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Abstract

Congenital Disorders of Glycosylation (CDG) compromise a rapidly expanding group of inherited metabolic disorders, with to date over 190 identified defects. SRD5A3-CDG is an ultra-rare subtype clinically presenting common CDG manifestations such as neurological, developmental impairments and failure to thrive. Characteristic for SRD5A3-CDG are severe ocular abnormalities, including retinitis pigmentosa and coloboma – which represent the clinical key features in the diagnostic. To date, the molecular mechanisms underlying the ocular manifestations remain poorly understood. CYP1B1, a cytochrome P450 enzyme, is primarily involved in detoxification processes and the regulation of steroid hormones, but it is also essential for physiological eye development. CYP1B1 deficiency is linked to congenital primary glaucoma, while overexpression in zebrafish has been shown to induce coloboma. My study aims to explore the potential connection between SRD5A3-CDG and CYP1B1. For this, I used fibroblasts from SRD5A3-CDG patients and generated and established a CRISPR-Cas9-based Srd5a3 knockout model in medaka (Oryzias latipes) embryos, representing clinical features of SRD5A3-CDG patients. My results reveal an upregulation of CYP1B1 expression in patient-derived fibroblasts and in Srd5a3 knockout medaka fish embryos – assessed by multiple experimental approaches, including qRT-PCR, nCounter targeted analysis, transcriptome analysis, and proteomic investigations (Western blotting and mass spectrometry). Elevated Cyp1b1 expression in Srd5a3-KO medaka fish embryos correlate strongly with the development of coloboma. Additionally, mass spectrometry and transcriptome analysis reveale elevated levels of CYP1B1 also in a broad variety of CDG types, including late and early CDG subtypes. My FACS analysis of SRD5A3-CDG fibroblasts indicate increased oxidative stress. This points to a disruption in the AhR-Nrf2 signaling pathway leading to elevated expression of CYP1B1 since AhR regulates its expression and Nrf2 plays a key role in the oxidative stress response. Downregulation of AhR and its repressor AhRR as well as potential decreased activity of Nrf2 propose instead that rather unconventional and yet unidentified pathway which are associated with general glycosylation defects are activated or prone to induce an elevated expression of CYP1B1, resulting in ophthalmological abnormalities – independent of the type of CDG. My findings provide valuable insights into the molecular mechanisms driving the ocular phenotype in SRD5A3-CDG, highlighting the elevated expression of CYP1B1 as a potential key factor. These results emphasize the critical role CYP1B1 among a variety of CDG types and opens opportunities for novel therapeutic strategies to treat ophthalmological abnormalities.