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Approximately 10% of sudden infant death syndrome (SIDS) may stem from potentially lethal cardiac channelopathies, with approximately half of channelopathic SIDS involving the NaV1.5 cardiac sodium channel. Recently, NaV beta subunits have been implicated in various cardiac arrhythmias. Thus, the four genes encoding NaV beta subunits represent plausible candidate genes for SIDS.
To determine the spectrum, prevalence and functional consequences of sodium channel beta subunit mutations in a SIDS cohort.
In this IRB-approved study, mutational analysis of the 4 beta subunit genes: SCN1B – 4B was performed using PCR, DHPLC, and direct DNA sequencing of DNA derived from 292 SIDS cases. Engineered mutations were co-expressed with SCN5A in HEK 293 cells, and whole cell patch clamped. One of the putative SIDS-associated mutations was similarly studied in adenovirally transduced adult rat ventricular myocytes.
3 rare (absent in 200–800 reference alleles) missense mutations (β3-V36M, β3-V54G and β4-S206L) were identified in 3/292 SIDS cases. Compared to SCN5A+β3-WT, β3-V36M significantly decreased peak INa and increased late INa while β3-V54G resulted in a marked loss-of-function. β4-S206L accentuated late INa and positively shifted the midpoint of inactivation compared to SCN5A+β4-WT. In native cardiomyocytes, β4-S206L accentuated late INa and increased the ventricular action potential duration (APD) compared to β4-WT.
This study provides the first molecular and functional evidence to implicate the NaV beta subunits in SIDS pathogenesis. Altered NaV1.5 sodium channel function due to beta subunit mutations may account for the molecular pathogenic mechanism underlying approximately 1% of SIDS.
Sudden infant death syndrome (SIDS) is defined as the sudden death of an infant under the age of one that remains unexplained after a thorough case investigation including medical autopsy, death scene investigation and detailed review of the clinical history.1 Despite the successes of the National “Back to Sleep” Campaigns, over 2000 infants each year die from SIDS.2 SIDS is rare during the first month of life and although it can occur in older infants, most SIDS deaths occur by the end of the sixth month, with the greatest number between 2 and 4 months of age.
While the pathogenic mechanisms of SIDS still remain largely unexplained, it is estimated that approximately 10% of SIDS stems from cardiac channelopathies, including congenital long QT syndrome (LQTS), short QT syndrome (SQTS), Brugada syndrome (BrS), and catecholaminergic polymorphic ventricular tachycardia (CPVT), which can result in dysrhythmia and sudden cardiac death.3–8 Approximately half of these channelopathic deaths involve the NaV1.5 cardiac sodium channel (SCN5A) encoded by SCN5A,3, 6, 8, 9 where gain-of-function mutations result in LQT310–12 and loss-of-function mutations result in BrS1.13
NaV1.5 is a highly regulated macromolecular complex, with associated partner proteins also implicated in disease pathogenesis including caveolin-3 in LQTS and SIDS,14, 15 GPD1L in BrS and SIDS,16, 17 α1-syntrophin in LQTS and SIDS,18, 19 and ankyrin G in BrS.20 NaV1.5 is also highly regulated by its beta subunits, which also have been recently implicated in arrhythmias including BrS,21, 22 cardiac conduction disease (CCD),22 atrial fibrillation (AF),23 idiopathic ventricular fibrillation (IVF),24 and LQTS.25
Based on these recent findings and considering that perturbations involving NaV1.5 are more common in channelopathic SIDS compared to LQTS, we hypothesized that mutations in the NaV beta subunits may result in some cases of SIDS through dysregulation of NaV1.5. Therefore, the objective of our study was to determine the spectrum and prevalence of mutations in the genes encoding the cardiac sodium channel beta subunits (β1–4) in a large cohort of SIDS.
292 SIDS cases derived from population-based cohorts of unexplained infant deaths (114 female infants, 177 male infants, 1 unknown; 203 white, 76 African American, 10 Hispanic, 2 Asian, 1 unknown; average age, 2.9 ± 1.9 months; range, 6 hours - 12 months) were submitted to the Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory for postmortem genetic testing. Enrollment criterion was autopsy-negative sudden unexplained death of an infant < 1 year of age. By definition, the death had to be unexplained even after a comprehensive medico-legal autopsy. The study was approved by the Mayo Foundation Institutional Review Board as an anonymous necropsy study. Therefore, only limited medical information such as the sex, ethnicity and age of the infant at the time of death was generally available.
DNA was extracted from autopsy blood with the Puregene DNA Isolation Kit (Gentra, Minneapolis, Minn) or from frozen necropsy tissue with the Qiagen DNeasy Tissue Kit (Qiagen, Inc, Valencia, Calif). Mutational analysis of all 19 translated exons of the beta subunit genes SCN1B, SCN2B, SCN3B, and SCN4B was performed using polymerase chain reaction (PCR) and denaturing high performance liquid chromatography (DHPLC) after design of intronic primers to amplify all translated exons. Abnormal DHPLC profiles were then sequenced to determine the nature of the amino-acid substitution. Reference alleles, derived from healthy adult control subjects whose ethnicity matched the decedent, were also examined by DHPLC to determine whether an identified amino acid variant was a common polymorphism. Primer sequences, PCR conditions and DHPLC conditions are available upon request.
To be considered a possible SIDS-associated mutation, the genetic variant had to (1) be a non-synonymous variant (synonymous single-nucleotide polymorphisms were excluded from consideration), (2) involve a highly conserved residue, (3) be absent in reference alleles from healthy ethnic-matched control subjects, and (4) yield a functionally altered, pro-arrhythmic cellular phenotype consistent with a potentially lethal arrhythmogenic substrate.16
The mutations in beta subunits were created by site-directed mutagenesis (mutagenesis kit from Stratagene, La Jolla, California, USA) using a PCR technique in the pcDNA3 vector (Invitrogen, Carlsbad, California, USA). Mutations were then subcloned into pIRES2-EGFP vector (Clontech Laboratories, Palo Alto, California, USA) and an entry vector of pENTR1A-IRES2EGFP.19 Integrity of the constructs was verified by DNA sequencing. The EGFP-tagged beta WTs and mutants were co-transfected with the most common splice variant SCN5A [lacking a glutamine at position 1077 (Genbank accession No. AY148488)] in a 1:1 ratio and transiently expressed in HEK 293 cells for functional study as described previously.26
Animal handling practices used in this study were approved by animal use committee at the University of Wisconsin. Single ventricular myocytes were digested enzymatically from adult female Sprague-Dawley rats with collagenase and hyaluronidase, as previously described.27 Briefly, the heart was quickly excised and mounted on a Langendorff apparatus. The heart was perfused retrogradely for 10 min at 37°C with oxygenated 1mM Ca+2 Ringers solution containing (in mM) 125 NaCl, 5 KCl, 2 NaH2PO4, 5 sodium pyruvate, 1.2 MgSO4, 11 glucose, 1 CaCl2 and 25 HEPES, followed by a 5mM Taurine of Ca+2 free Ringers solution for 5 min, and a 10 min perfusion with continuous recirculation of the same solution containing 0.5 mg/ml collagenase (type II, Worthington), and 0.35 mg/ml hyaluronidase (TypeI-S, Sigma). Then, a gradual Ca+2 add-back was performed to bring Ca+2 concentrations of the enzyme solution up to 0.5 mM. Lastly, the myocytes were maintained in 1 mM Ca+2 Ringers solution.
Isolated myocytes were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum, 50 U/ml penicillin and 50 μg/ml streptomycin and (in mM) 1 CaCl2, 5 taurine,5 L-carnitine, and 5 creatine. The cells were plated on laminin-coated coverslips by incubating at 37°C, 5% CO2-95% room air for 2 hours.27 After 2 hours of incubation, culture medium was replaced with serum-free DMEM along with the adenovirus (Invitrogen, Carlsbad, CA) per manufacturer’s instructions. Viruses were diluted in serum-free DMEM to yield a 100 MOI and cells were incubated for 24 hours. After 24 hours of incubation, the serum-free DMEM was changed and replenished every day. Two to three days later, IRES2-GFP-expressing cells, i.e. those infected by adenoviral recombinants of either β4-WT or β4-S206L, were selected for functional study.
Macroscopic INa was measured using a standard whole-cell patch clamp method at a temperature of 22°–24°C as previously reported.26 The extracellular (bath) solution contained, in mmol/L, NaCl 140, KCl 4, CaCl2 1.8, MgCl2 0.75 and HEPES 5 (pH 7.4 set with NaOH). The pipette solution contained, in mmol/L, CsF 120, CsCl 20, EGTA 2 and HEPES 5 (pH 7.4 set with CsOH). Pipettes had resistances between 1.0 and 2.0 MΩ when filled with recording solution. The standard voltage-clamp protocols are presented with the data and as described in detail previously.28 For cardiomyocyte studies, INa were recorded in a standard whole-cell voltage-clamp model and APs were recorded in current-clamp mode using patch-clamp configuration with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA) and pClamp 9.2 software (Axon Instruments Inc. Union City, CA). The extracellular (bath) solution contained (in mmol/L) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 2.0 MgCl2, 5.0 Glucose and 5 HEPES, pH 7.4. The pipette solution contained (in mmol/L) 10 NaCl, 10 KCl, 2.0 MgCl2, 125 K-aspartate, 5.0 Glucose and 5 HEPES, pH 7.3. Cardiac myocytes were stimulated with current pulses of 1 ms duration. The currents were low-pass filtered at 5 kHz, digitized at a sampling rate of 5 kHz. INa and APs was recorded at room temperature (22°C~24°C) with pipettes resistance of 1.5~3 MΩ.
All data points are shown as the mean value and the bars represent the standard error of the mean (S.E.M.). Determinations of statistical significance were performed using a Student t test for comparisons of two groups. A p-value < 0.05 was considered statistically significant. Curve fits were done using pClamp 9.2 (Axon Instruments). Additional non-linear curve fitting was performed with Origin 6.0 (Microcal Software).
Overall, mutational analysis of the four beta subunits revealed putative pathogenic mutations in 3 of 292 cases (~1%) of SIDS. Figure 1 details the molecular characterization of the 3 identified missense mutations. An abnormal DHPLC elution profile (Figure 1A) and subsequent DNA sequencing (Figure 1B) led to the identification of a nucleotide substitution (106 G→A) producing a methionine (M) for valine (V) substitution (V36M) in SCN3B in a 6-week–old white female infant. A nucleotide substitution (161 A→C) causing a V54G-SCN3B missense mutation was also identified in a 6-month–old white male infant. V54G-SCN3B has been implicated recently with IVF in a 20-year-old.24 Similarly, a nucleotide substitution (617 G→A) in SCN4B yielded an S206L missense mutation in a 5-month–old black male infant.
All three missense mutations involved residues conserved across a variety of species (Figure 1C) and were absent in ethnic-matched control populations, 800 reference alleles for the SCN3B mutations and 200 reference alleles for the SCN4B mutation. Figure 1D depicts the linear topology of the NaV β3 and β4 subunits and location of the mutations. Both NaV β3 missense mutations localized to the extracellular loop (amino acids 1–135) while the NaV β4 mutation localized to the C-terminal, cytoplasmic region of the protein. In addition, a nucleotide substitution (28 G→A) producing a G10S amino acid substitution in the NaV β1 subunit was identified in both an African American infant and an African American control (data not shown). Since β1-G10S was identified in both a case and a control, it was excluded from further functional studies. Due to the blinded, anonymous nature of the SIDS population-based cohort, it was not possible to obtain further information on families of the SIDS victims to establish sporadic versus familial status.
Transfected HEK-293 cells transiently expressing SCN5A + vector only, SCN5A + β3-WT, or SCN5A + β3-V36M were voltage clamped after 24h incubation. Compared to WT, β3-V36M demonstrated a marked decrease in peak INa density and accentuated persistent/late INa for β3-V36M compared to WT channels (Table 1, Figure 2A, C and Figure 3). β3-V54G precipitated a marked attenuation in peak INa by disrupting proper trafficking of the SCN5A-encoded alpha subunit (data not shown).24
Transfected HEK-293 cells transiently expressing SCN5A + vector only, SCN5A + β4-WT, or SCN5A + β4-S206L were voltage clamped after 24h incubation. Compared with WT, β4-S206L did not affect peak INa (Table 2). However, a 7 mV positive shift of inactivation midpoint was found for β4-S206L compared to β4-WT channels (Table 2, Figure 4C). In addition, the “window” current where the activation curve overlaps the inactivation curve was also increased for β4-S206L compared to WT channels (Figure 4D). Lastly, late INa was increased significantly for β4-S206L compared to WT channels (Figure 5).
To further validate the mutant phenotype for beta subunit mutations, β4-S206L was also studied in a more native environment. IRES2-GFP-expressing isolated adult rat ventricular myocytes were voltage clamped 2–3 days following infection by adenoviral recombinants of β4-WT or β4-S206L containing the IRES2-GFP. Compared to β4-WT, ventricular myocytes infected by β4-S206L showed an increase in late INa akin to the heterologous expression studies in non cardiac cells (Figure 6A, B, E). Further, there was a marked increase in APD for β4-S206L compared to β4-WT under the current-clamp mode (Figure 6C, D, F).
This is the first study exploring the spectrum and prevalence of beta subunit mutations in SIDS. In total, 3 distinct missense mutations (2 in SCN3B and 1 in SCN4B) were identified in 292 SIDS victims (1%). These mutations were all found in residues that were well conserved across a variety of species and absent in ethnic-matched control populations. β3-V54G has been reported recently as a NaV1.5 trafficking defective, loss-of-function mutation first implicated in the out-of-hospital cardiac arrest of a 20-year-old white male who was resuscitated from ventricular fibrillation during a basketball game.24 β3-V36M displayed both NaV1.5 gain- and loss-of-function properties. β4-S206L conferred a gain-of-function to NaV1.5 akin to the previously published LQTS-associated SCN4B missense mutation.25
NaV1.5 is responsible for initiating the cardiac action potential and consists of a pore-forming α subunit and, among other regulatory proteins, one or more auxiliary β subunits.29 Mutations within SCN5A-encoded NaV1.5 can result in various arrhythmia syndromes such as LQT3, BrS1, and autosomal recessive sick sinus syndrome. Mutations in sodium channel beta subunits have also been implicated in various diseases. For example, a mutation in SCN1B has been associated with febrile seizures.30 In addition, mutations in SCN1B and its splice variant were identified in a cohort of 282 BrS patients and 44 CCD patients which caused NaV1.5 loss-of-function in vitro.22 Mutations in SCN1B and SCN2B have been associated with AF23 while a SCN3B mutation has been linked to the BrS ECG phenotype.21 Moreover, a mutation in SCN4B was reported in a 21-month old with LQTS and an in vitro LQT3-like increase in late INa.25
Sodium channel beta subunits play critical roles in cell adhesion, signal transduction, sodium channel plasma membrane expression and modulation of channel gating and voltage dependence.29 Structurally, β1 and β3 subunits share sequence homology and non-covalently associate with the subunit, whereas the homologous β2 and β4 form disulfide bonds with the subunits.29 In the present study, the β3-V36M mutation resulted in both gain- and loss-of-function channel phenotypes when compared with β3-WT, in contrast to the recently characterized β3-V54G, which is purely loss-of-function.24 Similar to β3-V54G, β3-V36M localizes to the extracellular loop, which is important for proper β3 membrane trafficking.31 Deletion of the extracellular domain positively shifts steady-state inactivation and negatively shifts steady-state activation without affecting INa density.31 Moreover, given that the artificially engineered mutation, C96A, causes the complete loss of β3 membrane expression32 and that V54G yields a trafficking defective NaV1.5 as well, it is possible that V36M may affect alpha subunit trafficking, which would explain its loss-of-function effects.
β4-S206L demonstrated increased late INa in both a heterologous system and the more native environment of adult rat cardiomyocytes. It is interesting to note that serine 206 is in close proximity to the open-channel blocker sequence from position 184–197 in the full gene product. This short sequence (KKLITFILKKTREK) is replete with hydrophobic and lysine residues which serve to block the open-channel state and induce resurgent “late sodium current” in Purkinje neurons.32,33 This was also shown to be applicable to NaV1.5.32 It is thus conceivable that a mutation which adds another highly hydrophobic residue, leucine, close to this hydrophobic sequence may enhance its channel-blocking ability and produce a resurgent INa large enough to result in a lethal arrhythmia.
The functional studies in heterologous systems have some limitations for interpretation and application. Although localization studies have demonstrated selective targeting of the beta subunits in the cardiomyocyte,34 it is still unclear which combinations of subunits are most critical in impacting net sodium current. While individual mutations in each of the beta subunits have been implicated in various arrhythmogenic processes, it remains challenging when performing functional studies involving beta subunits to determine which of the mutant phenotypes displayed in vitro may be modulated in vivo in the presence of the other “normal” subunits. Given these limitations, it is therefore important that we were able to reproduce the late INa findings for the β4-S206L mutation in a more native environment of cardiocytes, as well as demonstrate a marked prolongation in the cardiocyte’s APD, which is the cellular substrate that predisposes to torsades de pointes, the hallmark dysrhythmia for the potentially lethal syndrome of LQTS. That these functionally abnormal mutations were absent in controls, in residues conserved across multiple species, and discovered in a sudden death cohort, we speculate that these infants succumbed to an infantile expression of NaV1.5 dysfunction: BrS/IVF in the case of β3-V54G, LQTS for β4-S206L, and possibly a mixed/overlapping phenotype for β3-V36M.
Lastly, it is interesting to note that all three beta subunit mutations were discovered in infants outside the classic risk period for SIDS, one before two months of age, and two after four months of age. Although the age range of our assembled SIDS cohort is appropriate (1–5 months of age), this observation is consistent with our findings overall that infants found to have perturbations in the NaV1.5 sodium channel complex tend to fall outside the classic two-to-four month age range.35 Again, this raises the issue of whether the infant deaths occurring outside the critical risk period for SIDS actually stem from fundamentally different substrates, and further studies comparing different subsets of SIDS victims may elucidate this further.
In conclusion, this is the first study to implicate the sodium channel beta subunits in the pathogenesis of channelopathic SIDS. All three mutations identified were absent in controls, conserved across various species, and in vitro imparted abnormal gain- or loss-of-function to NaV1.5. This study offers additional evidence for the crucial role of sodium channel beta subunits in the regulation of NaV1.5 and expands the genetic heterogeneity for this emerging subset of channelopathic SIDS. Whether or not this particular subset (~10%) of SIDS is identifiable and preventable requires much further investigation.
This work was supported by the University of Wisconsin Cellular and Molecular Arrhythmia Research Program (J.C.M.), the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program (M.J.A.), the Established Investigator Award from the American Heart Association (to M.J.A.), and the grants HD42569 (to M.J.A.) and HL71092 (to J.C.M.) from National Institutes of Health, USA.
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