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. While 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 examined the regulatory activity of 1,664 variants in linkage disequilibrium with 26 AIS lead variants identified through genome-wide association studies (GWAS). Candidate regulatory sequences containing the reference or alternative alleles were tested in two human chondrocyte cell lines, TC28a2 and SW1353, as chondrocytes are a major cell type implicated in AIS pathogenesis. We identified 92 variants with significant allele-specific regulatory activity, 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 ADGRG6 locus, which shows consistent differential regulatory activity in both cell lines. ADGRG6 is a key regulator of cartilage homeostasis, and its cartilage-specific knockout in mice results in a scoliosis-like phenotype. The AIS risk allele of rs9496392 (T) is predicted to strongly disrupt several TFBSs, including SP1. This study provides a foundational catalog of functional AIS-associated regulatory variants active in chondrocytes, offering crucial insights into the perturbed gene regulatory networks in AIS. These findings lay the groundwork for identifying biomarkers and potential therapeutic targets for this complex childhood disease.