Patient-derived induced pluripotent stem cells (hiPSC) are a valuable approach to model cardiovascular diseases. We nucleofected non-integrating episomal vectors in skin fibroblasts of four family members. Two of them carried the single nucleotide variant (SNV) SCN5A_c.287 T > C, leading to NaV1.5_p.L96P, and two were non-carrier family members. The resulting hiPSC cell lines differentiate into cells of the 3 germ layers, display normal karyotypes and express markers of the undifferentiated hPSC state. Thus, they are a reliable source to study the effect of the identified mutation in a physiologically relevant environment.
E. Selga, R. Martínez-Moreno, Albert Rigat Pujolàs et al.· Stem Cell Research· 0 citations
Molecular autopsy can identify candidate variants in unexplained sudden cardiac death (SUD), but functional evidence is often required to support pathogenic interpretation and family risk assessment. Here, we investigated the functional consequences of the SCN5A c.287 T > C (NaV1.5_p.Leu96Pro) variant, identified in a 29-year-old man who died of SUD. Cascade screening identified additional relatives carrying the variant, with variable clinical expression. Sodium current (INa) was analyzed in heterologously transfected human embryonic kidney (HEK) tsA201 cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from two variant carriers and two non-carrier relatives. SCN5A transcript levels, NaV1.5 membrane expression, and additional arrhythmia-associated genetic variation were also assessed. In HEK tsA201 cells, NaV1.5_p.Leu96Pro produced no measurable INa when expressed alone, whereas co-expression with wild-type SCN5A caused an approximately 50% reduction in peak INa density. Cell-surface biotinylation showed preserved total and membrane NaV1.5 expression, indicating that loss of current was not explained by impaired trafficking. hiPSC-CMs from both carriers showed reduced INa density compared with non-carrier relatives, despite no reduction in SCN5A transcript levels. Targeted sequencing did not identify variants explaining the differences between the two hiPSC-CM carriers, but revealed an additional SCN5A splice-site deletion in family members with more severe clinical manifestations. These findings show that NaV1.5_p.Leu96Pro causes severe loss of sodium channel function and support the value of combining molecular autopsy, family evaluation, and functional studies for variant interpretation in SUD.
Albert Rigat Pujolàs, R. Martínez-Moreno, David Carreras et al.· Forensic Science Internation...· 0 citations
