Combining molecular and functional genetics to decipher pathophysiological mechanisms in oculocutaneous albinism. – OCAGEN

Albinism is a rare disease associating generalized hypopigmentation and severe visual deficits in patients. Currently, the link between altered pigmentation and the retinal development anomalies that lead to impaired vision remain poorly understood. Albinism comprises a group of clinically and genetically heterogeneous disorders with 21 identified causative genes. The analysis of their coding regions has allowed the molecular diagnosis of ~70% of cases. For the remaining 30%, pathogenic variants may reside in the non-coding regions of the genes, or in novel genes as yet unidentified. In addition, many variants of unknown significance (VUS) exist, which also prevent diagnosis. The goal of our project is to address two major hurdles in albinism research. First, to improve genetic diagnosis by searching for variants in the regulatory elements of causative genes and by the functional characterization of VUS. Second, to establish the molecular links between genetic anomalies, functional pathways and retinal alterations. Towards this aim, we will focus on 5 forms of oculocutaneous albinism and their corresponding genes: OCA1 (TYR), 2 (OCA2), 3 (TYRP1), 4 (SLC45A2) and 8 (TYRP2/DCT).
The regulatory elements of these genes are poorly known. We will characterize these elements using a promoter capture Hi-C approach, which will complement a bioinformatics approach already performed in the laboratory. We will screen for pathogenic variants in the regulatory elements following whole genome sequencing of patients carrying heterozygous variants in one of the 5 genes, and then validate the variants by functional studies.
In parallel, we will develop a rapid functional assay based on pigmentation rescue in MNT-1 melanocyte cell lines to decipher the pathogenicity of VUS. To this end, the 5 genes will be knocked out by CRISPR-Cas9 to produce independent lines that can be transfected with candidate VUS cDNAs. The pathogenicity of each VUS will be established by comparing the test result to that of known pathogenic and benign variants. In addition to the simple pigmentation phenotype and melanin assay, additional studies such as intracellular trafficking and melanosome pH measurements will be performed, especially for VUS that confer an intermediate phenotype.
Furthermore, the knock-out lines will be exploited to answer the fundamental biological questions. In particular, we will perform qualitative and quantitative analyses of the different metabolic intermediates of the melanin biosynthesis pathway (including L-DOPA) to better understand the underlying signalling pathways and disease pathophysiology.
The same CRISPR-Cas9-mediated approach will be used to produce isogenic OCA knock-out induced pluripotent stem cell (iPSC) lines that we will differentiate into retinal pigment epithelium (RPE) cells and retinal organoids. In this way, for the first time, we will directly investigate the impact of OCA gene invalidation on the morphology and functionality of human RPE. In parallel, iPSC will be conjointly differentiated into retinal organoids, to study the impact of neighbouring OCA-null RPE on retinal development and thus to better understand the origin of visual anomalies in patients. Conversely, the iPSC-derived RPE knock-out lines will also serve to validate candidate RPE-specific regulatory elements and the pathogenicity of VUS, in addition to the studies performed in MNT-1 lines.