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Long QT syndrome (LQTS) is an inherited channelopathy that occurs in 1/2000 individuals (Schwartz et al.,2009). Pathogenic variants in at least 15 genes encoding ion channel proteins in cardiomyocytes have been associated with LQTS (Nakano & Shimizu, 2016). The most common types of LQTS are due to pathogenic variants in the genes KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3). Pathogenic variants in the LQTS genes result in a prolonged repolarization (QT interval), predisposing affected individuals to polymorphic ventricular tachycardia and sudden death before and after birth (Crotti et al.,2013; Schwartz, 2004). De novo pathogenic KCNH2 and SCN5A variants are more likely to manifest the signature LQTS rhythm of torsades de pointes and have a high mortality risk than familial LQTS (Cuneo et al., 2013; Strand et al., 2020). Mutations in the KCNH2 gene, which encodes hERG1, a voltage-gated potassium channel protein, account for 35–40 % of the diagnosed LQTS cases (Curran et al.,1995; Sanguinetti et al., 1995). As the genetic etiologies for unexplained stillbirth increase (Merc et al., 2024), patient-specific human induced pluripotent stem cells (hiPSCs) that can be differentiated into various cardiac lineage cells and used for cardiovascular disease modelling become more important (Sallam et al., 2014). Cardiomyocytes derived from hiPSCs (hiPSC-CMs) are emerging as a powerful system to study inherited LQTS caused by KCNH2 variants (Feng et al., 2021; Jain et al., 2023; Ukachukwu et al., 2022). Therefore, these three hiPSC lines generated in this study present a valuable platform for modelling LQTS pathogenesis and testing potential therapeutics. The clinical utility of this platform includes functionally assessing variants of uncertain pathogenicity and serving as an in vitro tool to determine the most effective pharmacotherapy for individuals.

In this study, we derived one hiPSC line from a fetus with congenital LQTS carrying a heterozygous c.1898A > G mutation in KCNH2 gene (USFi005-A). Two additional hiPSCs lines were derived from healthy biological parents of the fetus, including a 32-year-old female (USFi006-A) and a 34-year-old male (USFi007-A). Whole-genome sequencing demonstrated that the fetus carries c.1898A > G mutation, while both parents carry wild-type (WT) alleles of the KCNH2 gene. Amniotic fluid cells (AFCs) were collected from the fetus, and peripheral blood mononuclear cells (PBMCs) were obtained from her parents during a clinical visit. Somatic cell reprogramming was performed using non-integrated Sendai virus driven expression of human Yamanaka factors (OCT3/4, SOX2, KLF4, c-MYC). All three hiPSC lines exhibited typical stem cell morphology (Fig. 1A, Table 1) and expressed SOX2, NANOG, and OCT-3/4 pluripotency markers (Fig. 1B–D). The expression of these pluripotency markers was further quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). All three hiPSC lines showed comparable expression levels of NANOG and SOX2 to a control hiPSC line (CUSO-2) used in multiple previous studies (Fig. 1E) (Chi et al., 2019; Knight et al., 2021). Furthermore, all three iPSC lines can be differentiated into all three germ layers-endoderm, ectoderm, and mesoderm, demonstrating their pluripotency (Fig. 1F).

Cytogenetic analysis revealed USFi005-A and USFi006-A have normal 46XX karyotype, while USFi007-A has a 46XY karyotype (Fig. 1G). Sanger sequencing confirmed that the heterozygous KCNH2 mutation was presented only in USFi005-A, and not in USFi006-A or USFi007-A (Fig. 1H). Short Tandem Repeat (STR) analysis results verified that each iPSC line generated matched the identity of its respective somatic donor cell. Additionally, qPCR analysis demonstrated that expression of the non-integrating Sendai virus was present at low passage numbers (e.g., USFi006-A, p4), while absent at high passage numbers (e.g., USFi005-A, p13; USFi006-A, p10; and USFi007-A, p10) (Fig. 1I). Three hiPSC lines were negative for mycoplasma (Fig. 1J).