOPMed and UK Biobank will reveal common thematic and
mechanistic readouts, and these can be validated in iPS-CMs and computational molecular modeling. The
outcomes of this study will allow the field of functional genomics to begin to keep pace with rapidly evolving
genetic discovery through high integrity, high throughput, and highly reproducible and unbiased techniques. We
will create a methodologic template to catalog all other high-impact repolarization associated variants as a vital
step to transition from reactive to proactive classification. Moreover, we will help establish the methodology...

## Key facts

- **NIH application ID:** 10467096
- **Project number:** 1R01HL163987-01
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Lee Lochbaum Eckhardt
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $406,669
- **Award type:** 1
- **Project period:** 2022-04-01 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10467096

## Citation

> US National Institutes of Health, RePORTER application 10467096, Deep Mutational Scanning and Functional Analysis of Repolarization Determinants (1R01HL163987-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10467096. Licensed CC0.

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