Identification of SCN5a p.C335R Variant in a Large Family with Dilated Cardiomyopathy and Conduction Disease

Figure 1. Identifying SCN5a p.C335R and TTN truncating variants by using precision diagnostic methods. (A) Patient’s pedigree showing co-segregation of an SCN5a C335R variant. The index patient (III. 3) was diagnosed with DCM and sick sinus syndrome at age 59 and underwent CRT-D implantation. She only carried the SCN5a C335R variant. Patient IV.1 was diagnosed with DCM and conduction disease at age 24. He carried both SCN5a and TTN truncating variants. (B) Masson Trichrome stained left ventricular endomyocardial biopsy of patient IV.1 with SCN5a and TTN truncating variants. Histopathological examination demonstrated extensive cardiac fibrosis in this patient with DCM and conduction disease. (C) Schematic representation of the Nav1.5 cardiac sodium channel. The variant is located in Ploop between S5 and S6 of Domain-I.

Figure 2. SCN5a p.C335R carriers show conduction disease and reduced left ventricular ejection fraction (LV-EF). All family members with SCN5a p.C335R variant showed significantly longer PQ (A) and QRS intervals (B), lower left ventricular EF (C), and higher atrial fibrillation (AF) rates (D) than other family members without this variant. All 4 family members carrying SCN5a p.C335R who received CRT-D were non-responders (E,F). T-Test; * = p ≤ 0.05, ** = p ≤ 0.01. Data are presented as mean ± SD.

Figure 3. Functional analysis of SCN5a p.C335R variant. (A) Na+ current traces from Nav1.5 wild-type and p.C335R transfected Xenopus oocytes. This showed a loss of function in Nav1.5 p. C335R. Sodium channel currents (INa) were evoked from −80 mV to 70 mV. Western blot analysis confirmed that the wild-type and mutated Nav1.5 were expressed and that loss of Nav1.5 function was not due to absent expression of the protein. Anti-Rad50 used as a reference. (B,C) Representative traces of INa in iPSC-CMs with wild-type and SCN5a p.C335R variant. Sodium channel currents (n = 9) were reduced in iPSC-CMs of the DCM patients.

Figure 4. Disturbed sarcomeric z-disc regularity in iPSC-CMs with TTNtv. Sarcomeric structure is visualized by immunofluorescence staining against α-actinin (green) and TitinM8/M9 (red). Scale bar = 50 μm. Using fast Fourier transformation (FFT), the sarcomeric pattern regularity of the α-actinin channel was analyzed. Four cardiac differentiations for control (n = 72) and 3 cardiac differentiations for each patient-specific iPSC line (each n = 54) were analyzed. Data are presented as mean ± SEM. p < 0.001 by one-way ANOVA with Tukey’s correction. *** = p ≤ 0.001.