Abstract：
Pathogenic mutations in SCN5A could result in dysfunctions of Nav1.5 and consequently lead to a wide range of inherited cardiac diseases. However, the presence of numerous SCN5A-related variants with unknown significance (VUS) and the comprehensive genotype-phenotype relationship pose challenges to precise diagnosis and genetic counseling for affected families. Here, we functionally identified two novel compound heterozygous variants (L256del and L1621F) in SCN5A in a Chinese family exhibiting complex congenital cardiac phenotypes from sudden cardiac death to overlapping syndromes including sick sinus syndrome and dilated cardiomyopathy in an autosomal recessive pattern. In silico tools predicted decreased stability and hydrophobicity of the two mutated proteins due to conformational changes. Patch-clamp electrophysiology revealed slightly decreased sodium currents, accelerated inactivation, and reduced sodium window current in the Nav1.5-L1621F channels as well as no sodium currents in the Nav1.5-L256del channels. Western blotting analysis demonstrated decreased expression levels of mutated Nav1.5 on the plasma membrane, despite enhanced compensatory expression of the total Nav1.5 expression levels. Immunofluorescence imaging showed abnormal condensed spots of the mutated channels within the cytoplasm instead of normal membrane distribution, indicating impaired trafficking. Overall, we identified the loss-of-function characteristics exhibited by the two variants, thereby providing further evidence for their pathogenic nature. Our findings not only extended the variation and phenotype spectrums of SCN5A, but also shed light on the crucial role of patch-clamp electrophysiology in the functional analysis of VUS in SCN5A, which have significant implications for the clinical diagnosis, management, and genetic counseling in affected individuals with complex cardiac phenotypes.
摘要：
SCN5A的致病突变可能导致Nav1.5的功能障碍，从而导致广泛的遗传性心脏病。然而,大量意义未知的SCN5A相关变异体(VUS)的存在以及全面的基因型-表型关系给受影响家庭的精确诊断和遗传咨询带来了挑战.这里,我们在一个中国家族的SCN5A中从功能上鉴定了两个新的复合杂合变异体(L256del和L1621F),这些变异体表现出复杂的先天性心脏表型,包括心源性猝死和重叠综合征,包括常染色体隐性模式的病窦综合征和扩张型心肌病.计算机工具预测了由于构象变化导致的两种突变蛋白的稳定性和疏水性降低。膜片钳电生理学显示钠电流略有下降，加速失活,Nav1.5-L1621F通道中的钠窗口电流减少，而Nav1.5-L256del通道中没有钠电流。Western印迹分析显示质膜上突变的Nav1.5表达水平降低，尽管总Nav1.5表达水平的代偿性表达增强。免疫荧光成像显示细胞质内突变通道的异常凝聚点，而不是正常的膜分布。表明贩运受损。总的来说,我们确定了这两种变体所表现出的功能丧失特征，从而为其致病性提供了进一步的证据。我们的发现不仅扩展了SCN5A的变异和表型谱，但也揭示了膜片钳电生理学在SCN5A中VUS功能分析中的关键作用，这对临床诊断有重要意义，管理,以及患有复杂心脏表型的受影响个体的遗传咨询。
