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Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology

KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance....

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Autores principales: Ng, Chai-Ann, Farr, Jessica, Young, Paul, Windley, Monique J, Perry, Matthew D, Hill, Adam P, Vandenberg, Jamie I
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046900/
https://www.ncbi.nlm.nih.gov/pubmed/33884304
http://dx.doi.org/10.1093/biomethods/bpab003
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author Ng, Chai-Ann
Farr, Jessica
Young, Paul
Windley, Monique J
Perry, Matthew D
Hill, Adam P
Vandenberg, Jamie I
author_facet Ng, Chai-Ann
Farr, Jessica
Young, Paul
Windley, Monique J
Perry, Matthew D
Hill, Adam P
Vandenberg, Jamie I
author_sort Ng, Chai-Ann
collection PubMed
description KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-type (WT) and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3–4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.
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spelling pubmed-80469002021-04-20 Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology Ng, Chai-Ann Farr, Jessica Young, Paul Windley, Monique J Perry, Matthew D Hill, Adam P Vandenberg, Jamie I Biol Methods Protoc Methods Article KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-type (WT) and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3–4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant. Oxford University Press 2021-03-19 /pmc/articles/PMC8046900/ /pubmed/33884304 http://dx.doi.org/10.1093/biomethods/bpab003 Text en © The Author(s) 2021. Published by Oxford University Press. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) ), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Methods Article
Ng, Chai-Ann
Farr, Jessica
Young, Paul
Windley, Monique J
Perry, Matthew D
Hill, Adam P
Vandenberg, Jamie I
Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology
title Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology
title_full Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology
title_fullStr Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology
title_full_unstemmed Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology
title_short Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology
title_sort heterozygous kcnh2 variant phenotyping using flp-in hek293 and high-throughput automated patch clamp electrophysiology
topic Methods Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046900/
https://www.ncbi.nlm.nih.gov/pubmed/33884304
http://dx.doi.org/10.1093/biomethods/bpab003
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