Unveiling the evolutionary code of NOTCH3: mammalian bioinformatics sheds light on human pathogenicity.

NOTCH3 is a highly conserved transmembrane receptor implicated in CADASIL, a hereditary small vessel disease driven by mutations in its extracellular EGF-like repeats. The mechanism by which these mutations cause pathology remains unclear. We present the first large-scale comparative bioinformatic analysis of NOTCH3 across 113 mammalian species, uncovering three novel insights: i) a remarkable evolutionary conservation of all 204 cysteines, with the only exception being eight naturally occurring cysteine mutations in jaguar (EGFr13-15); ii) a unique deletion in Brandt's bat regulatory region, which may expose it to proteases, potentially altering signaling; iii) a rare human NOTCH3-X1 isoform, absent in most mammals but shared with select primates, a bat, and elephants, involving a cysteine-depleting deletion spanning EGFr20-22. These features provide novel evolutionary insights into human pathogenicity and suggest testable targets for in vivo experiments. Our study highlights the potential of comparative bioinformatics to identify previously hidden functional elements in disease-associated mammalian proteins.