Abstract
Adolescent idiopathic scoliosis (AIS), a 3-dimensional curvature of the spine with unknown aetiology exhibits a severe sex bias, with 9 out of 10 severe cases occurring in females. Genome wide association studies (GWAS) have identified numerous loci associated with AIS, although only one locus has been identified as sex-biased. GWAS identified a single nucleotide polymorphism (SNP) (rs6137473) significantly associated with female AIS risk. As this region is not conserved in mice, a mouse model was generated by deleting a conserved region nearby that we hypothesised is a sex-associated region (SAR) gene regulatory element. This was based on chromatin accessibility and the presence of relevant transcription factor-binding motifs. This study involved phenotyping this mouse line, focusing on sex-dependent changes in gene expression and degenerative changes arising in the intervertebral disc (IVD) that underlie a scoliotic-like phenotype.
Micro CT analysis showed a significant increase in vertebral rotational degree at T4 in the female Pax1-SARD mice at 4 months with evident rotational instability in the female wildtype (WT) and Pax1-SARD mice. It also showed an increase in vertebral rotation at T9 in the male Pax1-SARD mice at 6 months, showing much greater instability than the male WTs. Male Pax1-SARD mice also displayed lordotic sagittal curvature at 6 months, whereas female Pax1- SARD mice showed a more kyphotic sagittal curve, possibly relating to the site of maximal vertebral rotation. Male Pax1-SARD mice were found to have abnormal thoracic skeletal architecture, including smaller IVDs and vertebral bodies than their wild-type counterparts.
Histological observation of male WT mice showed healthy IVDs with minimal degenerative changes. In contrast, the female WT and male and female Pax1-SARD showed significant signs of degeneration with tears in the AF, disorganised AF structure, infiltration of hypertrophic chondrocytes, and a loss of clear AF/NP boundary which was supported by grading results for IVD degeneration. Both the male and female Pax1-SARD showed significant decreases in CS/DS content, as well as glycosaminoglycans (GAGs), underlying the degenerative changes observed in the IVD.
Gene expression analysis showed significant upregulation of Pax1, Stat3, Ar, and Nkx2.2 in the Pax1-SARD mice at E12.5 during the initial patterning of the IVD and VB. RNA-sequencing after IVD development (E18.5) showed sex-dependent changes in gene expression in response to Pax1-SARD with males showing a large number of differentially expressed genes to the male WT with significant downregulation of genes relevant in structural components of the extracellular matrix (Acan, Col2a1, Col11a2, Comp), immune and inflammatory response (Il- 10, Ccl2, Thbs1), and scoliosis (Slc26a2) and upregulation of genes relating to fibrinolysis (Fga, Fgg, Fgb, Plg), steroid hormone biosynthesis (Cyp3a16), and metabolism (Cycs). Female Pax1-SARD showed a small number of differentially expressed transcripts to the female WT with significant upregulation of genes relating to fibrinolysis (Fga, Fgg, Fgb) and scoliosis (DLL3).
The current results underscore the crucial role of the Pax1 sex-associated region, implicated in human AIS, in the development and maintenance of functional cartilage, ECM integrity, and disc health. These findings reveal the intricate mechanisms underlying spinal degeneration and instability, with notable sex-specific phenotypic variations.