Unravelling the pathophysiology of oculocutaneous albinism type 8 using patient iPSC-derived retinal pigment epithelium

Submission 81

PS2-64-Poster Presentation

Presented by: Daria Mamaeva

Daria Mamaeva ¹, Nejla Erkilic ^{1, 2}, Hassan Boukhaddaoui ¹, Maria Cruz-Santos ¹, Christel Vache ^{1, 3}, Laurent Guillou ¹, Emeline Nandrot ⁴, Anne-Françoise Roux ^{1, 3}, Isabelle Meunier ^{1, 2}, Marlin Marlin ^{5, 6}, Sophie Javerzat ⁷, Benoit Arveiler ^{7, 8}, Vasiliki Kalatzis ^{1, 2}

¹ Institute for Neurosciences of Montpellier, University of Montpellier, Inserm, Montpellier, France

² National Reference Center for Inherited Sensory Diseases, University of Montpellier, CHU, Montpellier, France

³ Molecular Genetics Laboratory, University of Montpellier, CHU Montpellier, Montpellier, France

⁴ Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012 Paris, France

⁵ National Reference Centre for Genetic Hearing Loss, Genomic Medicine Federation, Hôpital Necker-Enfants Malades, AP-HP, University Paris Cité, Paris, France

⁶ Laboratory of Genetics of rare ophthalmological, auditory and mitochondrial disorders, Imagine Institute, INSERM UMR 1163, University Paris Cité, Paris, France

⁷ Rare Diseases, Genetics and Metabolism, INSERM U1211, University of Bordeaux, Bordeaux, France

⁸ Molecular Genetics Laboratory, Bordeaux University Hospital, Bordeaux, France

Objective

Pathogenic variants in the Dopachrome tautomerase (DCT) gene have been linked to a newly identified oculocutaneous albinism type, OCA8. We generated a human induced pluripotent stem cell (iPSC) line from dermal fibroblasts of a compound heterozygous individual carrying two pathogenic DCT variants: a missense mutation in exon 1 (c.118T>A, p.Cys40Ser) and a 14-bp deletion in exon 9 (c.1406_1419del, p.Phe469*). This line was differentiated into a retinal pigment epithelium (RPE) to generate a physiologically relevant in vitro model for studying OCA8 pathophysiology.

Methods

Patient-derived fibroblasts were reprogrammed into iPSCs using a non-integrative Sendai virus method. Clones were validated via immunofluorescence, qPCR, and Sanger sequencing. iPSCs were differentiated into RPE, which was characterized using immunofluorescence, western blot and calcium imaging analyses. Ultrastructural analysis was performed by Transmission electron microscopy (TEM). To assess functional changes, we performed assays for photoreceptor outer segment phagocytosis, VEGF and PEDF secretion, and L-DOPA production.

Results

A DCT-OCA8-iPSC line was successfully generated and validated. Karyotyping showed no chromosomal abnormalities. Immunofluorescence confirmed nuclear expression of the pluripotency markers SOX2, NANOG, and OCT3/4. qPCR corroborated expression of NANOG, OCT3/4, and LIN28A. Trilineage differentiation potential was confirmed via embryoid body formation. Immunostaining showed expression of Nestin and GFAP (ectoderm), SMA and Vimentin (mesoderm), and AFP and FOXA2 (endoderm). Microsatellite DNA profiling across nine loci confirmed identity with the parental fibroblasts. The presence of the two DCT variants was verified by Sanger sequencing. RPE derived from DCT-OCA8-iPSCs showed altered expression of key RPE markers. TEM revealed ultrastructural changes in DCT-OCA8-RPE compared to wild-type RPE. Calcium imaging indicated altered calcium signaling in DCT-OCA8-RPE cells.

Conclusion

We have established and comprehensively characterized a DCT-OCA8-iPSC line, which was differentiated into RPE to provide a robust in vitro model for elucidating the molecular and cellular mechanisms underlying OCA8. This iPSC-derived platform offers a valuable resource for functional studies and may facilitate the development and evaluation of targeted therapeutic interventions.