Deletion of a conserved genomic region associated with adolescent idiopathic scoliosis leads to vertebral rotation in mice.

Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis, in which spinal curvature develops in adolescence, and 90% of patients are female. Scoliosis is a debilitating disease that often requires bracing or surgery in severe cases. AIS affects 2%-5.2% of the population; however, the biological origin of the disease remains poorly understood. In this study, we aimed to determine the function of a highly conserved genomic region previously linked to AIS using a mouse model generated by CRISPR-CAS9 gene editing to knockout this area of the genome to understand better its contribution to AIS, which we named AIS_CRMΔ. We also investigated the upstream factors that regulate the activity of this enhancer in vivo, whether the spatial expression of the LBX1 protein would change with the loss of AIS-CRM function, and whether any phenotype would arise after deletion of this region. We found a significant increase in mRNA expression in the developing neural tube at E10.5, and E12.5, for not only Lbx1 but also other neighboring genes. Adult knockout mice showed vertebral rotation and proprioceptive deficits, also observed in human AIS patients. In conclusion, our study sheds light on the elusive biological origins of AIS, by targeting and investigating a highly conserved genomic region linked to AIS in humans. These findings provide valuable insights into the function of the investigated region and contribute to our understanding of the underlying causes of this debilitating disease.

Characterization of a novel Lbx1 mouse loss of function strain.

Adolescent Idiopathic Scoliosis (AIS) is the most common type of spine deformity affecting 2-3% of the population worldwide. The etiology of this disease is still poorly understood. Several GWAS studies have identified single nucleotide polymorphisms (SNPs) located near the gene LBX1 that is significantly correlated with AIS risk. LBX1 is a transcription factor with roles in myocyte precursor migration, cardiac neural crest specification, and neuronal fate determination in the neural tube. Here, we further investigated the role of LBX1 in the developing spinal cord of mouse embryos using a CRISPR-generated mouse model expressing a truncated version of LBX1 (Lbx1Δ). Homozygous mice died at birth, likely due to cardiac abnormalities. To further study the neural tube phenotype, we used RNA-sequencing to identify 410 genes differentially expressed between the neural tubes of E12.5 wildtype and Lbx1Δ/Δ embryos. Genes with increased expression in the deletion line were involved in neurogenesis and those with broad roles in embryonic development. Many of these genes have also been associated with scoliotic phenotypes. In comparison, genes with decreased expression were primarily involved in skeletal development. Subsequent skeletal and immunohistochemistry analysis further confirmed these results. This study aids in understanding the significance of links between LBX1 function and AIS susceptibility.

Age and Sex-Related Changes to Gene Expression in the Mouse Spinal Cord.

The spinal cord is essential for neuronal communication between the brain and rest of the body. To gain further insight into the molecular changes underpinning maturation of the mouse spinal cord, we analysed gene expression differences between 4 weeks of age (prior to puberty onset) and adulthood (8 weeks). We found 800 genes were significantly differentially expressed between juvenile and adult spinal cords. Gene ontology analysis revealed an overrepresentation of genes with roles in myelination and signal transduction among others. The expression of a further 19 genes was sexually dimorphic; these included both autosomal and sex-linked genes. Given the presence of steroid hormone receptors in the spinal cord, we also looked at the impact of two major steroid hormones, oestradiol and dihydrotestosterone (DHT) on spinal cord gene expression for selected genes. In gonadectomised male animals, implants with oestradiol and DHT produced significant changes to spinal cord gene expression. This study provides an overview of the global gene expression changes that occur as the spinal cord matures, over a key period of maturation. This confirms that both age and sex are important considerations in studies involving the spinal cord.