# Decrypting Variants of Uncertain Significance in Long-QT Syndrome

> **NIH NIH R01** · NORTHWESTERN UNIVERSITY · 2022 · $1,304,424

## Abstract

SUMMARY
Clinical genetic testing has become standard-of-care for many diseases including the congenital long-QT
syndrome (LQTS). However, interpreting genetic test results is often confounded by the discovery of ‘variants
of uncertain significance’ (VUS) for which there are insufficient data to determine whether a particular variant is
pathogenic or benign. The goal of this project is to use a novel paradigm for distinguishing pathogenic from
benign variants in LQTS with a focus on KCNQ1, the most common cause of LQTS. During the prior periods of
support, we implemented high throughput strategies to determine the functional consequences of ~180
KCNQ1 variants, the functional consequences of all known disease-associated KCNE1 variants, and assessed
the stability, structure, and cell surface expression of several dozen KCNQ1 variants. We then integrated data
on KCNQ1 structure, function and sequence conservation with machine learning tools to build a gene-specific
algorithm in a web-based format to predict the likelihood that specific KCNQ1 variants are deleterious. In the
next funding period, we propose to continue this powerful and productive multidisciplinary paradigm to extend
our research. We used our machine learning approach incorporating an artificial neural network model to
predict the functional consequences of 136 KCNQ1 VUS from ClinVar. In Aim 1, we will experimentally
evaluate these predictions by determining functional consequences of the variants using automated patch
clamp recording. In separate experiments, we will perform deep mutational scanning (DMS) of major regions of
KCNQ1 (pore and voltage-sensing domains, C-terminus) to identify all possible single nucleotide variants that
cause impaired trafficking of the channel to the plasma membrane. In Aim 2, we will use our quantitative flow
cytometry-based method to evaluate cell surface expression of disease-associated KCNQ1 variants in regions
of the channel (pore domain, C-terminus) that not been fully investigated for trafficking, and to determine if
dysfunctional KCNE1 variants interfere with KCNQ1 trafficking. We will also employ biophysical methods
(nano-differential scanning fluorimetry, cellular thermal shift assay) to evaluate the stability of trafficking-
impaired KCNQ1 variants in the context of purified channel protein consisting of the voltage-sensor and pore
domains. In Aim 3, we will evolve our machine learning algorithm as a deep neural network and enhance
algorithm performance by using structural channel models built with a custom version of AlphaFold2.0,
computed free energy, and outputs from molecular dynamics simulations of KCNQ1-KCNE1 channels. Our
study will yield a large and unprecedented database of functional, structural, and biochemical properties of
hundreds of KCNQ1 and KCNE1 variants, along with an advanced, data-trained computational prediction
algorithm capable of accurately discriminating deleterious from benign variants. These results will contribute...

## Key facts

- **NIH application ID:** 10540127
- **Project number:** 2R01HL122010-09
- **Recipient organization:** NORTHWESTERN UNIVERSITY
- **Principal Investigator:** Alfred L. George
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $1,304,424
- **Award type:** 2
- **Project period:** 2014-08-01 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10540127

## Citation

> US National Institutes of Health, RePORTER application 10540127, Decrypting Variants of Uncertain Significance in Long-QT Syndrome (2R01HL122010-09). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10540127. Licensed CC0.

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