Article ID: CJ-25-0283
Background: Loss-of-function SCN5A variants are primarily associated with Brugada syndrome (BrS), but can also present with overlapping phenotypes. We investigated Cys1384Phe of SCN5A, a novel missense variant associated with BrS, sick sinus syndrome (SSS), and dilated cardiomyopathy (DCM).
Methods and Results: This study included a large 4-generation Japanese family consisting of 15 individuals (1 proband and 14 family members). Among them, the proband, a cousin, a second cousin and the second cousin’s father were diagnosed with BrS. Two of these 4 BrS patients experienced VF events, while the other 2 remained asymptomatic. Another cousin was diagnosed with DCM, and 3 additional family members exhibited complete right bundle branch block and/or SSS. Comprehensive genetic analysis using a target panel sequencing identified a novel missense variant, Cys1384Phe in SCN5A, in the proband and affected family members; however, the phenotypes were different. Whole-cell patch-clamp experiments using HEK293 cells transfected wild-type or Cys1384Phe plasmid demonstrated a complete loss-of-function in the sodium current of the Cys1384Phe cells. Furthermore, the heterozygous expression of Cys1384Phe and wild-type (WT) channels showed a significant reduction of peak sodium current compared with the WT, suggesting a dominant-negative suppression, but no trafficking defect was observed.
Conclusions: The novel Cys1384Phe variant in SCN5A is a complete loss-of-function mutation with dominant-negative suppression, and associated with overlapping phenotypes of BrS, SSS, and DCM.
Brugada syndrome (BrS) is characterized by coved-type ST-segment elevation in the right precordial leads on ECG and an increased risk of ventricular fibrillation (VF), potentially leading to sudden cardiac death (SCD).1 Loss-of-function variants in the SCN5A gene are identified in approximately 20% of BrS cases,2 and we have previously demonstrated that BrS patients with loss-of-function SCN5A variants are at higher risk of ventricular arrhythmias.3,4 Expression levels of cardiac sodium channels in the right ventricular outflow tract may be associated with the arrhythmogenicity in BrS.5,6 In BrS, SCN5A mutation carriers exhibit more pronounced epicardial electrical abnormalities and a more aggressive clinical presentation.7
On the other hand, pathogenic variants of SCN5A are associated not only with BrS but also other arrhythmic phenotypes, such as progressive cardiac conduction defect, sick sinus syndrome (SSS), atrial fibrillation, and dilated cardiomyopathy (DCM).8 Therefore, genetic testing of SCN5A is strongly recommended for an index case with BrS and variant-specific genetic testing is also recommended for family members.9 Here, we identified a large Japanese family with a novel SCN5A variant associated with an overlapping phenotype, including BrS, SSS, and DCM.
This study included a large Japanese family consisting of 15 individuals (1 proband and 14 family members, Figure 1). Written informed consent was given by all participants. The study conformed to the Declaration of Helsinki and received approval from the National Cerebral and Cardiovascular Center’s ethics committee (M29-167-10).
Family pedigree of patients with the SCN5A-Cys1384Phe variant. BrS, Brugada syndrome; DCM, dilated cardiomyopathy; RBBB, right bundle branch block; SICD, subcutaneous implantable cardioverter-defibrillator; SSS, sinus node dysfunction; VF/SCD, ventricular fibrillation/sudden cardiac death.
Genetic and Functional Analysis
Details of the genetic analysis have been described previously.10 In brief, genomic DNA was extracted from the patients’ blood lymphocytes. For the proband (III-3) and his cousin (III-4), next-generation sequencing (NGS) was performed using a customized targeted gene panel (HaloPlex HS custom panel [Agilent Technology]) (for proband) or TruSight Cardio panel (Illumina) (for his cousin) and sequenced on a MiSeq platform (Illumina) (Supplementary Table). Sanger sequencing was then conducted to confirm the variant in the proband and all enrolled family members.
Functional Analysis of the SCN5A VariantTo assess the variant’s pathogenicity, we performed a functional analysis using the whole-cell patch-clamp technique. Human SCN5A cDNA was subcloned into a pcDNA 3.1 vector. The target variant was generated using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, USA) according to the manufacturer’s instructions. For the INa patch-clamp recordings, HEK293 cells at 50–60% confluency in 35-mm cell culture dishes were transiently cotransfected with 0.5 μg of Nav1.5 plasmid (wild-type [WT] or p.Cys1384Phe) and 1.0 μg of pEGFP-C3 vector using the jetPEI transfection reagent, according to the manufacturer’s instructions. For the hetero mimicking condition, 0.5 μg of both WT and p.Cys1384Phe plasmid and 0.5 μg of pEGFP-C3 were cotransfected. Patch-clamp recordings were conducted 36–48 h post-transfection in the whole-cell configuration at 37℃. Ion currents were recorded using Patchmaster software (HEKA, Lambrecht, Germany). Patch pipettes were prepared for resistances of 0.5–1.2 MΩ when filled with the pipette solution. Currents were filtered at 10 kHz and digitized at 50 kHz. Data were acquired and analyzed with Fitmaster software (HEKA, Lambrecht, Germany). The internal pipette solution for INa contained (in mM): 5 NaCl, 2 MgCl2 6 H2O, 1 CaCl2, 130 CsCl, 15 EGTA, 10 HEPES, and 4 MgATP, with pH adjusted to 7.2 using CsOH. The bath solution contained (in mM): 80 NaCl, 50 CaCl, 2 CaCl2 10 glucose, 2.5 MgCl2, and 10 HEPES, with pH adjusted to 7.4 using CsOH.
Membrane Trafficking AnalysisMembrane trafficking was assessed by biotin binding assay. For this analysis, HEK293 cells cultured on 60-mm dishes were transfected with 3.0 μg WT- or C1384F-SCN5A plasmid with HA tag. Cell surface protein was biotinylated and precipitated by PierceTM NeutrAvidinTM Agarose. Cell surface protein fraction and total protein were migrated and transferred to PVDF membranes. Nav1.5 and GAPDH were probed by rabbit anti-HA antibody (#3724, Cell signaling) and rabbit anti-GAPDH (ab9485, abcam). Goat anti-rabbit secondary antibody and chemilumione (#07880, nacarai, Kyoto, Japan) were used to visualize the bands. The intensity of the protein bands was quantified using Image J software and normalized to the density of the GAPDH band.
Statistical AnalysisThe statistical analyses were performed using JMP Pro version 17 software (SAS Institute Inc., Tokyo, Japan) and R software (version 4.4.1; R Foundation for Statistical Computing, Vienna, Austria). Continuous data are expressed as the mean±standard deviation, and categorical data as number (%). For the patch-clamp analysis, the data are shown as the mean±standard error. Student’s t-test, Mann-Whitney U test, and χ2 test were performed as appropriate for statistical differences. P<0.05 was considered significant.
A 33-year-old man (III-3, proband) was first diagnosed with BrS at the age of 23 years. Although asymptomatic, his ECG showed a spontaneous type-1 Brugada pattern (Figure 2A), and he had a family history of BrS with VF. Genetic testing identified a novel missense variant, Cys1384Phe in SCN5A (Figure 3). Programmed electrical stimulation from the right ventricular outflow tract induced VF. Based on these findings, the patient opted for implantation of a subcutaneous implantable cardioverter-defibrillator (ICD) for primary prevention of SCD. To date, no VF or appropriate shock therapy have occurred during the past 8 years, and he remains off medication.
12-lead ECGs of the proband (A) and affected family members (B–E).
(A) Sequence analysis of the proband’s genomic DNA compared to the control. (B) Topology of Nav1.5, with the red circle indicating the location of the identified variant.
A 39-year-old man (III-4), the proband’s cousin, presented with chest pain and frequent premature ventricular contractions (PVCs), 35,643/day (35%). He did not exhibit a Brugada-ECG (Figure 2B). Coronary angiography showed no significant stenosis, but echocardiography revealed left ventricular dilatation (LVDd: 59 mm) and a reduced left ventricular ejection fraction (LVEF: 48%). He was diagnosed with DCM and treated with the β-blocker, carvedilol (5 mg/day).
Another of the proband’s cousins (III-6) first experienced VF at the age of 35 years and was subsequently diagnosed with BrS (Figure 2C,D), leading to implantation of a transvenous ICD. Two years later, he experienced 2 episodes of recurrent VF and was prescribed bepridil, but VF developed. Consequently, radiofrequency catheter ablation of the epicardium was performed, although he again received an appropriate ICD shock for recurrent VF.
A 37-year-old man (III-8), the proband’s second cousin, was asymptomatic but diagnosed with BrS during a medical checkup (Figure 2E). His close relatives had a history of sudden cardiac events; his father (II-7) experienced aborted SCD, and his father’s sister (II-6) died suddenly at the age of 20 years. The patient remains asymptomatic and is now under careful observation without ICD implantation.
In addition to these cases of BrS and SCD, the proband’s father (I-2), grandmother (II-1), and nephew (IV-1) exhibited SSS or right bundle branch block, suggesting a potential cardiac conduction defect.
Genetic AnalysisA comprehensive genetic analysis using the NGS-panel identified, and the Sanger sequencing confirmed, a novel SCN5A heterozygous variant, c.4151G>T, p.Cys1384Phe (NM_001099404) (Figure 3A) in the proband (III-3) and his cousin (III-4), located at the S5–S6 (pore) region in domain III of the sodium channel (Figure 3B). The variant was also identified in his cousin (III-6), a second cousin (III-8), and the second cousin’s father (II-7), as well as in the proband’s father (II-1), grandmother (I-2), sister (III-1), and her son (IV-1) (Figure 1). The genotype–phenotype association was not entirely consistent in this family, as the proband’s sister was asymptomatic, and his father, despite being middle-aged, exhibited no phenotype of BrS or DCM. The variant c.4151G>T, p. Cys1384Phe is classified as likely pathogenic based on PP3, PM2, and PM1 according to the Franklin ACMG classification (https://franklin.genoox.com/clinical-db/home). However, no information is available in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) or ToMMo (https://jmorp.megabank.tohoku.ac.jp/).
Functional AnalysisWhole-cell patch-clamp analysis revealed no measurable sodium (Na+) current in the Cys1384Phe mutant cells, suggesting it is a complete loss-of-function Na+ channel variant (Figure 4A). Therefore, the functional data added PS3, classifying the variant as pathogenic.
(A) Original sodium current traces of cells expressing wild-type (WT)-SCN5A, WT + Cys1384Phe-SCN5A, and Cys1384Phe-SCN5A only, with the pulse protocol shown in the inset. (B) Transfection conditions. In all groups, the total plasmid amount was adjusted to 1.5 µg. (C) Current–voltage (I–V) relationships for cells expressing SCN5A plasmids. The test pulse protocol is shown in the inset. *P<0.05. vs WT by Student’s t-test. Numbers in parentheses represent the number of samples (n). (D) Steady-state inactivation and activation curves. The test pulse protocol for inactivation is shown in the inset. Numbers in parentheses represent the number of samples (n).
We performed an additional patch-clamp study to verify the dominant-negative effect of the identified variant. HEK293 cells were transfected with either [0.5 μg WT SCN5A plasmid] or [0.5 μg Cys1384Phe- and 0.5 μg WT-SCN5A plasmid] (Figure 4B). This cotransfection condition was specifically chosen to evaluate the presence of a dominant-negative effect. Because a loss-of-function variant produces little to no current, the total INa in this setting would be expected to match that of WT 0.5 μg alone if there is no dominant-negative effect. Thus, a significant reduction in current density compared to WT 0.5 μg would indicate the presence of a dominant-negative effect. Under these conditions, we observed a significant (32.9%) reduction of peak INa density in cells coexpressing Cys1384Phe and WT channels (mimicking a heterozygous state) compared to WT, suggesting a dominant-negative effect by the variant (269.2±39.0 vs. 401.8±48.6 pA/pF at −40 mV, Figure 4C). The voltage dependence of steady-state activation and inactivation did not differ between the heterozygous expression of the Cys1384Phe and WT plasmid and WT only (Figure 4D).
Moreover, as shown in Figure 5, both WT and Cys1384Phe mutated Nav1.5 exhibited comparable cell surface expression in HEK293 cells, suggesting that the observed dominant-negative effect was not due to a trafficking defect, but rather to channel-channel interaction at the cell surface.
(A) Representative immunoblot images of biotinylated surface protein fractions and total protein fractions from cells transfected with wild-type or mutant SCN5A plasmids. WT (wild-type); NonTfx (Non transfected). (B) Bee swarm plots showing individual data points from the biotinylation assay (n=4). Box plots are overlaid to indicate the median, interquartile range (box), and data distribution (whiskers).
We identified a novel SCN5A variant, Cys1384Phe, in a large family spanning 4 generations, with multiple phenotypes such as BrS, SSS, and DCM. Functional analysis revealed a complete loss-of-function with a dominant-negative suppression. Thus, the SCN5A-Cys1384Phe variant is likely associated with overlapping phenotypes of BrS, SSS, and DCM.
The variant (c.4151G>T, p.Cys1384Phe) has not been reported in ClinVar or gnomAD. According to the ACMG guidelines,11 it was initially classified as a variant of uncertain significance based on 3 supporting and 1 moderate criterion (PM2, PP2, PP3, PP4). However, it also could be classified as likely pathogenic because Cys1384 is located in the pore region of Nav1.5 (PM1 in addition). The location of this variant may explain the complete loss of Na+ current, which contributes to the severe phenotype.2 Furthermore, the results of our functional study (i.e., complete loss-of-function of Nav1.5 current) could add a strong criterion (PS3) that reclassifies the variant as pathogenic.5
SCN5A loss-of-function variants with a dominant-negative effect have been reported in several studies to date.12,13 The phenotypes in those previous reports were mainly BrS. However, nearly 20% of pathogenic or likely pathogenic variants in SCN5A were the overlap syndrome, including BrS, long-QT syndrome, progressive cardiac conduction disorders and SSS.14 Moreover, Probst et al. reported that SCN5A variants were not directly causal to the occurrence of BrS-ECG in some cases,15 suggesting that the pathophysiology of BrS should be oligogenic rather than monogenic.16 In this report, we demonstrated only the loss-of-function variant in SCN5A in a large family with multiple phenotypes. However, the reason for these various phenotypes remains unclear. It might be due to age at diagnosis, sex, and differences in single-nucleotide polymorphisms associated with the BrS-ECG.17 Therefore, interpreting the SCN5A variant as the sole cause of the family’s phenotypic complexity may be inappropriate. Further research is needed for the appropriate interpretation of this effect.
The most frequently identified causative gene in patients with familial DCM is TTN, but when conduction defects (atrioventricular block) are familial, then LMNA is often responsible.9 The concept of SCN5A variants as a cause of DCM is uncommon,14 but multifocal ectopic premature Purkinje-related complexes (MEPPC) is a known SCN5A-related condition characterized by frequent Purkinje-related PVCs and reduced EF. However, MEPPC is typically associated with gain-of-function SCN5A variants.18 Furthermore, the PVCs in this case (III-4) did not appear to be Purkinje-related, which is not consistent with MEPPC but rather represents a loss-of-function SCN5A variant-related DCM.
BrS is male predominant, and the BrS phenotype tends to diminish in females after puberty.19 In addition, Aizawa et al. found that among SCN5A variant carriers, female patients exhibited a higher prevalence of sinus node dysfunction (SSS) compared to males.20 Consistent with this, in our pedigree, a sole female patient (I-2) presented SSS, but not BrS. Another potentially affected female patient (II-6) experienced SCD; however, genetic testing was not performed, so the cause of her SCD should be interpreted with caution.
In conclusion, the novel SCN5A Cys1384Phe variant is a complete loss-of-function mutation associated with overlapping phenotypes of BrS, SSS, and DCM.
The authors are grateful to Rieko Osawa, Kaori Kugo and Madoka Tanimoto for technical assistance with the genetic analysis.
None.
This study was approved by the Ethics Committee of the National Cerebral and Cardiovascular Center (Reference number: M29-167-10).
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https://doi.org/10.1253/circj.CJ-25-0283
