Abstract:
Inherited retinal degenerations (IRD) are among the leading causes of blindness in people under the age of 45 years. Crumbs homologue 1 (CRB1) is one of the major genes mutated in patients with autosomal-recessive Leber congenital amaurosis, the most severe form of IRD, and Retinitis pigmentosa (RP). There is currently no treatment available to stop vision loss in these patients. The aim of this study was to identify the molecular mechanisms and potential modifiers underlying CRB1-associated IRD. To address this aim, two siblings, who were diagnosed with early-onset RP due to a homozygous CRB1 c.2843G>A;p.C948Y mutation, were studied. Interestingly, while one of the siblings was blind (referred to as patient A), the other sibling was significantly less affected (referred to as patient B). Using western blot and immunofluorescence microscopy, results obtained revealed that canonical CRB1 protein levels are reduced in induced pluripotent stem cell (iPSC)-derived retinal organoids (RO) of both patients compared to control indicating that any process requiring the full-length CRB1 protein may be affected. To further investigate the function of CRB1, the retinal interactome was studied using a porcine retinal pull-down approach followed by mass spectrometry. Novel members of the retinal CRB1 complex were identified including regulators of the actin cytoskeleton, vesicular trafficking and lipid metabolism. To further identify potential candidate modifiers of disease severity, whole genome sequencing and full-proteome analysis of iPSC-derived RO were performed. One of the potential protective candidate modifiers identified was the Crumbs family member CRB2. CRB2 was significantly more abundant in RO of the less affected patient B compared to control and patient A RO. In addition, a heterozygous single nucleotide variant upstream of the CRB2 gene was identified in patient B. As several studies highlight the modulatory and rescue ability of CRB2 in CRB1-associated IRD, the relationship of CRB1 and CRB2 was further investigated. The results obtained show that CRB1 and CRB2 form homo- and heteromeric complexes. Furthermore, human CRB2 was able to pull-down endogenous CRB1 from porcine retinal lysates and the retinal interactome of CRB2 revealed high overlap with that of CRB1, suggesting that CRB1 and CRB2 may be involved in similar cellular processes.
Crumbs homologue 1