Runx2 Mutation Plays A Key Role in the Development of Scoliosis

Dear Editor,

Scoliosis is an abnormal lateral curvature of the spine with Cobb angle more than 10°.1 The detailed genetic etiology and underlying mechanisms of this disease are still unclear. We identified three pathogenic variants in the RUNX2 gene (p.Ala173LeufsTer3, p.Arg225Gln, and pArg190Trp) in three patients with scoliosis by whole-exome or -genome sequencing and confirmed by BAM files (Figure 1A). Through phenotype analysis, we found that patients are associated with anomalies in multiple organ systems, including trapezoidal shaped vertebral bodies, left thoracic scoliosis (75°), right thoracic scoliosis 45°), right lumbar scoliosis (65°), and thoracic kyphosis. Patients also exhibit midclavicular aplasia, absent right clavicle, and left clavicular hypoplasia. In addition, one patient showed phenotypes of abnormal rib cage morphology and open anterior fontanelle. These results suggest that patients with scoliosis may associate with some cleidocranial dysplasia phenotypes.

Subsequently, we collected human spine samples from different developmental stages (fertilization age from 5 to 17 weeks), isolated the spinal segment through micro-dissection, performed single-cell RNA sequencing (scRNA-seq) and generated a single-cell-based transcriptomic atlas. We recovered 31,647 cells, expressing an average of 2,000 genes per cell. Cells from scRNA-seq were integrated clustered by uniform manifold approximation and projection. In human fetal spine, we identified 34 cell types (Figure 1B). The results demonstrated relatively widespread RUNX2 expression, with higher expression observed in mature chondrocytes and osteoblast progenitors.

The global knockout (KO) of the Runx2 gene in mice leads to pulmonary atelectasis due to poor rib cage development, resulting in embryonic lethality,2 which poses challenges to study the role of Runx2 in hereditary bone diseases. Recent studies suggest that specific subpopulations of bone and cartilage cells derived from mesenchymal stem cells play distinct roles in the development of the musculoskeletal system.2 Gli1 is a key member of the hedgehog family and is involved in skeletal development.3 Through lineage tracing, we found that Gli1+ cells were mainly distributed in the endplate (EP) cartilage and annulus fibrosus (AF) of the intervertebral disc (IVD) (Figure 1C). We then generated Runx2Gli1CreER conditional KO (cKO) mice. The immunofluorescent data revealed that Runx2 expression was co-localized with Gli1 protein in the IVD (data not shown). Runx2 cKO mice exhibited smaller body size (Figure 1D). Typical scoliosis phenotype was observed in Runx2 cKO mice (Figures 1E and 1F) with 100% penetrance (we have analyzed 12 Runx2 cKO mice). Results of micro-CT analysis showed that the average Cobb angle reached 70° in the thoracic spine of Runx2 cKO mice (Figure 1G), recapitulating the patients’ scoliosis phenotype. In addition, spinal fusion was also observed in the lumbar spine of Runx2 cKO mice (Figure 1H). Subsequently, we analyzed changes in bone mass on the 8th thoracic vertebra (exhibiting spinal curvature). The results showed a significant decrease in bone mineral density in the 8th thoracic vertebra of Runx2 cKO mice (Figure 1I). There were no statistically significant differences in other bone parameters, including bone volume, trabecular number, trabecular thickness, and trabecular separation.