Cryo-EM Structure of a KCNQ1/CaM Complex Reveals Insights into Congenital Long QT Syndrome.

KCNQ1 is the pore-forming subunit of cardiac slow-delayed rectifier potassium (IKs) channels. Mutations in the kcnq1 gene are the leading cause of congenital long QT syndrome (LQTS). Here, we present the cryoelectron microscopy (cryo-EM) structure of a KCNQ1/calmodulin (CaM) complex. The conformation corresponds to an "uncoupled," PIP2-free state of KCNQ1, with activated voltage sensors and a closed pore. Unique structural features within the S4-S5 linker permit uncoupling of the voltage sensor from the pore in the absence of PIP2. CaM contacts the KCNQ1 voltage sensor through a specific interface involving a residue on CaM that is mutated in a form of inherited LQTS. Using an electrophysiological assay, we find that this mutation on CaM shifts the KCNQ1 voltage-activation curve. This study describes one physiological form of KCNQ1, depolarized voltage sensors with a closed pore in the absence of PIP2, and reveals a regulatory interaction between CaM and KCNQ1 that may explain CaM-mediated LQTS.

KCNQ1 is an essential mediator of the sex-dependent perception of moderate cold temperatures.

Low temperatures and cooling agents like menthol induce cold sensation by activating the peripheral cold receptors TRPM8 and TRPA1, cation channels belonging to the TRP channel family, while the reduction of potassium currents provides an additional and/or synergistic mechanism of cold sensation. Despite extensive studies over the past decades to identify the molecular receptors that mediate thermosensation, cold sensation is still not fully understood and many cold-sensitive peripheral neurons do not express the well-established cold sensor TRPM8. We found that the voltage-gated potassium channel KCNQ1 (Kv7.1), which is defective in cardiac LQT1 syndrome, is, in addition to its known function in the heart, a highly relevant and sex-specific sensor of moderately cold temperatures. We found that KCNQ1 is expressed in skin and dorsal root ganglion neurons, is sensitive to menthol and cooling agents, and is highly sensitive to moderately cold temperatures, in a temperature range at which TRPM8 is not thermosensitive. C-fiber recordings from KCNQ1-/- mice displayed altered action potential firing properties. Strikingly, only male KCNQ1-/- mice showed substantial deficits in cold avoidance at moderately cold temperatures, with a strength of the phenotype similar to that observed in TRPM8-/- animals. While sex-dependent differences in thermal sensitivity have been well documented in humans and mice, KCNQ1 is the first gene reported to play a role in sex-specific temperature sensation. Moreover, we propose that KCNQ1, together with TRPM8, is a key instrumentalist that orchestrates the range and intensity of cold sensation.

Pairwise biosynthesis of ion channels stabilizes excitability and mitigates arrhythmias.

Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many disorders including cardiac arrhythmias. Prior work revealed association of mRNAs encoding cardiac NaV1.5 (SCN5A) and hERG1 (KCNH2), but the functional significance of this association was not established. Here, we provide a more comprehensive picture of KCNH2, SCN5A, CACNA1C, and KCNQ1 transcripts collectively copurifying with nascent hERG1, NaV1.5, CaV1.2, or KCNQ1 channel proteins. Single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence reveals that the channel proteins are synthesized predominantly as heterotypic pairs from discrete molecules of mRNA, not as larger cotranslational complexes. Puromycin disrupted colocalization of mRNA with its encoded protein, as expected, but remarkably also pairwise mRNA association, suggesting that transcript association relies on intact translational machinery or the presence of the nascent protein. Targeted depletion of KCHN2 by specific shRNA resulted in concomitant reduction of all associated mRNAs, with a corresponding reduction in the encoded channel currents. This co-knockdown effect, originally described for KCNH2 and SCN5A, thus appears to be a general phenomenon among transcripts encoding functionally related proteins. In multielectrode array recordings, proarrhythmic behavior arose when IKr was reduced by the selective blocker dofetilide at IC50 concentrations, but not when equivalent reductions were mediated by shRNA, suggesting that co-knockdown mitigates proarrhythmic behavior expected from the selective reduction of a single channel species. We propose that coordinated, cotranslational association of functionally related ion channel mRNAs confers electrical stability by co-regulating complementary ion channels in macromolecular complexes.

Proof of concept for monoclonal antibody therapy in a cellular model of acquired long QT syndrome type 3.

Long QT syndrome (LQTS) type 3 although less common than the first two forms, differs in that arrhythmic events are less likely triggered by adrenergic stimuli and are more often lethal. Effective pharmacological treatment is challenged by interindividual differences, mutation dependence, and adverse effects, translating into an increased use of invasive measures (implantable cardioverter-defibrillator, sympathetic denervation) in patients with LQTS type 3. Previous studies have demonstrated the therapeutic potential of polyclonal KCNQ1 antibody for LQTS type 2. Here, we sought to identify a monoclonal KCNQ1 antibody that preserves the electrophysiological properties of the polyclonal form. Using hybridoma technology, murine monoclonal antibodies were generated, and patch clamp studies were performed for functional characterization. We identified a monoclonal KCNQ1 antibody able to normalize cardiac action potential duration and to suppress arrhythmias in a pharmacological model of LQTS type 3 using human-induced pluripotent stem cell-derived cardiomyocytes.NEW & NOTEWORTHY Long QT syndrome is a leading cause of sudden cardiac death in the young. Recent research has highlighted KCNQ1 antibody therapy as a new treatment modality for long QT syndrome type 2. Here, we developed a monoclonal KCNQ1 antibody that similarly restores cardiac repolarization. Moreover, the identified monoclonal KCNQ1 antibody suppresses arrhythmias in a cellular model of long QT syndrome type 3, holding promise as a first-in-class antiarrhythmic immunotherapy.

A mutation in the cardiac KV7.1 channel possibly disrupts interaction with Yotiao protein.

Here, we characterized the p.Arg583His (R583H) Kv7.1 mutation, identified in two unrelated families suffered from LQT syndrome. This mutation is located in the HС-HD linker of the cytoplasmic portion of the Kv7.1 channel. This linker, together with HD helix are responsible for binding the A-kinase anchoring protein 9 (AKAP9), Yotiao. We studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1 along with KCNE1 subunit and Yotiao protein, using the whole-cell patch-clamp technique. We found that R583H mutation, even at the heterozygous state, impedes IKs activation. Molecular modeling showed that HС and HD helixes of the C-terminal part of Kv7.1 channel are swapped along the C-terminus length of the channel and that R583 position is exposed to the outer surface of HC-HD tandem coiled-coil. Interestingly, the adenylate cyclase activator, forskolin had a smaller effect on the mutant channel comparing with the WT protein, suggesting that R583H mutation may disrupt the interaction of the channel with the adaptor protein Yotiao and, therefore, may impair phosphorylation of the KCNQ1 channel.

Genetic characterization of KCNQ1 variants improves risk stratification in type 1 long QT syndrome patients.

AIMS: KCNQ1 mutations cause QTc prolongation increasing life-threatening arrhythmias risks. Heterozygous mutations [type 1 long QT syndrome (LQT1)] are common. Homozygous KCNQ1 mutations cause type 1 Jervell and Lange-Nielsen syndrome (JLNS) with deafness and higher sudden cardiac death risk. KCNQ1 variants causing JLNS or LQT1 might have distinct phenotypic expressions in heterozygous patients. The aim of this study is to evaluate QTc duration and incidence of long QT syndrome-related cardiac events according to genetic presentation. METHODS AND RESULTS: We enrolled LQT1 or JLNS patients with class IV/V KCNQ1 variants from our inherited arrhythmia clinic (September 1993 to January 2023). Medical history, ECG, and follow-up were collected. Additionally, we conducted a thorough literature review for JLNS variants. Survival curves were compared between groups, and multivariate Cox regression models identified genetic and clinical risk factors. Among the 789 KCNQ1 variant carriers, 3 groups were identified: 30 JLNS, 161 heterozygous carriers of JLNS variants (HTZ-JLNS), and 550 LQT1 heterozygous carriers of non-JLNS variants (HTZ-Non-JLNS). At diagnosis, mean age was 3.4 ± 4.7 years for JLNS, 26.7 ± 21 years for HTZ-JLNS, and 26 ± 21 years for HTZ-non-JLNS; 55.3% were female; and the mean QTc was 551 ± 54 ms for JLNS, 441 ± 32 ms for HTZ-JLNS, and 467 ± 36 ms for HTZ-Non-JLNS. Patients with heterozygous JLNS mutations (HTZ-JLNS) represented 22% of heterozygous KCNQ1 variant carriers and had a lower risk of cardiac events than heterozygous non-JLNS variant carriers (HTZ-Non-JLNS) [hazard ratio (HR) = 0.34 (0.22-0.54); P < 0.01]. After multivariate analysis, four genetic parameters were independently associated with events: haploinsufficiency [HR = 0.60 (0.37-0.97); P = 0.04], pore localization [HR = 1.61 (1.14-1.2.26); P < 0.01], C-terminal localization [HR = 0.67 (0.46-0.98); P = 0.04], and group [HR = 0.43 (0.27-0.69); P < 0.01]. CONCLUSION: Heterozygous carriers of JLNS variants have a lower risk of cardiac arrhythmic events than other LQT1 patients.

Correlation Analysis of KCNQ1 Gene Polymorphism with Blood Indexes and Prognosis of Non-Small Cell Lung Cancer.

BACKGROUND: This study was conducted to investigate the correlation between KCNQ1 rs2237895 A/C gene polymorphism and blood indexes and prognosis in non-small cell lung cancer (NSCLC). METHODS: A total of 260 NSCLC patients were selected and classified into stage I - II (n = 109) and stage III - IV (n = 151) according to by American Joint Committee on Cancer Staging Manual. A control group was established with another 92 healthy subjects. The genotype distribution of rs2237895 was analyzed in all subjects. 2 analysis or Fisher's test was employed to analyze the association between genotype and allele distribution frequencies with carcinoembryonic antigen (CEA), squamous cell carcinoma antigen, and cytokeratin fragment 19 (CyfrA 21-1). Overall survival was compared by genotype stratification using Kaplan-Meier analysis. Univariate and multivariate Cox risk regression analyses were used to determine the prognostic value of allele C in NSCLC. RESULTS: AC/CC genotypes in NSCLC patients were associated with gender, hypertension, smoking, clinical TNM stage, lymph node metastasis, and distant metastasis. C allele was associated with higher risk levels of serum tumor markers. Patients with allele C (AC + CC) had lower overall survival than patients with genotype AA. Finally, clinical stage, lymph node metastasis, higher CEA and CyfrA 21-1 serum levels, and rs2237895 A/C gene poly-morphism were independent prognostic factors of NSCLC. CONCLUSIONS: rs2237895 A/C polymorphism of the KCNQ1 gene can be a prognostic predictor in patients with surgically treated NSCLC.

Modulating the voltage sensor of a cardiac potassium channel shows antiarrhythmic effects.

Cardiac arrhythmias are the most common cause of sudden cardiac death worldwide. Lengthening the ventricular action potential duration (APD), either congenitally or via pathologic or pharmacologic means, predisposes to a life-threatening ventricular arrhythmia, Torsade de Pointes. IKs (KCNQ1+KCNE1), a slowly activating K+ current, plays a role in action potential repolarization. In this study, we screened a chemical library in silico by docking compounds to the voltage-sensing domain (VSD) of the IKs channel. Here, we show that C28 specifically shifted IKs VSD activation in ventricle to more negative voltages and reversed the drug-induced lengthening of APD. At the same dosage, C28 did not cause significant changes of the normal APD in either ventricle or atrium. This study provides evidence in support of a computational prediction of IKs VSD activation as a potential therapeutic approach for all forms of APD prolongation. This outcome could expand the therapeutic efficacy of a myriad of currently approved drugs that may trigger arrhythmias.

KCNQ1 p.D446E Variant as a Risk Allele for Arrhythmogenic Phenotypes: Electrophysiological Characterization Reveals a Complex Phenotype Affecting the Slow Delayed Rectifier Potassium Current (IKs) Voltage Dependence by Causing a Hyperpolarizing Shift and a Lack of Response to Protein Kinase A Activation.

Genetic testing is crucial in inherited arrhythmogenic channelopathies; however, the clinical interpretation of genetic variants remains challenging. Incomplete penetrance, oligogenic, polygenic or multifactorial forms of channelopathies further complicate variant interpretation. We identified the KCNQ1/p.D446E variant in 2/63 patients with long QT syndrome, 30-fold more frequent than in public databases. We thus characterized the biophysical phenotypes of wildtype and mutant IKs co-expressing these alleles with the ß-subunit minK in HEK293 cells. KCNQ1 p.446E homozygosity significantly shifted IKs voltage dependence to hyperpolarizing potentials in basal conditions (gain of function) but failed to shift voltage dependence to hyperpolarizing potentials (loss of function) in the presence of 8Br-cAMP, a protein kinase A activator. Basal IKs activation kinetics did not differ among genotypes, but in response to 8Br-cAMP, IKs 446 E/E (homozygous) activation kinetics were slower at the most positive potentials. Protein modeling predicted a slower transition of the 446E Kv7.1 tetrameric channel to the stabilized open state. In conclusion, biophysical and modelling evidence shows that the KCNQ1 p.D446E variant has complex functional consequences including both gain and loss of function, suggesting a contribution to the pathogenesis of arrhythmogenic phenotypes as a functional risk allele.

Prediction of Kv11.1 potassium channel PAS-domain variants trafficking via machine learning.

Congenital long QT syndrome (LQTS) is characterized by a prolonged QT-interval on an electrocardiogram (ECG). An abnormal prolongation in the QT-interval increases the risk for fatal arrhythmias. Genetic variants in several different cardiac ion channel genes, including KCNH2, are known to cause LQTS. Here, we evaluated whether structure-based molecular dynamics (MD) simulations and machine learning (ML) could improve the identification of missense variants in LQTS-linked genes. To do this, we investigated KCNH2 missense variants in the Kv11.1 channel protein shown to have wild type (WT) like or class II (trafficking-deficient) phenotypes in vitro. We focused on KCNH2 missense variants that disrupt normal Kv11.1 channel protein trafficking, as it is the most common phenotype for LQTS-associated variants. Specifically, we used computational techniques to correlate structural and dynamic changes in the Kv11.1 channel protein PAS domain (PASD) with Kv11.1 channel protein trafficking phenotypes. These simulations unveiled several molecular features, including the numbers of hydrating waters and hydrogen bonding pairs, as well as folding free energy scores, that are predictive of trafficking. We then used statistical and machine learning (ML) (Decision tree (DT), Random forest (RF), and Support vector machine (SVM)) techniques to classify variants using these simulation-derived features. Together with bioinformatics data, such as sequence conservation and folding energies, we were able to predict with reasonable accuracy (≈75%) which KCNH2 variants do not traffic normally. We conclude that structure-based simulations of KCNH2 variants localized to the Kv11.1 channel PASD led to an improvement in classification accuracy. Therefore, this approach should be considered to complement the classification of variant of unknown significance (VUS) in the Kv11.1 channel PASD.

The utility of zebrafish cardiac arrhythmia model to predict the pathogenicity of KCNQ1 variants.

Genetic testing for inherited arrhythmias and discriminating pathogenic or benign variants from variants of unknown significance (VUS) is essential for gene-based medicine. KCNQ1 is a causative gene of type 1 long QT syndrome (LQTS), and approximately 30% of the variants found in type 1 LQTS are classified as VUS. We studied the role of zebrafish cardiac arrhythmia model in determining the clinical significance of KCNQ1 variants. We generated homozygous kcnq1 deletion zebrafish (kcnq1del/del) using the CRISPR/Cas9 and expressed human Kv7.1/MinK channels in kcnq1del/del embryos. We dissected the hearts from the thorax at 48 h post-fertilization and measured the transmembrane potential of the ventricle in the zebrafish heart. Action potential duration was calculated as the time interval between peak maximum upstroke velocity and 90% repolarization (APD90). The APD90 of kcnq1del/del embryos was 280 ± 47 ms, which was significantly shortened by injecting KCNQ1 wild-type (WT) cRNA and KCNE1 cRNA (168 ± 26 ms, P < 0.01 vs. kcnq1del/del). A study of two pathogenic variants (S277L and T587M) and one VUS (R451Q) associated with clinically definite LQTS showed that the APD90 of kcnq1del/del embryos with these mutant Kv7.1/MinK channels was significantly longer than that of Kv7.1 WT/MinK channels. Given the functional results of the zebrafish model, R451Q could be reevaluated physiologically from VUS to likely pathogenic. In conclusion, functional analysis using in vivo zebrafish cardiac arrhythmia model can be useful for determining the pathogenicity of loss-of-function variants in patients with LQTS.

Long QT syndrome-associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel.

Calmodulin (CaM) is a highly conserved mediator of calcium (Ca2+ )-dependent signalling and modulates various cardiac ion channels. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS). LQTS patients display prolonged ventricular recovery times (QT interval), increasing their risk of incurring life-threatening arrhythmic events. Loss-of-function mutations to Kv7.1 (which drives the slow delayed rectifier potassium current, IKs, a key ventricular repolarising current) are the largest contributor to congenital LQTS (>50% of cases). CaM modulates Kv7.1 to produce a Ca2+ -sensitive IKs, but little is known about the consequences of LQTS-associated CaM mutations on Kv7.1 function. Here, we present novel data characterising the biophysical and modulatory properties of three LQTS-associated CaM variants (D95V, N97I and D131H). We showed that mutations induced structural alterations in CaM and reduced affinity for Kv7.1, when compared with wild-type (WT). Using HEK293T cells expressing Kv7.1 channel subunits (KCNQ1/KCNE1) and patch-clamp electrophysiology, we demonstrated that LQTS-associated CaM variants reduced current density at systolic Ca2+ concentrations (1 µm), revealing a direct QT-prolonging modulatory effect. Our data highlight for the first time that LQTS-associated perturbations to CaM's structure impede complex formation with Kv7.1 and subsequently result in reduced IKs. This provides a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype. KEY POINTS: Calmodulin (CaM) is a ubiquitous, highly conserved calcium (Ca2+ ) sensor playing a key role in cardiac muscle contraction. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS), a life-threatening cardiac arrhythmia syndrome. LQTS-associated CaM variants (D95V, N97I and D131H) induced structural alterations, altered binding to Kv7.1 and reduced IKs. Our data provide a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype.

KCNE1 does not shift TMEM16A from a Ca2+ dependent to a voltage dependent Cl- channel and is not expressed in renal proximal tubule.

The TMEM16A (ANO1) Cl- channel is activated by Ca2+ in a voltage-dependent manner. It is broadly expressed and was shown to be also present in renal proximal tubule (RPT). KCNQ1 is an entirely different K+ selective channel that forms the cardiac IKS potassium channel together with its ß-subunit KCNE1. Surprisingly, KCNE1 has been claimed to interact with TMEM16A, and to be required for activation of TMEM16A in mouse RPT. Interaction with KCNE1 was reported to switch TMEM16A from a Ca22+-dependent to a voltage-dependent ion channel. Here we demonstrate that KCNE1 is not expressed in mouse RPT. TMEM16A expressed in RPT is activated by angiotensin II and ATP in a KCNE1-independent manner. Coexpression of KCNE1 does not change TMEM16A to a voltage gated Cl- channel and Ca2+-dependent regulation of TMEM16A is fully maintained in the presence of KCNE1. While overexpressed KCNE1 slightly affects Ca2+-dependent regulation of TMEM16A, the data provide no evidence for KCNE1 being an auxiliary functional subunit for TMEM16A.

The role of native cysteine residues in the oligomerization of KCNQ1 channels.

KCNQ1, the major component of the slow-delayed rectifier potassium channel, is responsible for repolarization of cardiac action potential. Mutations in this channel can lead to a variety of diseases, most notably long QT syndrome. It is currently unknown how many of these mutations change channel function and structure on a molecular level. Since tetramerization is key to proper function and structure of the channel, it is likely that mutations modify the stability of KCNQ1 oligomers. Presently, the C-terminal domain of KCNQ1 has been noted as the driving force for oligomer formation. However, truncated versions of this protein lacking the C-terminal domain still tetramerize. Therefore, we explored the role of native cysteine residues in a truncated construct of human KCNQ1, amino acids 100-370, by blocking potential interactions of cysteines with a nitroxide based spin label. Mobility of the spin labels was investigated with continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. The oligomerization state was examined by gel electrophoresis. The data provide information on tetramerization of human KCNQ1 without the C-terminal domain. Specifically, how blocking the side chains of native cysteines residues reduces oligomerization. A better understanding of tetramer formation could provide improved understanding of the molecular etiology of long QT syndrome and other diseases related to KCNQ1.

The second PI(3,5)P2 binding site in the S0 helix of KCNQ1 stabilizes PIP2-at the primary PI1 site with potential consequences on intermediate-to-open state transition.

The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a known regulator of membrane protein trafficking. PI(3,5)P2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P2 binding sites, the known PIP2 site PS1 and the newly identified N-terminal α-helix S0 as relevant for functional PIKfyve effects. Cd2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P2 to both sites.

Computational Study on Effect of KCNQ1 P535T Mutation in a Cardiac Ventricular Tissue.

Heart diseases such as arrhythmia are the main causes of sudden death. Arrhythmias are typically caused by mutations in specific genes, damage in the cardiac tissue, or due to some chemical exposure. Arrhythmias caused due to mutation is called inherited arrhythmia. Induced arrhythmias are caused due to tissue damage or chemical exposure. Mutations in genes that encode ion channels of the cardiac cells usually result in (dysfunction) improper functioning of the channel. Improper functioning of the ion channel may lead to major changes in the action potential (AP) of the cardiac cells. This further leads to distorted electrical activity of the heart. Distorted electrical activity will affect the ECG that results in arrhythmia. KCNQ1 P535T mutation is one such gene mutation that encodes the potassium ion channel (KV7.1) of the cardiac ventricular tissue. Its clinical significance is not known. This study aims to perform a simulation study on P535T mutation in the KCNQ1 gene that encodes the potassium ion channel KV7.1 in the ventricular tissue grid. The effect of P535T mutation on transmural tissue grids for three genotypes (wild type, heterozygous, and homozygous) of cells are studied and the generated pseudo-ECGs are compared. Results show the delayed repolarization in the cells of ventricular tissue grid. Slower propagation of action potential in the transmural tissue grid is observed in the mutated (heterozygous and homozygous) genotypes. Longer QT interval is also observed in the pseudo-ECG of heterozygous and homozygous genotype tissue grids. From the pseudo-ECGs, it is observed that KCNQ1 P535T mutation leads to Long QT Syndrome (LQTS) which may result in life-threatening arrhythmias, such as Torsade de Pointes (TdP), Jervell and Lange-Nielsen syndrome (JLNS), and Romano-Ward syndrome (RWS).

Continuous Bayesian variant interpretation accounts for incomplete penetrance among Mendelian cardiac channelopathies.

PURPOSE: The congenital Long QT Syndrome (LQTS) and Brugada Syndrome (BrS) are Mendelian autosomal dominant diseases that frequently precipitate fatal cardiac arrhythmias. Incomplete penetrance is a barrier to clinical management of heterozygotes harboring variants in the major implicated disease genes KCNQ1, KCNH2, and SCN5A. We apply and evaluate a Bayesian penetrance estimation strategy that accounts for this phenomenon. METHODS: We generated Bayesian penetrance models for KCNQ1-LQT1 and SCN5A-LQT3 using variant-specific features and clinical data from the literature, international arrhythmia genetic centers, and population controls. We analyzed the distribution of posterior penetrance estimates across 4 genotype-phenotype relationships and compared continuous estimates with ClinVar annotations. Posterior estimates were mapped onto protein structure. RESULTS: Bayesian penetrance estimates of KCNQ1-LQT1 and SCN5A-LQT3 are empirically equivalent to 10 and 5 clinically phenotype heterozygotes, respectively. Posterior penetrance estimates were bimodal for KCNQ1-LQT1 and KCNH2-LQT2, with a higher fraction of missense variants with high penetrance among KCNQ1 variants. There was a wide distribution of variant penetrance estimates among identical ClinVar categories. Structural mapping revealed heterogeneity among "hot spot" regions and featured high penetrance estimates for KCNQ1 variants in contact with calmodulin and the S6 domain. CONCLUSIONS: Bayesian penetrance estimates provide a continuous framework for variant interpretation.

Predicting the functional impact of KCNQ1 variants with artificial neural networks.

Recent advances in experimental and computational protein structure determination have provided access to high-quality structures for most human proteins and mutants thereof. However, linking changes in structure in protein mutants to functional impact remains an active area of method development. If successful, such methods can ultimately assist physicians in taking appropriate treatment decisions. This work presents three artificial neural network (ANN)-based predictive models that classify four key functional parameters of KCNQ1 variants as normal or dysfunctional using PSSM-based evolutionary and/or biophysical descriptors. Recent advances in predicting protein structure and variant properties with artificial intelligence (AI) rely heavily on the availability of evolutionary features and thus fail to directly assess the biophysical underpinnings of a change in structure and/or function. The central goal of this work was to develop an ANN model based on structure and physiochemical properties of KCNQ1 potassium channels that performs comparably or better than algorithms using only on PSSM-based evolutionary features. These biophysical features highlight the structure-function relationships that govern protein stability, function, and regulation. The input sensitivity algorithm incorporates the roles of hydrophobicity, polarizability, and functional densities on key functional parameters of the KCNQ1 channel. Inclusion of the biophysical features outperforms exclusive use of PSSM-based evolutionary features in predicting activation voltage dependence and deactivation time. As AI is increasingly applied to problems in biology, biophysical understanding will be critical with respect to 'explainable AI', i.e., understanding the relation of sequence, structure, and function of proteins. Our model is available at www.kcnq1predict.org.

Enhancing the interpretation of genetic observations in KCNQ1 in unselected populations: relevance to secondary findings.

AIMS: Rare variants in the KCNQ1 gene are found in the healthy population to a much greater extent than the prevalence of Long QT Syndrome type 1 (LQTS1). This observation creates challenges in the interpretation of KCNQ1 rare variants that may be identified as secondary findings in whole exome sequencing.This study sought to identify missense variants within sub-domains of the KCNQ1-encoded Kv7.1 potassium channel that would be highly predictive of disease in the context of secondary findings. METHODS AND RESULTS: We established a set of KCNQ1 variants reported in over 3700 patients with diagnosed or suspected LQTS sent for clinical genetic testing and compared the domain-specific location of identified variants to those observed in an unselected population of 140 000 individuals. We identified three regions that showed a significant enrichment of KCNQ1 variants associated with LQTS at an odds ratio (OR) >2: the pore region, and the adjacent 5th (S5) and 6th (S6) transmembrane (TM) regions. An additional segment within the carboxyl terminus of Kv7.1, conserved region 2 (CR2), also showed an increased OR of disease association. Furthermore, the TM spanning S5-Pore-S6 region correlated with a significant increase in cardiac events. CONCLUSION: Rare missense variants with a clear phenotype of LQTS have a high likelihood to be present within the pore and adjacent TM segments (S5-Pore-S6) and a greater tendency to be present within CR2. This data will enhance interpretation of secondary findings within the KCNQ1 gene. Further, our data support a more severe phenotype in LQTS patients with variants within the S5-Pore-S6 region.

Gene- and variant-specific efficacy of serum/glucocorticoid-regulated kinase 1 inhibition in long QT syndrome types 1 and 2.

AIMS: Current long QT syndrome (LQTS) therapy, largely based on beta-blockade, does not prevent arrhythmias in all patients; therefore, novel therapies are warranted. Pharmacological inhibition of the serum/glucocorticoid-regulated kinase 1 (SGK1-Inh) has been shown to shorten action potential duration (APD) in LQTS type 3. We aimed to investigate whether SGK1-Inh could similarly shorten APD in LQTS types 1 and 2. METHODS AND RESULTS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and hiPSC-cardiac cell sheets (CCS) were obtained from LQT1 and LQT2 patients; CMs were isolated from transgenic LQT1, LQT2, and wild-type (WT) rabbits. Serum/glucocorticoid-regulated kinase 1 inhibition effects (300 nM-10 µM) on field potential durations (FPD) were investigated in hiPSC-CMs with multielectrode arrays; optical mapping was performed in LQT2 CCS. Whole-cell and perforated patch clamp recordings were performed in isolated LQT1, LQT2, and WT rabbit CMs to investigate SGK1-Inh (3 µM) effects on APD. In all LQT2 models across different species (hiPSC-CMs, hiPSC-CCS, and rabbit CMs) and independent of the disease-causing variant (KCNH2-p.A561V/p.A614V/p.G628S/IVS9-28A/G), SGK1-Inh dose-dependently shortened FPD/APD at 0.3-10 µM (by 20-32%/25-30%/44-45%). Importantly, in LQT2 rabbit CMs, 3 µM SGK1-Inh normalized APD to its WT value. A significant FPD shortening was observed in KCNQ1-p.R594Q hiPSC-CMs at 1/3/10 µM (by 19/26/35%) and in KCNQ1-p.A341V hiPSC-CMs at 10 µM (by 29%). No SGK1-Inh-induced FPD/APD shortening effect was observed in LQT1 KCNQ1-p.A341V hiPSC-CMs or KCNQ1-p.Y315S rabbit CMs at 0.3-3 µM. CONCLUSION: A robust SGK1-Inh-induced APD shortening was observed across different LQT2 models, species, and genetic variants but less consistently in LQT1 models. This suggests a genotype- and variant-specific beneficial effect of this novel therapeutic approach in LQTS.