Biology subjects

Ng, C. A.

Publications and source records attributed to Ng, C. A..

3 recordsLinked to original sources

bioRxiv2021

A Massively Parallel Trafficking Assay Accurately Predicts Loss of Channel Function in KCNH2 Variants

High throughput genomics has greatly facilitated identification of genetic variants. However, determining which variants contribute to disease causation is challenging with more than half of all missense variants now classified as variants of uncertain significance (VUS). A VUS leaves patients and their clinicians unable to utilize the variant information in clinical decision-making. In long QT syndrome type 2, KCNH2 channel function is directly associated with disease presentation. Therefore, functional phenotyping of KCNH2 variants can provide direct evidence to aid variant classification. Here, we investigated the expression of all codon variants in exon 2 of KCNH2 using a massively parallel trafficking assay and for a subset of 458 single nucleotide variants compared the results with peak tail current density and gating using automated patch clamp electrophysiology. Trafficking could correctly classify loss of peak tail current density variants with an AUC reaching 0.94 compared to AUCs of 0.75 to 0.8 for in silico variant classifiers. We suggest massively parallel trafficking assays can provide prospective and accurate functional assessment for all missense variants in KCNH2 and most likely many other ion channels and membrane proteins.

biophysics↗

bioRxiv2021

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. 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 bicistronic vector to co-express 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 hours 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.

biophysics↗

bioRxiv2020

Modified N-linked glycosylation status predicts trafficking defective human Piezo1 channel mutations

Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like all membrane proteins, they pass through biosynthetic quality control in the endoplasmic reticulum and Golgi that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the cap region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for higher-order glycosylation in the propeller regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated protein. The higher order glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.

biophysics↗