Forward and Reverse Genetic Studies of AIS and Spine Development in Zebrafish

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Project Abstract Adolescent idiopathic scoliosis (AIS) is the most common spine disorder affecting nearly 3% of pediatric population worldwide, presenting in otherwise healthy children without overt structural defects of vertebral units. AIS is more common in young women. More severe cases require bracing or surgery. Despite its medical significance, understanding of the genetic bases and pathogenesis of AIS is just beginning and is driven by advances in human genetic studies, combined with forward and reverse genetic approaches in zebrafish and mouse, to which our collaborative Program Project team significantly contributed in the first funding period. During the previous funding period, our Project 2-Zebrafish screened 1,673 chemically-mutagenized F3 zebrafish families for mutations affecting spine development, yielding 73 recessive juvenile and adult scoliosis mutants. Whole genome and exome sequencing of 20 alleles, mapped them to 13 chromosomal loci, implicating numerous genes in normal spine development and indicating the screen is far from saturation. Our forward and reverse genetic efforts in collaboration with Project 1-Human and the Project 3-Genomics of this program identify (i) components of extracellular matrix, ii) inflammation, and (ii) pathways affecting the assembly of the Reissner fiber as culprits in scoliosis. Surprisingly, scoliosis phenotypes often result from hypomorphic mutations in otherwise essential genes, including adamts9 and scospondin. Leveraging the momentum of our productive forward genetic screen, here we propose to extend our morphologic scoliosis screen from 1 to 3 months post fertilization, allowing us to find new genes and pathways and monitor systematically sex distribution of the scoliosis phenotypes. New mutant loci molecularly characterized by whole exome or whole genome sequencing will become candidates for human genetic studies in Project 1-Human and for analyses of regulatory elements in Project 3-Genomics of this program. Applying highly efficient genome disruption and editing approaches, we will validate candidate loci identified by Project 1 in human AIS patients, including protein-altering mutations in RAPGEF3 and LBX1 genes. We will define the underlying tissue, and molecular mechanisms underlying scoliosis in the zebrafish mutants identified by our forward and reverse genetic approaches, through comprehensive assessment of skeletal morphology, bone density, inflammation pathways, the Reissner fiber formation and maintenance, transcriptomes and physiology of the spinal canal. We will test the ability of rationally chosen drugs to suppress the phenotypes. Our genetic efforts in Project 2-Zebrafish will complement and synergize with the Project 1-Human and Project 3-Genomics components of this program to provide the first atlas of genes and will define genetic pathways critical for proper spine development in general and to AIS specifically.