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Dan Roden
Faculty Member
Last active: 3/24/2020

Deep Mutational Scan of an Voltage Sensor.

Glazer AM, Kroncke BM, Matreyek KA, Yang T, Wada Y, Shields T, Salem JE, Fowler DM, Roden DM
Circ Genom Precis Med. 2020 13 (1): e002786

PMID: 31928070 · PMCID: PMC7031040 · DOI:10.1161/CIRCGEN.119.002786

BACKGROUND - Variants in ion channel genes have classically been studied in low throughput by patch clamping. Deep mutational scanning is a complementary approach that can simultaneously assess function of thousands of variants.

METHODS - We have developed and validated a method to perform a deep mutational scan of variants in , 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 in the S4 voltage sensor of domain IV and stably integrated the library into HEK293T cells.

RESULTS - In preliminary experiments, challenge with 3 drugs (veratridine, brevetoxin, and ouabain) could discriminate wild-type channels from gain- and loss-of-function pathogenic variants. High-throughput sequencing of the pre- and postdrug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The deep mutational scan scores identified 40 putative gain-of-function and 33 putative loss-of-function variants. For 8 of 9 variants, patch clamping data were consistent with the scores.

CONCLUSIONS - These experiments demonstrate the accuracy of a high-throughput in vitro scan of variant function, which can be used to identify deleterious variants in and other ion channel genes.

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