Molecular characterization of an iPSC line derived from a patient with cone‐rod dystrophy related to a mutation in the PROM1 gene

Abstract

Aims/Purpose: The PROM1 gene encodes the protein Prominin‐1, which plays a crucial role in the morphogenesis of photoreceptor outer segments. The c.1354dupT mutation, associated with various inherited retinal diseases (IRD) phenotypes, leads to a premature stop codon. This study aimed to assess the silencing efficiency of reprogramming vectors and the expression of endogenous pluripotency genes in the iPSC line [DCB]‐FiPSC1‐Ep5F‐2.Methods: The iPSC line [DCB]‐FiPSC1‐Ep5F‐2 was generated from a patient with Cone‐rod dystrophy (CRD) linked to the c.1354dupT mutation in the PROM1 gene. iPSC colonies were stained using alkaline phosphatase (AP) blue membrane substrate. The c.1354dupT mutation was Sanger sequenced. To confirm the absence of episomal vector integration, quantitative PCR (qPCR) targeting a region common to all reprogramming vectors (within the EBNA1 gene) was conducted. Vector‐specific primer‐based quantitative reverse transcription PCR (qRT‐PCR) was used to assess the expression of reprogramming genes from the episomal vectors. Additionally, qRT‐PCR with gene‐specific primers was utilized to evaluate the expression of endogenous pluripotency genes. Untransfected fibroblasts served as the negative control, while fibroblasts transfected with the episomal vectors 72 h earlier were used as the positive control.Results: The iPSC line [DCB]‐FiPSC1‐Ep5F‐2 exhibited positive AP activity. The presence of the c.1354dupT mutation was confirmed. Furthermore, the number of copies of the EBNA1 gene was significantly lower in the generated iPSC line compared to the control. The relative mRNA expression of the reprogramming vectors was also significantly lower in the iPSC line, while the relative mRNA expression of the endogenous pluripotency genes was significantly higher than in the control.Conclusions: While further immunocytochemical characterization is necessary to fully ascertain iPSC pluripotency, the patient‐derived iPSC line [DCB]‐FiPSC1‐Ep5F‐2 shows promise as a disease model for personalized therapy development.