Identification and validation of CARS1 p.E712V and NF1 p.Q2002X in sporadic Moyamoya disease across 30 trio pedigrees

Moyamoya disease (MMD) is a progressive cerebrovascular disorder with intracranial arterial stenosis and collateralization. Over 70% of sporadic cases lack known genetic drivers; RNF213 variants explain only 23% of Chinese cases, highlighting unmet diagnostic and therapeutic needs. Trio-based whole-exome sequencing (WES) of 126 Chinese sporadic MMD patients (30 pediatric-parent trios) underwent cross-platform validation, cohort screening (n = 268), and functional analysis in human brain microvascular endothelial cells (HBMECs). Variants were prioritized by population frequency, pathogenicity, and case-control comparisons. Endothelial function and oxidative stress were assessed via proliferation, migration, tube formation, and molecular markers. WES identified 42 rare sporadic and 15 de novo mutations; Sanger validation confirmed 11 sporadic/11 de novo variants including de novo NF1 p.Q2002X and recurrent CARS1 p.E712V. CARS1 p.E712V carriers showed early-onset stenosis (mean age 7.5 ± 4.4 years) and right-dominant Suzuki stage ≥4, while NF1 p.Q2002X correlated with severe bilateral stenosis in a child (onset age 3). CARS1 p.E712V showed significant patient enrichment (P = 0.004). Silencing NF1/CARS1 in HBMECs enhanced proliferation, migration, tube formation, and reduced GPX4 (CARS1-specific). CARS1 mutation augmented angiogenesis, indicating functional alteration. This study identifies NF1 and CARS1 as novel susceptibility genes for sporadic MMD in Chinese, expanding the genetic landscape beyond RNF213.