Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant genetic... more Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant genetic disorder of adrenergically-mediated polymorphic ventricular arrhythmias. Traditionally, it has been characterized by absence of structural heart disease. Recent case reports have linked CPVT due to exon 3 deletions in the ryanodine receptor (RYR2) gene to left ventricular noncompaction (LVNC). Hypothesis: We hypothesized that structural heart disease would be evident on cardiac MRI in patients with clinically confirmed CPVT, despite normal echocardiograms. Methods: We performed a retrospective observational analysis of families with CPVT, followed by the Stanford Inherited Arrhythmia Clinic, who had normal echocardiograms. Medical records, cardiac MRIs ordered for clinical indications, and genetic testing results were reviewed. Results: Of 13 patients identified with CPVT, 10 had bidirectional or polymorphic ventricular ectopy on treadmill testing and 69% (9 patients) had suffered an aborted cardiac arrest. G...
At sites of vascular injury, thrombin interacts with multiple procoagulant substrates, to mediate... more At sites of vascular injury, thrombin interacts with multiple procoagulant substrates, to mediate both fibrin clotting and platelet aggregation. But upon binding to thrombomodulin on the vascular endothelium, thrombin instead activates protein C, thereby functioning as an anticoagulant and attenuating clot formation. Upon infusion in vivo, both the procoagulant and anticoagulant effects of thrombin were observed. Preliminary studies indicating that thrombin's protein C activating and fibrinogen clotting activities could be dissociated by mutagenesis suggested to us that a thrombin variant that lacked procoagulant activity while retaining anticoagulant function might be an attractive antithrombotic agent. Using protein engineering, we introduced a single substitution, E229A, that substantially shifted thrombin's specificity in favour of the anticoagulant substrate, protein C. In monkeys, this modified thrombin functioned as an endogenous protein C activator demonstrating dose...
Background: ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linki... more Background: ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linking ACTN2 mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel ACTN2 mutations to reveal insights into the physiological function of ACTN2. Methods: Patients harboring ACTN2 protein-truncating variants were identified using a custom mutation pipeline. In patient-derived iPSC-cardiomyocytes, we investigated transcriptional profiles using RNA sequencing, contractile properties using video-based edge detection, and cellular hypertrophy using immunohistochemistry. Structural changes were analyzed through electron microscopy. For mechanistic studies, we used coimmunoprecipitation for ACTN2, followed by mass-spectrometry to investigate protein-protein interaction, and protein tagging followed by confocal microscopy to investigate introduction of truncated ACTN2 into the sarcomeres. Results: Patient-derived iPSC-cardiomyocytes were hypertrophic, displ...
Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant genetic... more Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant genetic disorder of adrenergically-mediated polymorphic ventricular arrhythmias. Traditionally, it has been characterized by absence of structural heart disease. Recent case reports have linked CPVT due to exon 3 deletions in the ryanodine receptor (RYR2) gene to left ventricular noncompaction (LVNC). Hypothesis: We hypothesized that structural heart disease would be evident on cardiac MRI in patients with clinically confirmed CPVT, despite normal echocardiograms. Methods: We performed a retrospective observational analysis of families with CPVT, followed by the Stanford Inherited Arrhythmia Clinic, who had normal echocardiograms. Medical records, cardiac MRIs ordered for clinical indications, and genetic testing results were reviewed. Results: Of 13 patients identified with CPVT, 10 had bidirectional or polymorphic ventricular ectopy on treadmill testing and 69% (9 patients) had suffered an ab...
Introduction: Perinatal LQTS represents a severe form of long-QT syndrome with poor outcomes and ... more Introduction: Perinatal LQTS represents a severe form of long-QT syndrome with poor outcomes and early genotype-specific treatment is limited by the 2 month turnaround time of standard panel genetic testing. Hypothesis: We aimed to provide a molecular diagnosis within a clinically actionable timeframe. Methods: We performed rapid CLIA-certified whole genome sequencing on two infants with perinatal long-QT syndrome delivering a molecular diagnosis at 10-days of life. Whole cell patch clamping and single cell genotyping were also performed. Results: In Case #1 we discovered a previously characterized variant in KCNH2 which was paternally inherited, however whole genome sequencing provided an unbiased assessment of the entire catalog of human genes revealing a second maternally inherited modifier variant in RNF207. In Case #2 we discovered a novel mutation leucine replacing valine (V1762L) at residue 1762 in the SNC5A sodium channel. Whole-cell patch clamping experiments show the V1762...
Alpha-actinin 2 (ACTN2) anchors actin within cardiac sarcomeres. The mechanisms linkingACTN2mutat... more Alpha-actinin 2 (ACTN2) anchors actin within cardiac sarcomeres. The mechanisms linkingACTN2mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novelACTN2mutations to reveal insights into the physiological function of ACTN2. Patient-derived iPSC-cardiomyocytes harboring ACTN2 protein-truncating variants were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca2+-signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc structure. In homozygous stop-gain cells, affinity-purification mass spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 and GJA1, two sarcolemma-associated proteins, that may lead to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene...
Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant genetic... more Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant genetic disorder of adrenergically-mediated polymorphic ventricular arrhythmias. Traditionally, it has been characterized by absence of structural heart disease. Recent case reports have linked CPVT due to exon 3 deletions in the ryanodine receptor (RYR2) gene to left ventricular noncompaction (LVNC). Hypothesis: We hypothesized that structural heart disease would be evident on cardiac MRI in patients with clinically confirmed CPVT, despite normal echocardiograms. Methods: We performed a retrospective observational analysis of families with CPVT, followed by the Stanford Inherited Arrhythmia Clinic, who had normal echocardiograms. Medical records, cardiac MRIs ordered for clinical indications, and genetic testing results were reviewed. Results: Of 13 patients identified with CPVT, 10 had bidirectional or polymorphic ventricular ectopy on treadmill testing and 69% (9 patients) had suffered an aborted cardiac arrest. G...
At sites of vascular injury, thrombin interacts with multiple procoagulant substrates, to mediate... more At sites of vascular injury, thrombin interacts with multiple procoagulant substrates, to mediate both fibrin clotting and platelet aggregation. But upon binding to thrombomodulin on the vascular endothelium, thrombin instead activates protein C, thereby functioning as an anticoagulant and attenuating clot formation. Upon infusion in vivo, both the procoagulant and anticoagulant effects of thrombin were observed. Preliminary studies indicating that thrombin's protein C activating and fibrinogen clotting activities could be dissociated by mutagenesis suggested to us that a thrombin variant that lacked procoagulant activity while retaining anticoagulant function might be an attractive antithrombotic agent. Using protein engineering, we introduced a single substitution, E229A, that substantially shifted thrombin's specificity in favour of the anticoagulant substrate, protein C. In monkeys, this modified thrombin functioned as an endogenous protein C activator demonstrating dose...
Background: ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linki... more Background: ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linking ACTN2 mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel ACTN2 mutations to reveal insights into the physiological function of ACTN2. Methods: Patients harboring ACTN2 protein-truncating variants were identified using a custom mutation pipeline. In patient-derived iPSC-cardiomyocytes, we investigated transcriptional profiles using RNA sequencing, contractile properties using video-based edge detection, and cellular hypertrophy using immunohistochemistry. Structural changes were analyzed through electron microscopy. For mechanistic studies, we used coimmunoprecipitation for ACTN2, followed by mass-spectrometry to investigate protein-protein interaction, and protein tagging followed by confocal microscopy to investigate introduction of truncated ACTN2 into the sarcomeres. Results: Patient-derived iPSC-cardiomyocytes were hypertrophic, displ...
Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant genetic... more Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant genetic disorder of adrenergically-mediated polymorphic ventricular arrhythmias. Traditionally, it has been characterized by absence of structural heart disease. Recent case reports have linked CPVT due to exon 3 deletions in the ryanodine receptor (RYR2) gene to left ventricular noncompaction (LVNC). Hypothesis: We hypothesized that structural heart disease would be evident on cardiac MRI in patients with clinically confirmed CPVT, despite normal echocardiograms. Methods: We performed a retrospective observational analysis of families with CPVT, followed by the Stanford Inherited Arrhythmia Clinic, who had normal echocardiograms. Medical records, cardiac MRIs ordered for clinical indications, and genetic testing results were reviewed. Results: Of 13 patients identified with CPVT, 10 had bidirectional or polymorphic ventricular ectopy on treadmill testing and 69% (9 patients) had suffered an ab...
Introduction: Perinatal LQTS represents a severe form of long-QT syndrome with poor outcomes and ... more Introduction: Perinatal LQTS represents a severe form of long-QT syndrome with poor outcomes and early genotype-specific treatment is limited by the 2 month turnaround time of standard panel genetic testing. Hypothesis: We aimed to provide a molecular diagnosis within a clinically actionable timeframe. Methods: We performed rapid CLIA-certified whole genome sequencing on two infants with perinatal long-QT syndrome delivering a molecular diagnosis at 10-days of life. Whole cell patch clamping and single cell genotyping were also performed. Results: In Case #1 we discovered a previously characterized variant in KCNH2 which was paternally inherited, however whole genome sequencing provided an unbiased assessment of the entire catalog of human genes revealing a second maternally inherited modifier variant in RNF207. In Case #2 we discovered a novel mutation leucine replacing valine (V1762L) at residue 1762 in the SNC5A sodium channel. Whole-cell patch clamping experiments show the V1762...
Alpha-actinin 2 (ACTN2) anchors actin within cardiac sarcomeres. The mechanisms linkingACTN2mutat... more Alpha-actinin 2 (ACTN2) anchors actin within cardiac sarcomeres. The mechanisms linkingACTN2mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novelACTN2mutations to reveal insights into the physiological function of ACTN2. Patient-derived iPSC-cardiomyocytes harboring ACTN2 protein-truncating variants were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca2+-signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc structure. In homozygous stop-gain cells, affinity-purification mass spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 and GJA1, two sarcolemma-associated proteins, that may lead to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene...
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