Adolescent idiopathic scoliosis (AIS) is a common pediatric musculoskeletal disorder characterized by lateral curvature of the spine and associated with chronic pain and deformity. A substantial genetic contribution to AIS has been established by genome-wide association studies (GWAS), yet the functional consequences of most associated variants—particularly those located in non-coding regions—remain unclear. This study aimed to functionally characterize candidate regulatory variants in genomic regions linked to AIS risk, assaying their allele-specific regulatory activity in a disease-relevant cell type, chondrocytes, to map perturbed gene-regulatory elements and prioritize variants for follow-up.
The authors compiled 1,664 variant positions that are in linkage disequilibrium with 26 AIS lead variants reported by GWAS. From these positions they generated a library of 7,173 candidate regulatory sequences encompassing both reference and alternate allele contexts. Using a massively parallel reporter assay (MPRA) framework, the library enabled direct comparison of 1,664 reference alleles versus 4,708 alternate alleles to detect allele-dependent differences in regulatory activity.
The MPRA experiments were performed in two human chondrocyte cell lines: TC28a2 and SW1353. Chondrocytes were chosen because they are a major cell type implicated in AIS pathogenesis and because cartilage biology is relevant to spinal development and maintenance. The paired-cell-line design allowed assessment of consistency of regulatory effects across different chondrocyte contexts.
From the comparisons of reference and alternate alleles across the two chondrocyte cell lines, the analysis identified 92 variants that showed significant differential regulatory activity between alleles. Of these 92 variants, 79 were predicted to disrupt transcription factor binding sites (TFBS). The predicted TFBS disruptions often correlated with the observed direction and magnitude of regulatory change measured in the MPRA, suggesting that altered transcription factor binding is a common mechanism by which these non-coding variants influence regulatory activity in chondrocytes.
A notable finding highlighted by the authors is rs9496392, a single-nucleotide variant located near the ADGRG6 locus. This variant exhibited consistent differential regulatory activity in both TC28a2 and SW1353 cell lines in the MPRA. The AIS-associated risk allele at rs9496392 (T) is predicted to strongly disrupt multiple transcription factor binding sites, including a predicted disruption of SP1 binding. The consistent allelic effect across both chondrocyte models supports the variant’s functional relevance in cartilage-related regulatory contexts.
The pattern that most MPRA-identified variants are predicted to alter TFBSs points to perturbation of transcription factor–mediated regulatory networks as a major mechanism underlying the impact of non-coding AIS-associated variants. Correlations between predicted TFBS disruption and measured regulatory activity suggest specific transcription factors may mediate allelic effects, though the preprint reports prediction-based associations rather than direct in vivo TF binding measurements.
ADGRG6 (the locus near which rs9496392 maps) is described as a key regulator of cartilage homeostasis. Supporting its relevance to spinal morphology, cartilage-specific knockout of ADGRG6 in mice produces a scoliosis-like phenotype. The functional evidence for rs9496392 altering regulatory activity near ADGRG6 therefore provides a plausible mechanistic link between a GWAS-associated locus, altered gene regulation in chondrocytes, and a phenotype relevant to AIS.
The authors present a catalog of candidate regulatory variants active in chondrocytes and note concordant predictions linking many variants to TFBS disruption. The study is based on MPRA assays in cell lines and TFBS predictions; the preprint does not report orthogonal in vivo validation for most variants beyond the highlighted rs9496392. Additional functional follow-up, direct TF binding assays, and in vivo studies would be required to establish causal links between specific regulatory changes and AIS pathogenesis. Supplementary materials accompanying the preprint provide further experimental details and data.
The authors declare no competing interests. Funding is reported from the Scoliosis Research Society. The preprint and associated supplementary material were posted on bioRxiv, and the manuscript is made available under a CC-BY-NC-ND 4.0 International license.