Background: The D1275N SCN5A mutation is associated with dilated cardiomyopathy (DCM) in humans. ... more Background: The D1275N SCN5A mutation is associated with dilated cardiomyopathy (DCM) in humans. We have previously reported that in 12 week old mice carrying humanized SCN5A alleles, DCM is evident by echo in those homozygous for D1275N (DN/DN) compared to those homozygous for the wild-type allele (H/H), with striking conduction slowing and disorganization by ECG and mapping. Here we compared cellular structure, expression of sodium channel partners, and calcium handling in young (3-week) DN/DN versus H/H mice to identify early changes and thereby illuminate mechanisms for DCM in this model. Methods and Results: By 3 weeks, QRS duration was markedly prolonged and disorganized in DN/DN mice (HH, n=11, 9.8 ± 0.2; DN/DN, n=9, 22.3 ± 2.2 P ratio ) and SR calcium stores (H/H, 0.85±0.08; DN/DN, 0.92±0.07, F ratio ) were not statistically significantly different in DN/DN compared to H/H, arguing against aberrant calcium handling as a primary driver of DCM in these mice. Similarly, there w...
IntroductionUp to 30% of patients with Brugada Syndrome (BrS) carry loss-of-function (LoF) varian... more IntroductionUp to 30% of patients with Brugada Syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel geneSCN5A. Recent studies have suggested that theSCN5Aprotein product NaV1.5 can form dimers and exert dominant negative effects.MethodsWe identified 35 LoF variants (<10% peak current compared to wild type (WT)) and 15 partial LoF variants (10-50% peak current compared to WT) that we assessed for dominant negative behavior.SCN5Avariants were studied in HEK293T cells alone or in heterozygous co-expression with WTSCN5Ausing automated patch clamp. To assess clinical risk, we compared the prevalence of dominant negative vs. putative haploinsufficient (frameshift/splice site) variants in a BrS case consortium and the gnomAD population database.ResultsIn heterozygous expression with WT, 32/35 LoF variants and 6/15 partial LoF showed reduction to <75% of WT-alone peak INa, demonstrating a dominant negative effect. Carriers of dominant negative LoF missens...
RationalePartial or complete loss of function variants inSCN5Aare the most common genetic cause o... more RationalePartial or complete loss of function variants inSCN5Aare the most common genetic cause of the arrhythmia disorder Brugada Syndrome (BrS1). However, the pathogenicity ofSCN5Avariants is often unknown or disputed; 80% of the 1,390SCN5Amissense variants observed in at least one individual to date are variants of uncertain significance (VUS). The designation of VUS is a barrier to the use of sequence data in clinical care.ObjectiveWe selected 83 variants for study: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada Syndrome-associated variants, selected on the basis of their frequency in the general population and in patients with Brugada Syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data.Methods and ResultsTen previously studied variants had functional properties concordant with published manual patch clamp data. All 10 susp...
Hereditary hemochromatosis (HH) is an autosomal recessive disorder of excess iron absorption. The... more Hereditary hemochromatosis (HH) is an autosomal recessive disorder of excess iron absorption. The most common form, HH1, is caused by loss of function variants in HFE. HFE encodes a cell surface protein that binds to the Transferrin Receptor (TfR1), reducing TfR1's affinity for the transferrin/iron complex and thereby limiting cellular iron uptake. Two common missense alleles for HH1 have been identified, HFE C282Y and HFE H63D; H63D is considered to be a less penetrant allele. When we deployed Phenotype Risk Scores (PheRS), a method that aggregates multiple symptoms together in Electronic Health Records (EHRs), we identified HFE E168Q as a novel variant associated with HH. E168Q is on the same haplotype as H63D, and in a crystal structure HFE E168 lies at the interface of the HFE-TfR1 interaction and makes multiple salt bridge connections with TfR1. In in vitro cell surface abundance experiments, the HFE E168Q+H63D double mutation surprisingly increased cell surface abundance o...
Background Genome‐wide association studies have implicated variants in SCN 10A , which encodes Na... more Background Genome‐wide association studies have implicated variants in SCN 10A , which encodes Nav1.8, as modulators of cardiac conduction. Follow‐up work has indicated the SCN 10A sequence includes an intronic enhancer for SCN 5A . Yet the role of the Nav1.8 protein in the myocardium itself is still unclear. To investigate this , we use homozygous knockout mice ( Scn10a −/− ) generated by disruption of exons 4 and 5, leaving the Scn5a enhancer intact. Methods and Results We previously reported that pharmacologic blockade of Nav1.8 in wild‐type animals blunts action potential prolongation by ATX ‐ II at slow drive rates (≤1 Hz). Here we present evidence of the same blunting in Scn10a −/− compared to wild‐type ventricular myocytes, supporting the conclusion that Nav1.8 contributes to late sodium current at slow rates. In contrast to earlier studies, we found no differences in electrocardiographic parameters between genotypes. Low‐dose ATX ‐ II exposure in lightly anesthetized animals...
Background— The D1275N SCN5A mutation has been associated with a range of unusual phenotypes, inc... more Background— The D1275N SCN5A mutation has been associated with a range of unusual phenotypes, including conduction disease and dilated cardiomyopathy, as well as atrial and ventricular tachyarrhythmias. However, when D1275N is studied in heterologous expression systems, most studies show near-normal sodium channel function. Thus, the relationship of the variant to the clinical phenotypes remains uncertain. Methods and Results— We identified D1275N in a patient with atrial flutter, atrial standstill, conduction disease, and sinus node dysfunction. There was no major difference in biophysical properties between wild-type and D1275N channels expressed in Chinese hamster ovary cells or tsA201 cells in the absence or presence of β1 subunits. To determine D1275N function in vivo, the Scn5a locus was modified to knock out the mouse gene, and the full-length wild-type (H) or D1275N (DN) human SCN5A cDNAs were then inserted at the modified locus by recombinase mediated cassette exchange. Mic...
Deficiency of carbamoyl phosphate synthetase I (CPSI) results in hyperammonemia ranging from neon... more Deficiency of carbamoyl phosphate synthetase I (CPSI) results in hyperammonemia ranging from neonatally lethal to environmentally induced adult-onset disease. Over 24 years, analysis of tissue and DNA samples from 205 unrelated individuals diagnosed with CPSI deficiency (CPSID) detected 192 unique CPS1 gene changes, of which 130 are reported here for the first time. Pooled with the already reported mutations, they constitute a total of 222 changes, including 136 missense, 15 nonsense, 50 changes of other types resulting in enzyme truncation, and 21 other changes causing in-frame alterations. Only ~10% of the mutations recur in unrelated families, predominantly affecting CpG dinucleotides, further complicating the diagnosis because of the "private" nature of such mutations. Missense changes are unevenly distributed along the gene, highlighting the existence of CPSI regions having greater functional importance than other regions. We exploit the crystal structure of the CPSI allosteric domain to rationalize the effects of mutations affecting it. Comparative modeling is used to create a structural model for the remainder of the enzyme. Missense changes are found to directly correlate, respectively, with the one-residue evolutionary importance and inversely correlate with solvent accessibility of the mutated residue. This is the first large-scale report of CPS1 mutations spanning a wide variety of molecular defects highlighting important regions in this protein.
Background: The D1275N SCN5A mutation is associated with dilated cardiomyopathy (DCM) in humans. ... more Background: The D1275N SCN5A mutation is associated with dilated cardiomyopathy (DCM) in humans. We have previously reported that in 12 week old mice carrying humanized SCN5A alleles, DCM is evident by echo in those homozygous for D1275N (DN/DN) compared to those homozygous for the wild-type allele (H/H), with striking conduction slowing and disorganization by ECG and mapping. Here we compared cellular structure, expression of sodium channel partners, and calcium handling in young (3-week) DN/DN versus H/H mice to identify early changes and thereby illuminate mechanisms for DCM in this model. Methods and Results: By 3 weeks, QRS duration was markedly prolonged and disorganized in DN/DN mice (HH, n=11, 9.8 ± 0.2; DN/DN, n=9, 22.3 ± 2.2 P ratio ) and SR calcium stores (H/H, 0.85±0.08; DN/DN, 0.92±0.07, F ratio ) were not statistically significantly different in DN/DN compared to H/H, arguing against aberrant calcium handling as a primary driver of DCM in these mice. Similarly, there w...
IntroductionUp to 30% of patients with Brugada Syndrome (BrS) carry loss-of-function (LoF) varian... more IntroductionUp to 30% of patients with Brugada Syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel geneSCN5A. Recent studies have suggested that theSCN5Aprotein product NaV1.5 can form dimers and exert dominant negative effects.MethodsWe identified 35 LoF variants (<10% peak current compared to wild type (WT)) and 15 partial LoF variants (10-50% peak current compared to WT) that we assessed for dominant negative behavior.SCN5Avariants were studied in HEK293T cells alone or in heterozygous co-expression with WTSCN5Ausing automated patch clamp. To assess clinical risk, we compared the prevalence of dominant negative vs. putative haploinsufficient (frameshift/splice site) variants in a BrS case consortium and the gnomAD population database.ResultsIn heterozygous expression with WT, 32/35 LoF variants and 6/15 partial LoF showed reduction to <75% of WT-alone peak INa, demonstrating a dominant negative effect. Carriers of dominant negative LoF missens...
RationalePartial or complete loss of function variants inSCN5Aare the most common genetic cause o... more RationalePartial or complete loss of function variants inSCN5Aare the most common genetic cause of the arrhythmia disorder Brugada Syndrome (BrS1). However, the pathogenicity ofSCN5Avariants is often unknown or disputed; 80% of the 1,390SCN5Amissense variants observed in at least one individual to date are variants of uncertain significance (VUS). The designation of VUS is a barrier to the use of sequence data in clinical care.ObjectiveWe selected 83 variants for study: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada Syndrome-associated variants, selected on the basis of their frequency in the general population and in patients with Brugada Syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data.Methods and ResultsTen previously studied variants had functional properties concordant with published manual patch clamp data. All 10 susp...
Hereditary hemochromatosis (HH) is an autosomal recessive disorder of excess iron absorption. The... more Hereditary hemochromatosis (HH) is an autosomal recessive disorder of excess iron absorption. The most common form, HH1, is caused by loss of function variants in HFE. HFE encodes a cell surface protein that binds to the Transferrin Receptor (TfR1), reducing TfR1's affinity for the transferrin/iron complex and thereby limiting cellular iron uptake. Two common missense alleles for HH1 have been identified, HFE C282Y and HFE H63D; H63D is considered to be a less penetrant allele. When we deployed Phenotype Risk Scores (PheRS), a method that aggregates multiple symptoms together in Electronic Health Records (EHRs), we identified HFE E168Q as a novel variant associated with HH. E168Q is on the same haplotype as H63D, and in a crystal structure HFE E168 lies at the interface of the HFE-TfR1 interaction and makes multiple salt bridge connections with TfR1. In in vitro cell surface abundance experiments, the HFE E168Q+H63D double mutation surprisingly increased cell surface abundance o...
Background Genome‐wide association studies have implicated variants in SCN 10A , which encodes Na... more Background Genome‐wide association studies have implicated variants in SCN 10A , which encodes Nav1.8, as modulators of cardiac conduction. Follow‐up work has indicated the SCN 10A sequence includes an intronic enhancer for SCN 5A . Yet the role of the Nav1.8 protein in the myocardium itself is still unclear. To investigate this , we use homozygous knockout mice ( Scn10a −/− ) generated by disruption of exons 4 and 5, leaving the Scn5a enhancer intact. Methods and Results We previously reported that pharmacologic blockade of Nav1.8 in wild‐type animals blunts action potential prolongation by ATX ‐ II at slow drive rates (≤1 Hz). Here we present evidence of the same blunting in Scn10a −/− compared to wild‐type ventricular myocytes, supporting the conclusion that Nav1.8 contributes to late sodium current at slow rates. In contrast to earlier studies, we found no differences in electrocardiographic parameters between genotypes. Low‐dose ATX ‐ II exposure in lightly anesthetized animals...
Background— The D1275N SCN5A mutation has been associated with a range of unusual phenotypes, inc... more Background— The D1275N SCN5A mutation has been associated with a range of unusual phenotypes, including conduction disease and dilated cardiomyopathy, as well as atrial and ventricular tachyarrhythmias. However, when D1275N is studied in heterologous expression systems, most studies show near-normal sodium channel function. Thus, the relationship of the variant to the clinical phenotypes remains uncertain. Methods and Results— We identified D1275N in a patient with atrial flutter, atrial standstill, conduction disease, and sinus node dysfunction. There was no major difference in biophysical properties between wild-type and D1275N channels expressed in Chinese hamster ovary cells or tsA201 cells in the absence or presence of β1 subunits. To determine D1275N function in vivo, the Scn5a locus was modified to knock out the mouse gene, and the full-length wild-type (H) or D1275N (DN) human SCN5A cDNAs were then inserted at the modified locus by recombinase mediated cassette exchange. Mic...
Deficiency of carbamoyl phosphate synthetase I (CPSI) results in hyperammonemia ranging from neon... more Deficiency of carbamoyl phosphate synthetase I (CPSI) results in hyperammonemia ranging from neonatally lethal to environmentally induced adult-onset disease. Over 24 years, analysis of tissue and DNA samples from 205 unrelated individuals diagnosed with CPSI deficiency (CPSID) detected 192 unique CPS1 gene changes, of which 130 are reported here for the first time. Pooled with the already reported mutations, they constitute a total of 222 changes, including 136 missense, 15 nonsense, 50 changes of other types resulting in enzyme truncation, and 21 other changes causing in-frame alterations. Only ~10% of the mutations recur in unrelated families, predominantly affecting CpG dinucleotides, further complicating the diagnosis because of the "private" nature of such mutations. Missense changes are unevenly distributed along the gene, highlighting the existence of CPSI regions having greater functional importance than other regions. We exploit the crystal structure of the CPSI allosteric domain to rationalize the effects of mutations affecting it. Comparative modeling is used to create a structural model for the remainder of the enzyme. Missense changes are found to directly correlate, respectively, with the one-residue evolutionary importance and inversely correlate with solvent accessibility of the mutated residue. This is the first large-scale report of CPS1 mutations spanning a wide variety of molecular defects highlighting important regions in this protein.
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Papers by Lynn Hall