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Papers by Kyla Dunn

Genetics in Medicine, Mar 1, 2018

Purpose: To describe the frequency and nature of differences in variant classifications between c... more Purpose: To describe the frequency and nature of differences in variant classifications between clinicians and genetic testing laboratories. Methods: Retrospective review of variants identified through genetic testing ordered in routine clinical care by clinicians in the Stanford Center for Inherited Cardiovascular Disease. We compared classifications made by clinicians, the testing laboratory, and other laboratories in ClinVar. Results: Of 688 laboratory classifications, 124 (18%) differed from the clinicians' classifications. Most differences in classification would probably affect clinical care of the patient and/or family (83%, 103/124). The frequency of discordant classifications differed depending on the testing laboratory (P o 0.0001) and the testing laboratory's classification (P o 0.00001). For the majority (82/124, 66%) of discordant classifications, clinicians were more conservative (less likely to classify a variant pathogenic or likely pathogenic). The clinicians' classification was discordant with one or more submitter in ClinVar in 49.1% (28/57) of cases, while the testing laboratory's classification was discordant with a ClinVar submitter in 82.5% of cases (47/57, P = 0.0002). Conclusion: The clinical team disagreed with the laboratory's classification at a rate similar to that of reported disagreements between laboratories. Most of this discordance was clinically significant, with clinicians tending to be more conservative than laboratories in their classifications.

Heart Rhythm, Oct 1, 2014

Background-The advent of clinical next generation sequencing is rapidly changing the landscape of... more Background-The advent of clinical next generation sequencing is rapidly changing the landscape of rare disease medicine. Molecular diagnosis of long QT syndrome (LQTS) can impact clinical management, including risk stratification and selection of pharmacotherapy based on the type of ion channel affected, but results from current gene panel testing requires 4 to 16 weeks before return to clinicians. Objective-A term female infant presented with 2:1 atrioventricular block and ventricular arrhythmias consistent with perinatal LQTS, requiring aggressive treatment including epicardial pacemaker, and cardioverter-defibrillator implantation and sympathectomy on day of life two. We sought to provide a rapid molecular diagnosis for optimization of treatment strategies. Methods-We performed CLIA-certified rapid whole genome sequencing (WGS) with a speedoptimized bioinformatics platform to achieve molecular diagnosis at 10 days of life. Results-We detected a known pathogenic variant in KCNH2 that was demonstrated to be paternally inherited by followup genotyping. The unbiased assessment of the entire catalog of human genes provided by whole genome sequencing revealed a maternally inherited variant of unknown significance in a novel gene. Conclusions-Rapid clinical WGS provides faster and more comprehensive diagnostic information by 10 days of life than standard gene-panel testing. In selected clinical scenarios such as perinatal LQTS, rapid WGS may be able to provide more timely and clinically actionable information than a standard commercial test.

Circulation, Nov 1, 2019

Atrial fibrillation (AF) is the most common sustained arrhythmia, affecting approximately 34 mill... more Atrial fibrillation (AF) is the most common sustained arrhythmia, affecting approximately 34 million worldwide. The pathophysiology of AF remains incompletely understood but is clearly complex with multiple underlying genetic, physiologic and environmental factors. Very early-onset AF (vEAF) (defined here as onset <45 years and without significant comorbidities), while rare (only ~0.5-3% of AF cases), is highly heritable, with a greater prevalence of rare variants in genes previously associated with AF. Patients with vEAF, therefore, represent an ideal population for discovering novel genes involved in the underlying genetic basis of AF. Notably, the Framingham study showed that patients with AF without comorbidities have a threefold higher risk for heart failure. Conversely, several forms of inherited cardiomyopathy have been strongly associated with AF suggestive of a shared etiology.

Analytical Biochemistry, Feb 1, 1996

with chelating agents (1). The use of IMAC for protein Protein purification has been made signifi... more with chelating agents (1). The use of IMAC for protein Protein purification has been made significantly easpurification has expanded recently due to the developier by the use of affinity tags that can be genetically ment of improved chelating agents that permit highengineered at either the amino-or carboxyl-terminus affinity coordination of metal ions by both the immobiof recombinant proteins. One of the most widely used lized chelation agent and the protein (2). In addition, tags is six consecutive histidine residues or 6His tag. genetic engineering has provided the means to intro-These residues bind with high affinity to metal ions duce high-affinity metal binding sites into any recombiimmobilized on chelating resins even in the presence nant protein. One of the most versatile purification of denaturing agents and can be mildly eluted with methods utilizes affinity tags containing six consecuimidazole. We report the methodology for the immobitive histidine residues (6His) engineered into the lization of six histidine-containing proteins onto amino-or carboxyl-terminus of recombinant proteins microtiter plates. A derivative of nitrilotriacetic and Ni 2/ ions immobilized on commercially available acid (NTA) was prepared. This derivative, N,Nresins. The resins are coupled with an active derivative bis[carboxymethyl]lysine (BCML), was easily coupled of the chelating moiety nitrilotriacetic acid (NTA, also to a maleic anhydride-activated polystyrene microticalled N,N-bis[carboxymethyl]glycine). ter plate. The plate was then charged with Ni 2/ for the Resins coupled with NTA derivatives are most suitcapture of the 6His-tagged proteins. Using two differable for IMAC using metal ions with coordination nument recombinant proteins with the 6His tag at either bers of six such as Ni 2/ because the quadridentate NTA the Nor C-terminus, we demonstrated that the bindoccupies four coordination positions, leaving two posiing to the Ni 2/-NTA plate was specific for six histidinetions available for tight but reversible interactions with containing proteins. Proteins lacking the 6His tags did proteins (Fig. 1). Thus, NTA represents an optimal not bind to the plate. The plate was used in a modified enzyme-linked immunoabsorbent assay format to compromise between the tridentate iminodiacetic acid quantitate protein concentrations and determine the (IDA) derivatives which bind Ni 2/ weakly through affinity of protein-ligand interactions. The technology three coordination positions and the pentadentate can also be extended to include high-throughput tris(carboxymethyl) ethylenediamine which occupies screening assays for antagonists of protein-protein infive coordination positions on Ni 2/ , leaving only one teractions. ᭧ 1996 Academic Press, Inc. free for protein binding (2). The high affinity of Ni 2/-NTA resins for proteins with 6His tags (K D É 10 013) facilitates their purification from complex mixtures be-The affinity of proteins containing exposed histidine cause nonspecifically bound proteins can be removed by and cysteine side chains for metal ions has long been washing with high salt (up to 1 M), nonionic detergents exploited to purify proteins by immobilized metal ion-(Triton X-100 or Tween 20 up to 1%), organic solvents affinity chromatography (IMAC) 2 on resins coupled (ethanol or glycerol up to 30%), and reducing agents

Archives of Cardiovascular Diseases Supplements, Sep 1, 2018

Objective The aim of the study was to compare the short-term outcome after patent ductus arterios... more Objective The aim of the study was to compare the short-term outcome after patent ductus arteriosus (PDA) percutaneous closure versus surgical ligation in extremely preterm infants. Methods We retrospectively performed a mono-centric matched case-control study. Cases were preterm infants born before 28 weeks of gestation with PDA percutaneous closure performed in Nantes University hospital. Each case was matched, for gestational age and post-natal age at intervention, with 2 controls who had PDA surgical ligation. The main criterion was the rate of extubated patients 2 days after the procedure. Results We included in the analysis 14 percutaneous closures and 28 surgical ligations performed between January 2006 and December 2017. Two days after the procedure, 7.1% patients were off mechanical ventilation in the surgical group versus 50% in the percutaneous group (P = 0.001). Mean duration of mechanical ventilation was 5.4 days (± 5.0) in the percutaneous group versus 12.5 days (± 11.1) in the surgical group (P = 0.009). No complication of percutaneous closure was noticed. Conclusion Percutaneous closure of PDA allowed a faster pulmonary recovery and mechanical ventilation cessation in preterm infant born before 28 weeks of gestation with the same efficacy and without any related complication than surgery. Disclosure of interest The authors have not supplied their declaration of competing interest.

Journal of Biological Chemistry, Jul 1, 1995

Journal of Biological Chemistry, Aug 1, 1995

Circulation, Nov 11, 2016

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...

Circulation, Nov 20, 2012

Biochemistry, 1996

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52... more Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in Vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in ViVo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in ViVo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

Circulation: Cardiovascular Genetics, 2015

Circulation, Nov 20, 2012

Nature, Jan 23, 1995

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...

Circulation: Genomic and Precision Medicine

Biochemistry, 1996

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52... more Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in Vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in ViVo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in ViVo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

New England Journal of Medicine

Circulation: Genomic and Precision Medicine

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...

Genetics in Medicine, Mar 1, 2018

Purpose: To describe the frequency and nature of differences in variant classifications between c... more Purpose: To describe the frequency and nature of differences in variant classifications between clinicians and genetic testing laboratories. Methods: Retrospective review of variants identified through genetic testing ordered in routine clinical care by clinicians in the Stanford Center for Inherited Cardiovascular Disease. We compared classifications made by clinicians, the testing laboratory, and other laboratories in ClinVar. Results: Of 688 laboratory classifications, 124 (18%) differed from the clinicians' classifications. Most differences in classification would probably affect clinical care of the patient and/or family (83%, 103/124). The frequency of discordant classifications differed depending on the testing laboratory (P o 0.0001) and the testing laboratory's classification (P o 0.00001). For the majority (82/124, 66%) of discordant classifications, clinicians were more conservative (less likely to classify a variant pathogenic or likely pathogenic). The clinicians' classification was discordant with one or more submitter in ClinVar in 49.1% (28/57) of cases, while the testing laboratory's classification was discordant with a ClinVar submitter in 82.5% of cases (47/57, P = 0.0002). Conclusion: The clinical team disagreed with the laboratory's classification at a rate similar to that of reported disagreements between laboratories. Most of this discordance was clinically significant, with clinicians tending to be more conservative than laboratories in their classifications.

Heart Rhythm, Oct 1, 2014

Background-The advent of clinical next generation sequencing is rapidly changing the landscape of... more Background-The advent of clinical next generation sequencing is rapidly changing the landscape of rare disease medicine. Molecular diagnosis of long QT syndrome (LQTS) can impact clinical management, including risk stratification and selection of pharmacotherapy based on the type of ion channel affected, but results from current gene panel testing requires 4 to 16 weeks before return to clinicians. Objective-A term female infant presented with 2:1 atrioventricular block and ventricular arrhythmias consistent with perinatal LQTS, requiring aggressive treatment including epicardial pacemaker, and cardioverter-defibrillator implantation and sympathectomy on day of life two. We sought to provide a rapid molecular diagnosis for optimization of treatment strategies. Methods-We performed CLIA-certified rapid whole genome sequencing (WGS) with a speedoptimized bioinformatics platform to achieve molecular diagnosis at 10 days of life. Results-We detected a known pathogenic variant in KCNH2 that was demonstrated to be paternally inherited by followup genotyping. The unbiased assessment of the entire catalog of human genes provided by whole genome sequencing revealed a maternally inherited variant of unknown significance in a novel gene. Conclusions-Rapid clinical WGS provides faster and more comprehensive diagnostic information by 10 days of life than standard gene-panel testing. In selected clinical scenarios such as perinatal LQTS, rapid WGS may be able to provide more timely and clinically actionable information than a standard commercial test.

Circulation, Nov 1, 2019

Atrial fibrillation (AF) is the most common sustained arrhythmia, affecting approximately 34 mill... more Atrial fibrillation (AF) is the most common sustained arrhythmia, affecting approximately 34 million worldwide. The pathophysiology of AF remains incompletely understood but is clearly complex with multiple underlying genetic, physiologic and environmental factors. Very early-onset AF (vEAF) (defined here as onset <45 years and without significant comorbidities), while rare (only ~0.5-3% of AF cases), is highly heritable, with a greater prevalence of rare variants in genes previously associated with AF. Patients with vEAF, therefore, represent an ideal population for discovering novel genes involved in the underlying genetic basis of AF. Notably, the Framingham study showed that patients with AF without comorbidities have a threefold higher risk for heart failure. Conversely, several forms of inherited cardiomyopathy have been strongly associated with AF suggestive of a shared etiology.

Analytical Biochemistry, Feb 1, 1996

with chelating agents (1). The use of IMAC for protein Protein purification has been made signifi... more with chelating agents (1). The use of IMAC for protein Protein purification has been made significantly easpurification has expanded recently due to the developier by the use of affinity tags that can be genetically ment of improved chelating agents that permit highengineered at either the amino-or carboxyl-terminus affinity coordination of metal ions by both the immobiof recombinant proteins. One of the most widely used lized chelation agent and the protein (2). In addition, tags is six consecutive histidine residues or 6His tag. genetic engineering has provided the means to intro-These residues bind with high affinity to metal ions duce high-affinity metal binding sites into any recombiimmobilized on chelating resins even in the presence nant protein. One of the most versatile purification of denaturing agents and can be mildly eluted with methods utilizes affinity tags containing six consecuimidazole. We report the methodology for the immobitive histidine residues (6His) engineered into the lization of six histidine-containing proteins onto amino-or carboxyl-terminus of recombinant proteins microtiter plates. A derivative of nitrilotriacetic and Ni 2/ ions immobilized on commercially available acid (NTA) was prepared. This derivative, N,Nresins. The resins are coupled with an active derivative bis[carboxymethyl]lysine (BCML), was easily coupled of the chelating moiety nitrilotriacetic acid (NTA, also to a maleic anhydride-activated polystyrene microticalled N,N-bis[carboxymethyl]glycine). ter plate. The plate was then charged with Ni 2/ for the Resins coupled with NTA derivatives are most suitcapture of the 6His-tagged proteins. Using two differable for IMAC using metal ions with coordination nument recombinant proteins with the 6His tag at either bers of six such as Ni 2/ because the quadridentate NTA the Nor C-terminus, we demonstrated that the bindoccupies four coordination positions, leaving two posiing to the Ni 2/-NTA plate was specific for six histidinetions available for tight but reversible interactions with containing proteins. Proteins lacking the 6His tags did proteins (Fig. 1). Thus, NTA represents an optimal not bind to the plate. The plate was used in a modified enzyme-linked immunoabsorbent assay format to compromise between the tridentate iminodiacetic acid quantitate protein concentrations and determine the (IDA) derivatives which bind Ni 2/ weakly through affinity of protein-ligand interactions. The technology three coordination positions and the pentadentate can also be extended to include high-throughput tris(carboxymethyl) ethylenediamine which occupies screening assays for antagonists of protein-protein infive coordination positions on Ni 2/ , leaving only one teractions. ᭧ 1996 Academic Press, Inc. free for protein binding (2). The high affinity of Ni 2/-NTA resins for proteins with 6His tags (K D É 10 013) facilitates their purification from complex mixtures be-The affinity of proteins containing exposed histidine cause nonspecifically bound proteins can be removed by and cysteine side chains for metal ions has long been washing with high salt (up to 1 M), nonionic detergents exploited to purify proteins by immobilized metal ion-(Triton X-100 or Tween 20 up to 1%), organic solvents affinity chromatography (IMAC) 2 on resins coupled (ethanol or glycerol up to 30%), and reducing agents

Archives of Cardiovascular Diseases Supplements, Sep 1, 2018

Objective The aim of the study was to compare the short-term outcome after patent ductus arterios... more Objective The aim of the study was to compare the short-term outcome after patent ductus arteriosus (PDA) percutaneous closure versus surgical ligation in extremely preterm infants. Methods We retrospectively performed a mono-centric matched case-control study. Cases were preterm infants born before 28 weeks of gestation with PDA percutaneous closure performed in Nantes University hospital. Each case was matched, for gestational age and post-natal age at intervention, with 2 controls who had PDA surgical ligation. The main criterion was the rate of extubated patients 2 days after the procedure. Results We included in the analysis 14 percutaneous closures and 28 surgical ligations performed between January 2006 and December 2017. Two days after the procedure, 7.1% patients were off mechanical ventilation in the surgical group versus 50% in the percutaneous group (P = 0.001). Mean duration of mechanical ventilation was 5.4 days (± 5.0) in the percutaneous group versus 12.5 days (± 11.1) in the surgical group (P = 0.009). No complication of percutaneous closure was noticed. Conclusion Percutaneous closure of PDA allowed a faster pulmonary recovery and mechanical ventilation cessation in preterm infant born before 28 weeks of gestation with the same efficacy and without any related complication than surgery. Disclosure of interest The authors have not supplied their declaration of competing interest.

Journal of Biological Chemistry, Jul 1, 1995

Journal of Biological Chemistry, Aug 1, 1995

Circulation, Nov 11, 2016

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...

Circulation, Nov 20, 2012

Biochemistry, 1996

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52... more Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in Vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in ViVo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in ViVo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

Circulation: Cardiovascular Genetics, 2015

Circulation, Nov 20, 2012

Nature, Jan 23, 1995

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...

Circulation: Genomic and Precision Medicine

Biochemistry, 1996

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52... more Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in Vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in ViVo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in ViVo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

New England Journal of Medicine

Circulation: Genomic and Precision Medicine

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...