Massively parallel characterization of adolescent idiopathic scoliosis risk variants.

Adolescent idiopathic scoliosis (AIS) is a common pediatric musculoskeletal disorder characterized by lateral spinal curvature, often leading to chronic pain and deformity. Although a significant genetic component to AIS is recognized, the functional impact of most associated genetic variants, particularly those in noncoding regions, remains largely unknown. Using massively parallel reporter assays, we characterize 1664 variant positions in linkage disequilibrium with 26 AIS lead variants identified by genome-wide association studies (GWASs) in chondrocytes, a major cell type implicated in AIS pathogenesis. Using a library of 7173 candidate regulatory sequences, we compare the 1664 reference alleles against 4708 alternate alleles in two human chondrocyte cell lines (TC28a2 and SW1353). Our analysis identifies 92 variants that exhibit significant differential regulatory activity between their reference and alternate alleles, 79 of which are predicted to disrupt transcription factor binding sites, often correlating with their observed regulatory effect. Notably, we validate rs9496392, a single-nucleotide variant near the