p.D372H: A novel SCN5A mutation associated with Brugada syndrome

Abstract

BackgroundBrugada syndrome (BrS) is a genetic cardiac arrhythmia disorder inherited in an autosomal dominant manner, characterized by ST-segment elevation in the right precordial leads (V1-V3) on electrocardiograms (ECGs). This syndrome predominantly affects young individuals with structurally normal hearts and significantly increases the risk of ventricular arrhythmias and sudden cardiac death (SCD). The most common genotype found among BrS patients is caused by mutations in the SCN5A gene, which lead to a loss of function of the cardiac sodium (Na+) channel (Nav1.5) by different mechanisms. MethodsPlasmids containing SCN5A were constructed using PCR and site-directed mutagenesis to create the D372H mutation. HEK293 cells were cultured and transfected with the wild-type and mutant constructs. Patch-clamp recordings assessed sodium current characteristics. Confocal microscopy visualized channel localization. Quantitative RT-PCR analyzed mRNA expression levels, while Western blot evaluated protein expression using specific antibodies. We identified a novel missense mutation, D372H, in the SCN5A gene associated with Brugada syndrome. Functional assays in HEK293 cells expressing the D372H mutant revealed a near-complete loss of sodium currents. Subsequent experiments with co-transfection of WT and D372H plasmids demonstrated that co-expression led to a significant reduction in current density in WT-expressing cells (P < 0.05). The D372H mutation also resulted in a hyperpolarizing shift of approximately 20 mV in the voltage dependence of inactivation, while activation and recovery kinetics remained unaffected. Additionally, confocal microscopy showed reduced membrane localization of the D372H mutant, with a significant decrease in protein expression levels confirmed by Western blot and RT-qPCR analyses. ConclusionIn summary, our findings indicate that the D372H mutation in the Nav1.5 sodium channel leads to significant reductions in sodium current density, altered channel expression, and impaired membrane localization. These changes contribute to the pathophysiology of Brugada syndrome by disrupting cardiac action potential dynamics.