Valdivia, Carmen R.

Introduction: Approximately 10 – 15% of sudden infant death syndrome (SIDS) may be caused by cardiac channelopathies including Brugada syndrome (BrS). Type 1 BrS (BrS1), due to mutations in the SCN5A-encoded sodium channel, accounts for approximately 20% of BrS. Recently, a novel mutation in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) disrupted trafficking of SCN5A in a multi-generational family with BrS. We hypothesized that mutations in GPD1-L may be responsible for some cases of SIDS. Methods: Using DHPLC and direct DNA sequencing, we performed comprehensive open reading frame/splice site mutational analysis of GPD1-L on genomic DNA extracted from necropsy tissue of 228 anonymous cases of SIDS (86 females, 142 males, average age = 3 + 2 months, range 6 hours to 12 months). Results: Three putative, SIDS-associated GPD1-L missense mutations, E83K, I124V, and R273C, were discovered in a three-month-old white male, a five-week-old white female, and a one-month-old white male, respectively. All mutations occurred in highly conserved residues and were absent in 600 reference alleles. Compared to wild type GPD1-L, GPD1-L mutations co-expressed with SCN5A in heterologous HEK cells produced a 60% reduction in peak sodium current density (p<0.004). Adenovirus-mediated gene transfer of the E83K-GPD1-L mutation into neonatal mouse myocytes markedly attenuated the sodium current as well (p<0.01). These decreases in current density are consistent with sodium channel loss-of-function diseases like BrS. Conclusion: This study is the first to report mutations in GPD1-L as a pathogenic cause for a small subset (~1%) of SIDS via a secondary loss-of-function mechanism whereby perturbations in GPD1-L precipitate a marked decrease in the peak sodium current and a potentially lethal, BrS-like pro-arrhythmic substrate.

Background: Genes encoding sodium channel interacting proteins (ChIPs) have been identified as novel long QT syndrome (LQTS)-susceptibility genes. Approximately 25% of patients with LQTS remain genotype negative. ChIPs comprising the sodium channel macromolecular complex represent attractive candidates for disease susceptibility. Previously, in human jejunal circular smooth muscle, we demonstrated that syntrophin γ2 regulates sodium channel gating by a PDZ-domain interaction involving the C-terminus. In the heart, syntrophin α is more highly expressed than γ2, therefore, syntrophin α was evaluated as a possible LQTS-susceptibility ChIP. Methods: Comprehensive open reading frame/splice mutational analysis of syntrophin α was performed using PCR, DHPLC, and direct DNA sequencing on a cohort of 50 unrelated patients with LQT1–10 negative/phenotype positive LQTS (34 females; average age at diagnosis 26 ± 16 years; average QTc 531 ± 60.4 ms; QTc range 480 –759 ms). Mutations were engineered by site-directed mutagenesis and transiently expressed in HEK293 cells containing the stably-expressed SCN5A- encoded sodium channel α subunit (hNaV1.5). Results: Analysis of syntrophin α, encoded by SNTA1 , revealed 3 missense mutations in 2 patients (4%). A female diagnosed with LQTS at 13 years (QTc, 480 ms) with family history of autopsy-negative unexplained sudden death, was compound heterozygous for P74L/A257G while a male, diagnosed with LQTS at 18 years following syncopal episodes (QTc, 529 ms), hosted the A390V mutation. These mutations were absent in 600 reference alleles and A257G and A390V involved highly conserved residues. Functional studies revealed a marked leftward shift in sodium current activation for A257G and a significant rightward shift in channel inactivation for A390V-SNTA1. Both kinetic perturbations precipitated a ``gain-of-function” increase in the sodium channel window current. Conclusion: We provide the sentinel report of SNTA1 as a novel LQTS-susceptibility gene with a putative pathogenic mechanism involving a secondary ``gain-of-function” in the NaV1.5 sodium channel. Thus, CAV3-LQTS (LQT9), SCN4B-LQTS (LQT10), and now SNTA1-LQTS represent functional homologues with LQT3-like perturbations.

An estimated 10–15% of sudden infant death syndrome (SIDS) may be channelopathic with approximately half of the SIDS-associated channel mutations involving the sodium channel (NaV1.5) macromolecular complex. Recently, SNTA1, which encodes the sodium channel interacting protein (ChIP), syntrophin alpha, was discovered to be a novel long QT syndrome (LQTS) susceptibility gene (LQT12) with mutations causing increased late sodium current secondary to increased s-nitrosylation of the sodium channel’s alpha subunit. Therefore, SNTA1 is a plausible candidate gene for SIDS. In this IRB-approved study, mutational analysis of SNTA1’s eight translated exons was performed using PCR, DHPLC, and direct DNA sequencing of DNA derived from 286 SIDS cases (112 females, 197 white, average age 2.9 ± 1.9 months, range 3 days -12 months). The anonymized study design precluded a determination of mutation status as familial or sporadic. Overall, 8/286 cases of SIDS harbored missense mutations in SNTA1 that were absent in 800 reference alleles. Six distinct, rare SNTA1 missense mutations were detected in 7/112 (6.25%) female infants but only 1/174 (0.6%) male infants (p < 0.01). Comprehensive open reading frame analysis of all 800 reference alleles also revealed rare genetic variants in 1% of the control subjects. A common pair of non-synonymous single nucleotide polymorphisms (P74L/A257G), with a heterozygous frequency of 1%, was identified similarly in both cases and controls. This study provides the first molecular evidence implicating mutations in SNTA1 as a novel pathogenic substrate for channelopathic SIDS. However, given the ~1% background rate of rare variants in controls, mechanistic studies of each putative SIDS-associated SNTA1 mutation will be necessary to confirm that the mutant ChIP yields a functionally perturbed NaV1.5 macromolecular complex akin to the investigations that established SNTA1 as a novel LQTS-susceptibility gene.

Background: Mutations in the Na V 1.5 sodium channel macromolecular complex have been identified in some cases classified as idiopathic ventricular fibrillation (IVF). IVF and Brugada syndrome (BrS) are partially overlapping syndromes. Here, we report a mutation in SCN3B- encoded sodium channel β3 subunit as a novel pathogenic mechanism for IVF. Methods: Comprehensive open reading frame mutational analysis of SCN5A, GPD1L, and the beta subunit genes ( SCN1–4B ) was performed using PCR, DHPLC, and direct DNA sequencing of DNA extracted from a 20-year-old patient diagnosed with IVF. The SCN3B mutation was made by site directed mutagenesis and co-transfected with SCN5A into HEK-293 cells for functional chraracterization using the patch clamp technique. Results: A novel missense mutation, V54G-SCN3B, was identified in a 20-year-old male following collapse and external defibrillation from VF. After recovery, there was no detectable electrocardiographic abnormality. Imaging studies demonstrated a structurally normal heart, and the patient was diagnosed with IVF. The mutation was absent in 800 reference alleles and involved a highly conserved residue in the extracellular domain of the beta 3 subunit. No other mutations were identified in the 5 other genes. HEK cells expressing SCN5A and either WT-, or V54G-SCN3B were studied 24 hours after transfection. Cells expressing V54G-SCN3B showed significant decrease in sodium current density of 60±20 pA/pF compared to 203±35 pA/pF in WT-SCN3B (n=14–19). In addition V54G-SCN3B significantly shifted the activation curve +5 mV without affecting inactivation. Conclusions: This study provides the first molecular and cellular evidence implicating SCN3B in IVF. Given the marked loss-of-function to the sodium channel by V54G-SCN3B and the overlap between IVF and BrS, it will be interesting to determine whether mutations in SCN3B explain some cases of genotype negative Brugada syndrome.

Hypertrophic cardiomyopathy (HCM) is triggered mainly by mutations in genes encoding sarcomeric proteins, but a significant proportion of patients lack a genetic diagnosis. We identified a potentially novel mutation in ryanodine receptor 2, RyR2-P1124L, in a patient from a genotype-negative HCM cohort. The aim of this study was to determine whether RyR2-P1124L triggers functional and structural alterations in isolated RyR2 channels and whole hearts. We found that P1124L induces significant conformational changes in the SPRY2 domain of RyR2. Recombinant RyR2-P1124L channels displayed a cytosolic loss-of-function phenotype, which contrasted with a higher sensitivity to luminal [Ca 2+ ], indicating a luminal gain of function. Homozygous mice for RyR2-P1124L showed mild cardiac hypertrophy, similar to the human patient. This phenotype, evident at 1 year of age, was accompanied by an increase in the expression of calmodulin (CaM). P1124L mice also showed higher susceptibility to arrhythmia at 8 months of age, before the onset of hypertrophy. RyR2-P1124L has a distinct cytosolic loss-of-function and a luminal gain-of-function phenotype. This bifunctionally divergent behavior triggers arrhythmias and structural cardiac remodeling, and it involves overexpression of CaM as a potential hypertrophic mediator. This study is relevant to continue elucidating the possible causes of genotype-negative HCM and the role of RyR2 in cardiac hypertrophy.

Background— A 2% to 5% background rate of rare SCN5A nonsynonymous single nucleotide variants (nsSNVs) among healthy individuals confounds clinical genetic testing. Therefore, the purpose of this study was to enhance interpretation of SCN5A nsSNVs for clinical genetic testing using estimated predictive values derived from protein-topology and 7 in silico tools. Methods and Results— Seven in silico tools were used to assign pathogenic/benign status to nsSNVs from 2888 long-QT syndrome cases, 2111 Brugada syndrome cases, and 8975 controls. Estimated predictive values were determined for each tool across the entire SCN5A -encoded Na v 1.5 channel as well as for specific topographical regions. In addition, the in silico tools were assessed for their ability to correlate with cellular electrophysiology studies. In long-QT syndrome, transmembrane segments S3–S5+S6 and the DIII/DIV linker region were associated with high probability of pathogenicity. For Brugada syndrome, only the transmembrane spanning domains had a high probability of pathogenicity. Although individual tools distinguished case- and control-derived SCN5A nsSNVs, the composite use of multiple tools resulted in the greatest enhancement of interpretation. The use of the composite score allowed for enhanced interpretation for nsSNVs outside of the topological regions that intrinsically had a high probability of pathogenicity, as well as within the transmembrane spanning domains for Brugada syndrome nsSNVs. Conclusions— We have used a large case/control study to identify regions of Na v 1.5 associated with a high probability of pathogenicity. Although topology alone would leave the variants outside these identified regions in genetic purgatory, the synergistic use of multiple in silico tools may help promote or demote a variant’s pathogenic status.

SCN5A encodes the α-subunit of the ion channel that carries Na current in human heart. From a human cardiac cDNA library we recloned SCN5A. The new clone hH1b differed from existing clones hH1 in four and from hH1a in three positions. The common polymorphism H558R was uniquely present in hH1b. Voltage clamp study showed minor but potentially important kinetic differences between hH1b and the other clones. More dramatically, when the LQT3 mutation M1766L was introduced into the different clones, Na current was markedly reduced in the hH1 and hH1a backgrounds, whereas in hH1b the Na current was not reduced. Immunocytochemistry experiments showed a trafficking defect for M1766L Na channels in hH1 and hH1a but not in hH1b. The double-mutation M1766L/H558R in the hH1a background restored normal trafficking and current including persistent late current, suggesting the disease phenotype was the result of a “double hit” that included the common polymorphism, H558R. These results show that the choice of background clone must be carefully considered in mutagenesis studies. This also represents an example of intragenic complementation, the first for such a large protein.

The common polymorphism SCN5A-S1103Y (∼13% allelic frequency in African Americans) is a risk factor for arrhythmia, sudden unexplained death (SUD), and sudden infant death syndrome. Prompted by a case of autopsy-negative SUD in a 23-year-old African American man who collapsed while playing football, we hypothesized that S1103Y interacted with other SCN5A variants to pathologically modify sodium current ( I Na ). Mutational analysis of arrhythmia-associated genes in the victim revealed the variants SCN5A-R680H and SCN5A-S1103Y. These variants were made both separately and in the same cDNA construct of the alternative splice variant backgrounds (SCN5A-Q1077del and Q1077) and expressed in HEK293 cells. In the most abundant SCN5A-Q1077del, late I Na for S1103Y alone was not significantly different from wild type (WT). However, late I Na for R680H, R680H+S1103Y (coexpressed), and R680H/S1103Y (on the same cDNA) was increased 2.1-, 3.4-, and 3.6-fold, respectively, compared with WT. Intracellular acidosis (pH 6.7) increased late I Na for S1103Y, R680H, R680H+S1103Y, and R680H/S1103Y by 2.2-, 2.4-, 5.0-, and 5.5-fold, respectively, compared with WT at pH 6.7. Expression in the less abundant SCN5A-Q1077 showed no increased late I Na . This is the initial report of a functional interaction for the common polymorphism S1103Y with another mutation in the major transcript Q1077del of SCN5A. The “double hit” and environmental factor of acidosis may have converged to cause arrhythmic sudden death in this case.

Background— Sudden infant death syndrome (SIDS) is a leading cause of death during the first 6 months after birth. About 5% to 10% of SIDS may stem from cardiac channelopathies such as long-QT syndrome. We recently implicated mutations in α1-syntrophin (SNTA1) as a novel cause of long-QT syndrome, whereby mutant SNTA1 released inhibition of associated neuronal nitric oxide synthase by the plasma membrane Ca-ATPase PMCA4b, causing increased peak and late sodium current ( I Na ) via S-nitrosylation of the cardiac sodium channel. This study determined the prevalence and functional properties of SIDS-associated SNTA1 mutations. Methods and Results— Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing of SNTA1 ’s open reading frame, 6 rare (absent in 800 reference alleles) missense mutations (G54R, P56S, T262P, S287R, T372M, and G460S) were identified in 8 (≈3%) of 292 SIDS cases. These mutations were engineered using polymerase chain reaction–based overlap extension and were coexpressed heterologously with SCN5A, neuronal nitric oxide synthase, and PMCA4b in HEK293 cells. I Na was recorded using the whole-cell method. A significant 1.4- to 1.5-fold increase in peak I Na and 2.3- to 2.7-fold increase in late I Na compared with controls was evident for S287R-, T372M-, and G460S-SNTA1 and was reversed by a neuronal nitric oxide synthase inhibitor. These 3 mutations also caused a significant depolarizing shift in channel inactivation, thereby increasing the overlap of the activation and inactivation curves to increase window current. Conclusions— Abnormal biophysical phenotypes implicate mutations in SNTA1 as a novel pathogenic mechanism for the subset of channelopathic SIDS. Functional studies are essential to distinguish pathogenic perturbations in channel interacting proteins such as α1-syntrophin from similarly rare but innocuous ones.

The SCN5A-encoded cardiac sodium channel underlies excitability in the heart, and dysfunction of sodium current ( INa) can cause fatal ventricular arrhythmia in maladies such as long QT syndrome, Brugada syndrome (BrS), and sudden infant death syndrome (SIDS). The gene GPD1L encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1), but the function for this enzyme is unknown. Mutations in GPD1L have been associated with BrS and SIDS and decrease INa through an unknown mechanism. Using a heterologous expression system, we show that GPD1L associated with SCN5A and that the BrS- and SIDS-related mutations in GPD1L caused a loss of enzymatic function resulting in glycerol-3-phosphate PKC-dependent phosphorylation of SCN5A at serine 1503 (S1503) through a GPD1L-dependent pathway. The direct phosphorylation of S1503 markedly decreased INa. These results show a function for GPD1L in cell physiology and a mechanism linking mutations in GPD1L to sudden cardiac arrest. Because the enzymatic step catalyzed by GPD1L depends upon nicotinamide adenine dinucleotide, this GPD1L pathway links the metabolic state of the cell to INa and excitability and may be important more generally in cardiac ischemia and heart failure.