Alphafold, Foldseek and MD in NOTCH3 variants: a cohort study

Background and ObjectivesNotch homolog 3 (NOTCH3) gene variants were fully penetrant to produce the disease phenotype of CADASIL. Aberrant NOTCH3 protein leads to degeneration of vascular SMCs and pericytes, targeting microcirculation dysfunction and blood-brain barrier (BBB) leakage. MethodsWe evaluated neuroimaging data of forty patients with NOTCH3 gene variants including eighteen missense/insertion mutations in epidermal growth factor repeat (EGF), negative regulatory region (NRR), and disordered region (Dis). We performed an AI-driven pipeline integrating AlphaFold3, Foldseek, and molecular dynamics simulations to elucidate clinical and molecular consequences. ResultsDistinct domain mutations exhibited characteristic patterns: EGFs 1, 2, 13-15, 32 and Dis correlated with microbleeds/macro-bleeds, lacunes, perivascular spaces, and acute cerebral microinfarcts; EGFs 2, 3, 13-15, 25 with disrupted disulfide bonds or binding motif of protein O-glucosyltransferase 1 (POGLUT1) were predicted to undergo greater structural and functional deteriorations in Notch signaling pathways. NRR/Fab (antigen-binding fragment) destabilized dominant motions and single apo-Dis exhibited low structural disorder. Agreement between computational and experimental data for wild-type EGFs/POGLUT1 and C49F, R75Q, R141C mutants suggests testable hypotheses that advance understanding of cerebral small-vessel disease. DiscussionTargeting POGLUT1 to modulate EGF-like domains and using the Fab region to stabilize NRR complexes may be a promising therapeutic approach deserving rigorous exploration.