A massively parallel assay accurately discriminates between functionally normal and abnormal variants in a hotspot domain of KCNH2.

Many genes, including KCNH2, contain "hotspot" domains associated with a high density of variants associated with disease. This has led to the suggestion that variant location can be used as evidence supporting classification of clinical variants. However, it is not known what proportion of all potential variants in hotspot domains cause loss of function. Here, we have used a massively parallel trafficking assay to characterize all single-nucleotide variants in exon 2 of KCNH2, a known hotspot for variants that cause long QT syndrome type 2 and an increased risk of sudden cardiac death. Forty-two percent of KCNH2 exon 2 variants caused at least 50% reduction in protein trafficking, and 65% of these trafficking-defective variants exerted a dominant-negative effect when co-expressed with a WT KCNH2 allele as assessed using a calibrated patch-clamp electrophysiology assay. The massively parallel trafficking assay was more accurate (AUC of 0.94) than bioinformatic prediction tools (REVEL and CardioBoost, AUC of 0.81) in discriminating between functionally normal and abnormal variants. Interestingly, over half of variants in exon 2 were found to be functionally normal, suggesting a nuanced interpretation of variants in this "hotspot" domain is necessary. Our massively parallel trafficking assay can provide this information prospectively.

A calibrated functional patch-clamp assay to enhance clinical variant interpretation in KCNH2-related long QT syndrome.

Modern sequencing technologies have revolutionized our detection of gene variants. However, in most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUSs). The aim of this study was to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in KCNH2. The assay was designed according to recommendations proposed by the Clinical Genome Sequence Variant Interpretation Working Group. Thirty-one variants (17 pathogenic/likely pathogenic, 14 benign/likely benign) were classified internally as variant controls. They were heterozygously expressed in Flp-In HEK293 cells for assessing the effects of variants on current density and channel gating in order to determine the sensitivity and specificity of the assay. All 17 pathogenic variant controls had reduced current density, and 13 of 14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying evidence of moderate or supporting strength for VUS reclassification. Inclusion of functional assay evidence enabled us to reclassify 6 out of 44 KCNH2 VUSs as likely pathogenic. The high-throughput patch-clamp assay can provide moderate-strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value of developing automated patch-clamp assays for functional characterization of ion channel gene variants.

Effects of cohort, genotype, variant, and maternal ß-blocker treatment on foetal heart rate predictors of inherited long QT syndrome.

AIMS: In long QT syndrome (LQTS), primary prevention improves outcome; thus, early identification is key. The most common LQTS phenotype is a foetal heart rate (FHR) < 3rd percentile for gestational age (GA) but the effects of cohort, genotype, variant, and maternal ß-blocker therapy on FHR are unknown. We assessed the influence of these factors on FHR in pregnancies with familial LQTS and developed a FHR/GA threshold for LQTS. METHODS AND RESULTS: In an international cohort of pregnancies in which one parent had LQTS, LQTS genotype, familial variant, and maternal ß-blocker effects on FHR were assessed. We developed a testing algorithm for LQTS using FHR and GA as continuous predictors. Data included 1966 FHRs at 7-42 weeks' GA from 267 pregnancies/164 LQTS families [220 LQTS type 1 (LQT1), 35 LQTS type 2 (LQT2), and 12 LQTS type 3 (LQT3)]. The FHRs were significantly lower in LQT1 and LQT2 but not LQT3 or LQTS negative. The LQT1 variants with non-nonsense and severe function loss (current density or ß-adrenergic response) had lower FHR. Maternal ß-blockers potentiated bradycardia in LQT1 and LQT2 but did not affect FHR in LQTS negative. A FHR/GA threshold predicted LQT1 and LQT2 with 74.9% accuracy, 71% sensitivity, and 81% specificity. CONCLUSION: Genotype, LQT1 variant, and maternal ß-blocker therapy affect FHR. A predictive threshold of FHR/GA significantly improves the accuracy, sensitivity, and specificity for LQT1 and LQT2, above the infant's a priori 50% probability. We speculate this model may be useful in screening for LQTS in perinatal subjects without a known LQTS family history.

The S1 helix critically regulates the finely tuned gating of Kv11.1 channels.

Congenital mutations in the cardiac Kv11.1 channel can cause long QT syndrome type 2 (LQTS2), a heart rhythm disorder associated with sudden cardiac death. Mutations act either by reducing protein expression at the membrane and/or by perturbing the intricate gating properties of Kv11.1 channels. A number of clinical LQTS2-associated mutations have been reported in the first transmembrane segment (S1) of Kv11.1 channels, but the role of this region of the channel is largely unexplored. In part, this is due to problems defining the extent of the S1 helix, as a consequence of its low sequence homology with other Kv family members. Here, we used NMR spectroscopy and electrophysiological characterization to show that the S1 of Kv11.1 channels extends seven helical turns, from Pro-405 to Phe-431, and is flanked by unstructured loops. Functional analysis suggests that pre-S1 loop residues His-402 and Tyr-403 play an important role in regulating the kinetics and voltage dependence of channel activation and deactivation. Multiple residues within the S1 helix also play an important role in fine-tuning the voltage dependence of activation, regulating slow deactivation, and modulating C-type inactivation of Kv11.1 channels. Analyses of LQTS2-associated mutations in the pre-S1 loop or S1 helix of Kv11.1 channels demonstrate perturbations to both protein expression and most gating transitions. Thus, S1 region mutations would reduce both the action potential repolarizing current passed by Kv11.1 channels in cardiac myocytes, as well as the current passed in response to premature depolarizations that normally helps protect against the formation of ectopic beats.

Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation.

Slow deactivation of Kv11.1 channels is critical for its function in the heart. The S4-S5 linker, which joins the voltage sensor and pore domains, plays a critical role in this slow deactivation gating. Here, we use NMR spectroscopy to identify the membrane-bound surface of the S4S5 linker, and we show that two highly conserved tyrosine residues within the KCNH subfamily of channels are membrane-associated. Site-directed mutagenesis and electrophysiological analysis indicates that Tyr-542 interacts with both the pore domain and voltage sensor residues to stabilize activated conformations of the channel, whereas Tyr-545 contributes to the slow kinetics of deactivation by primarily stabilizing the transition state between the activated and closed states. Thus, the two tyrosine residues in the Kv11.1 S4S5 linker play critical but distinct roles in the slow deactivation phenotype, which is a hallmark of Kv11.1 channels.

Rescue of protein expression defects may not be enough to abolish the pro-arrhythmic phenotype of long QT type 2 mutations.

KEY POINTS: Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression. Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2-associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored. Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics. Co-expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels. For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein. ABSTRACT: In the heart, Kv11.1 channels pass the rapid delayed rectifier current (IKr ) which plays critical roles in repolarization of the cardiac action potential and in the suppression of arrhythmias caused by premature stimuli. Over 500 inherited mutations in Kv11.1 are known to cause long QT syndrome type 2 (LQTS2), a cardiac electrical disorder associated with an increased risk of life threatening arrhythmias. Most missense mutations in Kv11.1 reduce the amount of channel protein expressed at the membrane and, as a consequence, there has been considerable interest in developing pharmacological agents to rescue the expression of these channels. However, pharmacological chaperones will only have clinical utility if the mutant Kv11.1 channels function normally after membrane expression is restored. The aim of this study was to characterize the gating phenotype for a subset of LQTS2 mutations to assess what proportion of mutations may be suitable for rescue. As an initial screen we used reduced temperature to rescue expression defects of mutant channels expressed in Xenopus laevis oocytes. Over half (∼56%) of Kv11.1 mutants exhibited functional gating defects that either dramatically reduced the amount of current contributing to cardiac action potential repolarization and/or reduced the amount of protective current elicited in response to premature depolarizations. Our data demonstrate that if pharmacological rescue of protein expression defects is going to have clinical utility in the treatment of LQTS2 then it will be important to assess the gating phenotype of LQTS2 mutations before attempting rescue.

Getting to the heart of hERG K(+) channel gating.

Potassium ion channels encoded by the human ether-a-go-go related gene (hERG) form the ion-conducting subunit of the rapid delayed rectifier potassium current (IKr ). Although hERG channels exhibit a widespread tissue distribution they play a particularly important role in the heart. There has been considerable interest in hERG K(+) channels for three main reasons. First, they have very unusual gating kinetics, most notably rapid and voltage-dependent inactivation coupled to slow deactivation, which has led to the suggestion that they may play a specific role in the suppression of arrhythmias. Second, mutations in hERG are the cause of 30-40% of cases of congenital long QT syndrome (LQTS), the commonest inherited primary arrhythmia syndrome. Third, hERG is the molecular target for the vast majority of drugs that cause drug-induced LQTS, the commonest cause of drug-induced arrhythmias and cardiac death. Drug-induced LQTS has now been reported for a large range of both cardiac and non-cardiac drugs, in which this side effect is entirely undesired. In recent years there have been comprehensive reviews published on hERG K(+) channels (Vandenberg et al. 2012) and we will not re-cover this ground. Rather, we focus on more recent work on the structural basis and dynamics of hERG gating with an emphasis on how the latest developments may facilitate translational research in the area of stratifying risk of arrhythmias.

Multiple interactions between cytoplasmic domains regulate slow deactivation of Kv11.1 channels.

The intracellular domains of many ion channels are important for fine-tuning their gating kinetics. In Kv11.1 channels, the slow kinetics of channel deactivation, which are critical for their function in the heart, are largely regulated by the N-terminal N-Cap and Per-Arnt-Sim (PAS) domains, as well as the C-terminal cyclic nucleotide-binding homology (cNBH) domain. Here, we use mutant cycle analysis to probe for functional interactions between the N-Cap/PAS domains and the cNBH domain. We identified a specific and stable charge-charge interaction between Arg(56) of the PAS domain and Asp(803) of the cNBH domain, as well an additional interaction between the cNBH domain and the N-Cap, both of which are critical for maintaining slow deactivation kinetics. Furthermore, we found that positively charged arginine residues within the disordered region of the N-Cap interact with negatively charged residues of the C-linker domain. Although this interaction is likely more transient than the PAS-cNBD interaction, it is strong enough to stabilize the open conformation of the channel and thus slow deactivation. These findings provide novel insights into the slow deactivation mechanism of Kv11.1 channels.

Role of the cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) domain in trafficking and stabilization of Kv11.1 channels.

The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking.

Pore helices play a dynamic role as integrators of domain motion during Kv11.1 channel inactivation gating.

Proteins that form ion-selective pores in the membrane of cells are integral to many rapid signaling processes, including regulating the rhythm of the heartbeat. In potassium channels, the selectivity filter is critical for both endowing an exquisite selectivity for potassium ions, as well as for controlling the flow of ions through the pore. Subtle rearrangements in the complex hydrogen-bond network that link the selectivity filter to the surrounding pore helices differentiate conducting (open) from nonconducting (inactivated) conformations of the channel. Recent studies suggest that beyond the selectivity filter, inactivation involves widespread rearrangements of the channel protein. Here, we use rate equilibrium free energy relationship analysis to probe the structural changes that occur during selectivity filter gating in Kv11.1 channels, at near atomic resolution. We show that the pore helix plays a crucial dynamic role as a bidirectional interface during selectivity filter gating. We also define the molecular bases of the energetic coupling between the pore helix and outer helix of the pore domain that occurs early in the transition from open to inactivated states, as well as the coupling between the pore helix and inner helix late in the transition. Our data demonstrate that the pore helices are more than just static structural elements supporting the integrity of the selectivity filter; instead they play a crucial dynamic role during selectivity filter gating.

Trafficking defects in PAS domain mutant Kv11.1 channels: roles of reduced domain stability and altered domain-domain interactions.

Loss of Kv11.1 potassium channel function is the underlying cause of pathology in long-QT syndrome type 2, one of the commonest causes of sudden cardiac death in the young. Previous studies have identified the cytosolic PAS (Per/Arnt/Sim) domain as a hotspot for mutations that cause Kv11.1 trafficking defects. To investigate the underlying basis of this observation, we have quantified the effect of mutants on domain folding as well as interactions between the PAS domain and the remainder of the channel. Apart from R56Q, all mutants impaired the thermostability of the isolated PAS domain. Six mutants, located in the vicinity of a hydrophobic patch on the PAS domain surface, also affected binding of the isolated PAS domain to an N-terminal truncated hERG (human ether-a-go-go-related gene) channel. Conversely, four other surface mutants (C64Y, T65P, A78P and I96T) and one buried mutant (L86R) did not prevent the isolated PAS domain binding to the truncated channels. Our results highlight a critical role for interactions between the PAS domain and the remainder of the channel in the hERG assembly and that mutants that affect PAS domain interactions with the remainder of the channel have a more severe trafficking defect than that caused by domain unfolding alone.

Clinical interpretation of KCNH2 variants using a robust PS3/BS3 functional patch-clamp assay.

Long QT syndrome (LQTS), caused by the dysfunction of cardiac ion channels, increases the risk of sudden death in otherwise healthy young people. For many variants in LQTS genes, there is insufficient evidence to make a definitive genetic diagnosis. We have established a robust functional patch-clamp assay to facilitate classification of missense variants in KCNH2, one of the key LQTS genes. A curated set of 30 benign and 30 pathogenic missense variants were used to establish the range of normal and abnormal function. The extent to which variants reduced protein function was quantified using Z scores, the number of standard deviations from the mean of the normalized current density of the set of benign variant controls. A Z score of -2 defined the threshold for abnormal loss of function, which corresponds to 55% wild-type function. More extreme Z scores were observed for variants with a greater loss-of-function effect. We propose that the Z score for each variant can be used to inform the application and weighting of abnormal and normal functional evidence criteria (PS3 and BS3) within the American College of Medical Genetics and Genomics variant classification framework. The validity of this approach was demonstrated using a series of 18 KCNH2 missense variants detected in a childhood onset LQTS cohort, where the level of function assessed using our assay correlated to the Schwartz score (a scoring system used to quantify the probability of a clinical diagnosis of LQTS) and the length of the corrected QT (QTc) interval.

Multisite Validation of a Functional Assay to Adjudicate SCN5A Brugada Syndrome-Associated Variants.

BACKGROUND: Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging, and ≈79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification. METHODS: An in vitro SCN5A-Brugada syndrome automated patch clamp assay was generated and independently studied at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function. RESULTS: Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values yielded 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic. CONCLUSIONS: This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A-Brugada syndrome variants of uncertain significance.

Multiplexed Assays of Variant Effect and Automated Patch-clamping Improve KCNH2-LQTS Variant Classification and Cardiac Event Risk Stratification.

Background: Long QT syndrome (LQTS) is a lethal arrhythmia syndrome, frequently caused by rare loss-of-function variants in the potassium channel encoded by KCNH2. Variant classification is difficult, often owing to lack of functional data. Moreover, variant-based risk stratification is also complicated by heterogenous clinical data and incomplete penetrance. Here, we sought to test whether variant-specific information, primarily from high-throughput functional assays, could improve both classification and cardiac event risk stratification in a large, harmonized cohort of KCNH2 missense variant heterozygotes. Methods: We quantified cell-surface trafficking of 18,796 variants in KCNH2 using a Multiplexed Assay of Variant Effect (MAVE). We recorded KCNH2 current density for 533 variants by automated patch clamping (APC). We calibrated the strength of evidence of MAVE data according to ClinGen guidelines. We deeply phenotyped 1,458 patients with KCNH2 missense variants, including QTc, cardiac event history, and mortality. We correlated variant functional data and Bayesian LQTS penetrance estimates with cohort phenotypes and assessed hazard ratios for cardiac events. Results: Variant MAVE trafficking scores and APC peak tail currents were highly correlated (Spearman Rank-order ρ = 0.69). The MAVE data were found to provide up to pathogenic very strong evidence for severe loss-of-function variants. In the cohort, both functional assays and Bayesian LQTS penetrance estimates were significantly predictive of cardiac events when independently modeled with patient sex and adjusted QT interval (QTc); however, MAVE data became non-significant when peak-tail current and penetrance estimates were also available. The area under the ROC for 20-year event outcomes based on patient-specific sex and QTc (AUC 0.80 [0.76-0.83]) was improved with prospectively available penetrance scores conditioned on MAVE (AUC 0.86 [0.83-0.89]) or attainable APC peak tail current data (AUC 0.84 [0.81-0.88]). Conclusion: High throughput KCNH2 variant MAVE data meaningfully contribute to variant classification at scale while LQTS penetrance estimates and APC peak tail current measurements meaningfully contribute to risk stratification of cardiac events in patients with heterozygous KCNH2 missense variants.

Insights into hERG K+ channel structure and function from NMR studies.

The unique gating kinetics of hERG K(+) channels are critical for normal cardiac repolarization, and patients with mutations in hERG have a markedly increased risk of cardiac arrhythmias and sudden cardiac arrest. HERG K(+) channels are also remarkably promiscuous with respect to drug binding, which has been a very significant problem for the pharmaceutical industry. Here, we review the progress that has been made in understanding the structure and function of hERG K(+) channels with a particular focus on nuclear magnetic resonance studies of the domains of the hERG K(+) channel.

Development of automated patch clamp assays to overcome the burden of variants of uncertain significance in inheritable arrhythmia syndromes.

Advances in next-generation sequencing have been exceptionally valuable for identifying variants in medically actionable genes. However, for most missense variants there is insufficient evidence to permit definitive classification of variants as benign or pathogenic. To overcome the deluge of Variants of Uncertain Significance, there is an urgent need for high throughput functional assays to assist with the classification of variants. Advances in parallel planar patch clamp technologies has enabled the development of automated high throughput platforms capable of increasing throughput 10- to 100-fold compared to manual patch clamp methods. Automated patch clamp electrophysiology is poised to revolutionize the field of functional genomics for inheritable cardiac ion channelopathies. In this review, we outline i) the evolution of patch clamping, ii) the development of high-throughput automated patch clamp assays to assess cardiac ion channel variants, iii) clinical application of these assays and iv) where the field is heading.

Multi-site validation of a functional assay to adjudicate SCN5A Brugada Syndrome-associated variants.

Brugada Syndrome (BrS) is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in the cardiac sodium channel gene, SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging and ~79% of SCN5A missense variants in ClinVar are currently classified as Variants of Uncertain Significance (VUS). An in vitro SCN5A-BrS automated patch clamp assay was generated for high-throughput functional studies of NaV1.5. The assay was independently studied at two separate research sites - Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute - revealing strong correlations, including peak INa density (R2=0.86). The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. The assay accurately distinguished benign controls (24/25) from pathogenic controls (23/24). Odds of Pathogenicity values derived from the experimental results yielded 0.042 for normal function (BS3 criterion) and 24.0 for abnormal function (PS3 criterion), resulting in up to strong evidence for both ACMG criteria. The calibrated assay was then used to study SCN5A VUS observed in four families with BrS and other arrhythmia phenotypes associated with SCN5A loss-of-function. The assay revealed loss-of-function for three of four variants, enabling reclassification to likely pathogenic. This validated APC assay provides clinical-grade functional evidence for the reclassification of current VUS and will aid future SCN5A-BrS variant classification.

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 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.

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

Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like most plasma membrane ion channel proteins they must pass through biosynthetic quality control in the endoplasmic reticulum 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 N-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 Piezo1 protein. Thus the N-linked glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.

High-throughput phenotyping of heteromeric human ether-à-go-go-related gene potassium channel variants can discriminate pathogenic from rare benign variants.

BACKGROUND: KCNH2 encodes the human ether-à-go-go-related gene potassium channel, which passes the rapid delayed rectifier potassium current. Loss-of-function variants in KCNH2 cause long QT syndrome type 2, which is associated with a markedly increased risk of cardiac arrhythmias. The majority of rare KCNH2 variants, however, are likely to be benign. OBJECTIVE: The purpose of this study was to develop a high-throughput assay for discriminating pathogenic from benign KCNH2 variants. METHODS: Nonsynonymous homozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 cell lines were phenotyped using an automated patch-clamp platform and a cell surface enzyme-linked immunosorbent assay. Functional phenotyping of heterozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 cell lines using a bicistronic vector was performed using an automated patch-clamp platform. RESULTS: In homozygous KCNH2 variant cell lines, discrepancies between current density and cell surface expression levels measured using an enzyme-linked immunosorbent assay can be explained by changes in gating properties of the variant channels. For the 30 heterozygous KCNH2 variant cell lines studied, the assay correctly predicted the ClinVar ascribed classification for 17/17 pathogenic/likely pathogenic/benign variants. Of the 13 pore-domain variants studied, 11 had a dominant-negative expression defect while the remaining 2 had enhanced inactivation gating, resulting in a dominant-negative phenotype. CONCLUSION: High-throughput electrophysiological phenotyping of heterozygous KCNH2 variants can accurately distinguish between dominant-negative, haploinsufficient loss-of-function, and benign variants. This assay will help with future classification of KCNH2 variants.