FishCOLler 2.0: an in vivo functional platform to assess variants pathogenicity and validate targetable patho-mechanisms underlying COL4A1/A2 cerebrovascular disease

COL4A1/A2 syndrome is a severe multisystem disorder with variable age of onset, no cure and limited guidelines to prevent life-threatening complications. It is caused by inherited or new mutations in COL4A1 and COL4A2, encoding key structural components of the basement membrane — an essential matrix supporting cells in all tissues, including the brain vasculature and the eyes. Most frequent manifestations are intracerebral hemorrhage (ICH), stroke, epilepsy, and ophthalmological impairment, which can arise even prenatally. Although genetic testing now identifies mutations more rapidly, determining their clinical significance remains a major challenge: many variants are unclassified, leaving patients and families without a clear diagnosis or guidance. Research in mice has been fundamental, but rodent studies are slow and costly, and incompatible with the pace at which new variants awaiting functional validation are identified. Furthermore, identifying disease mechanisms and molecules in vivo remains critical to initiate pre-clinical studies. Within FishCOLler, the first Telethon project on the disease, we developed in vivo disease models in the zebrafish — a rapid, genetically tractable vertebrate system — and demonstrated their ability to functionally test variant pathogenicity and highlight patho-mechanisms. FishCOLler 2.0 now builds on these tools and, supported by expert collaborators in genetics, disease modelling and clinical aspects, will: 1. functionally assess a clinician-prioritised variants panel to determine pathogenicity and severity across brain and eye phenotypes; 2. Model for the first time how brain circuit activity is altered and the risk associated with epilepsy; 3. validate emerging targetable disease molecules and test proof-of-principle rescue with approved drugs. Together, these results will concretely support patient diagnosis and management, provide evidence for ongoing natural history studies and accelerate pre-clinical developments.