Adolescent idiopathic scoliosis (AIS) affects ~3% of the population worldwide and is estimated to cost several billion dollars annually in surgeries alone in the US. The causes of AIS remain largely unknown. While mutations in genes leading to syndromic scoliosis (associated with other symptoms) have been discovered, the identification of mutations causing non-syndromic/isolated AIS (only AIS without any other symptoms), have been less successful. Several genome-wide association studies (GWAS) have identified AIS-associated single nucleotide polymorphisms (SNPs) in noncoding regions adjacent to promising candidate genes, suggesting a role for gene regulatory sequences, such as enhancers, in AIS. In our preliminary results for example, we show that the activity of a somitic muscle and spinal cord enhancer near PAX1, a gene known to be involved in spinal development, is abolished by AIS-associated SNPs identified by GWAS. Here, we plan to take advantage of functional enhancer assays in zebrafish and mice combined with mouse CRISPR/Cas9 knockouts to characterize gene regulatory sequences that are associated with AIS. These sequences will be selected from GWAS studies, both from the literature and Project 1 (Human) and near genes shown to cause AIS in zebrafish from our Project 2 (Zebrafish). In addition, using RNA-seq and ChIP-seq for a repressive mark (H3K27me3), active marks (H3K27ac, H3K4me1, RNAPII) and candidate transcription factors on AIS- associated tissues, we will identify gene regulatory elements that could be associated with AIS, thus revealing additional sequence candidates whose alteration could lead to AIS. While there is no specific tissue whose aberration is widely known to cause AIS, there is now ample evidence linking chondrocytes to AIS. Our collaborators in Project 2 have shown that chondrocyte-specific deletion of the Gpr126 gene (a AIS GWAS gene) in mice, leads to scoliosis beginning at 20 days of age. We thus plan to initially carry out RNA-seq and ChIP-seq on mouse chondrocytes at early time points. Later on in the project we plan to use RNA-seq and ChIP-seq to characterize additional cell types/tissues where AIS candidate genes identified in Project 1 (Human) and Project 2 (Zebrafish) are expressed. Combined, our work will allow for the functional characterization of regulatory regions that are important in AIS pathogenesis, and begin to provide a genomic encyclopedia of regulatory elements that could be associated with this disease, thus providing additional candidate regions for mutation screening in individuals with AIS. In addition, our work will serve as a model for the functional characterization of gene regulatory elements involved in additional subtypes of scoliosis, musculoskeletal and other human disease.
