Abstract
Introduction Several mutations in ABHD12 gene have been linked to PHARC syndrome, and nonsense mutations represent almost one third of the reported mutations in PHARC. The use of induced pluripotent cells (iPSC) helps to create faithful models to investigate the pathological mechanisms operating in the retina of PHARC patients, and thus facilitates the assessment of potential therapeutic interventions.
Aims We aimed to characterise human dermal fibroblasts (HDF) from a PHARC patient harbouring a nonsense mutation in ABHD12 gene; and to generate one human iPSC line from the PHARC HDF.
Methods HDF from a PHARC patient harbouring a nonsense mutation in ABHD12 gene were isolated and cultured. Gene expression was assessed by qPCR. ABHD12 presence was detected by SDS-PAGE and immunoblotting. Proteins involved in primary cilia morphogenesis were examined by immunofluorescence. HDF were reprogrammed into iPSC using integration free episomal reprogramming plasmids.
Results ABHD12 possess two alternative splicing isoforms (ABHD12-a and ABHD12-b). At a protein level, WT HDF express both of them, while PHARC HDF only express ABHD12-b. Cilia in PHARC HDF are less numerous and shorter than in WT. The transcript levels of GRP78/BiP, involved in unfolded protein response activation, are downregulated in PHARC compared to WT HDF. The iPSC line generated exhibits pluripotency markers and differentiation potential in vitro.
Conclusion A proportion of ABHD12 transcripts are escaping the nonsense mediated decay in PHARC HDF. Moreover, we report for the first time a defective cilia formation and a reduction in GRP78/BiP mRNA levels in PHARC HDF.
