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Int J Mol Sci
2021 Nov 30;2223:. doi: 10.3390/ijms222312990.
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Identification of SCN5a p.C335R Variant in a Large Family with Dilated Cardiomyopathy and Conduction Disease.
Sedaghat-Hamedani F
,
Rebs S
,
El-Battrawy I
,
Chasan S
,
Krause T
,
Haas J
,
Zhong R
,
Liao Z
,
Xu Q
,
Zhou X
,
Akin I
,
Zitron E
,
Frey N
,
Streckfuss-Bömeke K
,
Kayvanpour E
.
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INTRODUCTION: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease.
METHODS: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed.
RESULTS: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na+ channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM.
CONCLUSION: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.
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.
Supplemental Figure S1: Exercise-induced high grade atrioventricular (AV) block in patient with SCN5a C335R and TTNtv (IV.1).
Supplemental Figure S2 : Generated patient-specific induced pluripotent stem cells (iPSC) show full pluripotency characteristics (A-D). Pluripotency characterization is shown exemplary for iPSC clones from patient IV.14. A: The iPSC exhibited typical round colony-forming stem cell morphology (left) and were positive for alkaline phosphatase activity (right). Scale bars: 200 μm. B: The generated iPSC express pluripotency markers OCT4, SOX2, LIN28 and GDF3 on the mRNA level in similar amounts to an already published iPSC line (PC = positive control). Somatic cells like fibroblasts (FB) and mouse embryonic fibroblasts (MEF) are negative for expression of these pluripotency genes. GAPDH was used as a housekeeping gene. iPSC-IV.14 c1 and iPSC-IV.14 c2 represent iPSC clone 1 and 2 of patient IV.14. C: The iPSC cell lines express pluripotency markers on the protein level, which is visualized by immunoflurescent stainings against OCT4, SOX2, NANOG, LIN28 and TRA1-60. Scale bar: 100 μm. D: In vitro embryoid body formation assay confirmed differentiation capacity into all three germlayers. Postive signals were obtained after immunofluorescent staining against endodermal marker AFP, mesodermal marker α- SMA and ectodermal marker β-tubulin (β-TUB). Scale bars: 100 μm.
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