Functional investigation of a novel familial SCN5A mutation causative of Long QT syndrome.

Federica Diofano (Ulm)1, K. Weinmann-Emhardt (Ulm)2, M. Däuwel (Ulm)1, R. Pfluger (Ulm)1, L. Hartmann (Ulm)1, W. Rottbauer (Ulm)2, S. Just (Ulm)3

1Uniklinik Ulm Internal Medicine 2 Ulm, Deutschland; 2Universitätsklinikum Ulm Klinik für Innere Medizin II Ulm, Deutschland; 3Universitätsklinikum Ulm Molekulare Kardiologie, Klinik für Innere Medizin II Ulm, Deutschland


 Background: Genetic testing of a 37-year-old female patient with severe reduced left ventricular ejection fraction, recurrent ventricular tachycardia and a family history of heart failure and sudden cardiac death, rendered a heterogeneous missense mutation in Exon 9 in the SCN5A gene (c.1072T>C) with a predicted amino acid exchange of phenylalanine to leucine (p.PHE358LEU).

Methods and Results: This study utilized patient-derived iPSC cells and the isogenic control generated by CRISPR-Cas9 technology. Both iPSC cell lines were further differentiated into ventricular cardiomyocytes (CMs) and analyzed in detail. In addition, a transgenic zebrafish line overexpressing the patient´s mutation was established and functionally characterized.

Compared to the isogenic control, patient derived-CMs did not exhibit any significant alteration in protein or mRNA levels. Furthermore, they maintained a normal sarcomeric structure. However, functional analysis revealed altered calcium (Ca2+) dynamics consistent with a Long QT syndrome phenotype: a reduced time of peak (isogenic ctrl:1505±483,1ms; SCN5A1072T>C:703,7±230,5ms  ****p=<0,0001), reduced half width (isogenic ctrl:957±181,4ms; SCN5A1072T>C: 701,6±43,8ms ****p=<0,0001), reduced rise time (isogenic ctrl: 231,8±105,2ms; SCN5A1072T>C: 147,6±63,03ms  ****p=<0,0001) and reduced decay time (isogenic ctrl: 1277± 414,7ms; SCN5A1072T>C :556,1± 198,5ms  ****p=<0,0001). These findings were corroborated by ECG recordings from zebrafish transgenic embryos overexpressing the mutation. At 72 hours post fertilization the embryos overexpressing the mutation show a prolonged QTc (measured using the Fridericia formula) compared to wild-type siblings (wild-type:442,4±35,55; TgSCN5A:489,3±36,5 ***p=0,0008) together with an altered ST duration (wild-type: 183,6±31,44; TgSCN5A: 211,9±30,11 **p=0,0073) suggesting an impaired repolarization process.

Conclusion: This study, demonstrated for the first time, using the zebrafish model and patient-derived iPSC-CMs,  a novel SCN5A mutation responsible for Long QT syndrome.

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