Mutations in SCN10A are responsible for a large fraction of cases of Brugada syndrome

Abstract

Background: BrS is an inherited sudden cardiac death syndrome. Less than 35% of BrS probands have genetically identified pathogenic variants. Recent evidence has implicated SCN10A, a neuronal sodium channel gene encoding Nav1.8, in the electrical function of the heart.

Objectives: The purpose of this study was to test the hypothesis that SCN10A variants contribute to the development of Brugada syndrome (BrS).

Methods: Clinical analysis and direct sequencing of BrS susceptibility genes were performed for 150 probands and family members as well as >200 healthy controls. Expression and coimmunoprecipitation studies were performed to functionally characterize the putative pathogenic mutations.

Results: We identified 17 SCN10A mutations in 25 probands (20 male and 5 female); 23 of the 25 probands (92.0%) displayed overlapping phenotypes. SCN10A mutations were found in 16.7% of BrS probands, approaching our yield for SCN5A mutations (20.1%). Patients with BrS who had SCN10A mutations were more symptomatic and displayed significantly longer PR and QRS intervals compared with SCN10A-negative BrS probands. The majority of mutations localized to the transmembrane-spanning regions. Heterologous coexpression of wild-type (WT) SCN10A with WT-SCN5A in HEK cells caused a near doubling of sodium channel current compared with WT-SCN5A alone. In contrast, coexpression of SCN10A mutants (R14L and R1268Q) with WT-SCN5A caused a 79.4% and 84.4% reduction in sodium channel current, respectively. The coimmunoprecipitation studies provided evidence for the coassociation of Nav1.8 and Nav1.5 in the plasma membrane.

Conclusions: Our study identified SCN10A as a major susceptibility gene for BrS, thus greatly enhancing our ability to genotype and risk stratify probands and family members.

Keywords: Brugada syndrome; cardiac arrhythmias; cardiac conduction disease; electrophysiology; genetics; sudden cardiac death.

Figure 1. Representative Cases of the Different Brugada Syndrome (BrS) Phenotypes Associated with the SCN10A Mutations/Rare Variants Identified

Each panel shows the ECG phenotype, amino acid alignments of the mutated residue position in a number of mammalian species, and DNA chromatogram of wild-type (WT) and mutant SCN10A. For the pedigrees in panels D&E, +/- denotes heterozygous for the mutation; circles represent female subjects and squares represent male subjects. The arrow denotes the proband. Clinically affected and unaffected subjects are labeled as black and white, respectively. CCD: cardiac conduction disease; ERS= early repolarization syndrome; RBBB= right bundle branch block; SCD= sudden cardiac disease.

Figure 2. Clinical and Genetic Prevalence of SCN10A Mutations/Rare Variants in probands with Brugada Syndrome (BrS) Identified in the Present Study

A: Schematic showing topology of Na_v1.8, the pore-forming α subunit encoded by SCN10A and location of putative BrS-causing variants. B: Frequency distribution of SCN10A mutations/rare variants in BrS cases (green). C: Percentage of mutations/rare variants in BrS cases (green) by location. D: Mutation detection yield by gene in Masonic Medical Research Laboratory BrS cases. E&F: Bar graph showing age and gender distribution of BrS cases. CCD= cardiac conduction disease.

Figure 3. Electrophysiology Effect of SCN10A on Cardiac Sodium Channel Current (I_Na) when Co-Expressed with SCN5A and SCN3B in HEK293 Cells

A&B: Superimposed traces and bar graph depicting peak I_Na recorded from co-expression of SCN10A/wild type (WT)+SCN3B/WT, SCN5A/WT+SCN3B/WT and SCN5A/WT+SCN10A/WT+SCN3B/WT. **P<0.01 vs. SCN5A/WT+SCN10A/WT+SCN3B/WT, ^##P<0.01 vs. SCN5A/WT+SCN3B/WT. C-E: Representative I_Na traces, current-voltage relationship and voltage dependence of activation for SCN10A/WT, SCN10A/R14L and SCN10A/R1268Q when co-expressed with SCN5A/WT+SCN3B/WT. F&G: Representative steady-state inactivation and recovery traces recorded from WT and mutant channels. H&I: Boltzmann distributions of voltage-dependent channel inactivation and recovery curve with a double-exponential fit for the 3 groups. All related values and the number of cells used are presented in Table S2 in the Supplemental Materials.

Figure 4. Representative Experiments Demonstrating Physical Interaction between Na_v1.8 and Na_v1.5

Panel A shows protein input and panel B shows protein isolated by the antibody pull-down co-immunoprecipitation (Co-IP). Lanes 1-6 correspond to the following experimental conditions: (1) non-transfected, (2) Na_v1.8 expressed alone, (3) Na_v1.5 expressed alone, (4 and 5) Na_v1.8 and Na_v1.5 co-expressed, (6) mixed lysates from lanes 3 and 4. Panel B shows the pull-down of Na_v1.5 is specific to Na_v1.8 cellular co-expression. Central Illustration: SCN5A and SCN10A, genes encoding cardiac and neuronal sodium channels, are found in close proximity on chromosome 3 (A). Our study suggests that mutations in SCN10A can lead to a loss of function in sodium channel current (INa) and thus contribute to the manifestation of Brugada syndrome (BrS), a sudden cardiac death syndrome. The data suggest physical association of the two channel proteins (NaV1.5 and NaV1.8) in the plasma membrane (B). Our study identifies SCN10A as a major susceptibility gene for BrS, thus greatly enhancing our capability to genotype and risk stratify probands and family members (C).

Figure 4. Representative Experiments Demonstrating Physical Interaction between Na_v1.8 and Na_v1.5

Panel A shows protein input and panel B shows protein isolated by the antibody pull-down co-immunoprecipitation (Co-IP). Lanes 1-6 correspond to the following experimental conditions: (1) non-transfected, (2) Na_v1.8 expressed alone, (3) Na_v1.5 expressed alone, (4 and 5) Na_v1.8 and Na_v1.5 co-expressed, (6) mixed lysates from lanes 3 and 4. Panel B shows the pull-down of Na_v1.5 is specific to Na_v1.8 cellular co-expression. Central Illustration: SCN5A and SCN10A, genes encoding cardiac and neuronal sodium channels, are found in close proximity on chromosome 3 (A). Our study suggests that mutations in SCN10A can lead to a loss of function in sodium channel current (INa) and thus contribute to the manifestation of Brugada syndrome (BrS), a sudden cardiac death syndrome. The data suggest physical association of the two channel proteins (NaV1.5 and NaV1.8) in the plasma membrane (B). Our study identifies SCN10A as a major susceptibility gene for BrS, thus greatly enhancing our capability to genotype and risk stratify probands and family members (C).