Pathogenic MYRF ICA Domain Variant is Aberrantly Trafficked in the RPE and Results in Isolated Nanophthalmos

Abstract

Purpose: Myelin regulatory factor is a transcription factor essential for RPE development and serves as a critical "GO" signal for eye growth. Pathogenic variants in MYRF manifest in a spectrum of clinical phenotypes ranging from isolated nanophthalmos to severe syndromic disease, in a gene dosage dependent manner. In this study, we aimed to determine the pathogenicity and molecular mechanism of a novel variant in the MYRF ICA domain, c.2083C>T (p.R695C), that leads to isolated nanophthalmos and mild retinal pigment epithelial disruption. Methods: Whole genome sequencing was performed and identified a novel MYRF variant. Pathogenecity and in silico modeling were evaluated using the Ensembl VEP tool and PyMOL. Human primary RPE and/or ARPE-19 cells were transduced with FLAG-tagged variant and wildtype MYRF or double-tagged FLAG-MYRF-MYC proteins to evaluate transcriptional activity, localization, and protein trafficking. Results: In silico modeling supports variant pathogenicity and altered homotrimer conformation. Functional studies in human primary RPE (hRPE) and/or ARPE-19 cells confirmed that MYRF p.R695C is cleavage deficient, mislocalized to the apical surface of polarized RPE, and demonstrated reduced synergistic transcriptional activity with co-factor SOX10 in HEK293T cells. Protein trafficking studies demonstrated that wildtype MYRF is trafficked to the apical cell surface and plasma membrane and that endogenous MYRF requires ER to Golgi trafficking pathways for proper nuclear localization and cleavage dynamics. Conclusions: Together, these results confirm that p.R695C is pathogenic for nanophthalmos, implicate trafficking as a regulatory mechanism of MYRF in the hRPE, and support that RPE disruption as a feature of MYRF-associated nanophthalmos.