Heritable fragile bone disorders (FBDs), ranging from multifactorial to rare monogenic conditions, are characterized by an elevated fracture risk. Validating causative genes and understanding their mechanisms remain challenging. We assessed a semi-high throughput zebrafish screening platform for rapid in vivo functional testing of candidate FBD genes. Six genes linked to severe recessive osteogenesis imperfecta (OI) and four associated with bone mineral density (BMD) from genome-wide association studies were analyzed using CRISPR/Cas9-based crispant screening in F0 mosaic founder zebrafish. Next-generation sequencing confirmed high indel efficiency (mean 88%), mimicking stable knock-out models. Skeletal phenotyping at 7, 14, and 90 days post-fertilization (dpf) using microscopy, Alizarin Red S staining, and microCT was performed. Larval crispants showed variable osteoblast and mineralization phenotypes, while adult crispants displayed consistent skeletal defects, including malformed neural and haemal arches, vertebral fractures and fusions, and altered bone volume and density. In addition, aldh7a1 and mbtps2 crispants experienced increased mortality due to severe skeletal deformities. RT-qPCR revealed differential expression of osteogenic markers bglap and col1a1a, highlighting their biomarker potential. Our results establish zebrafish crispant screening as a robust tool for FBD gene validation, combining skeletal and molecular analyses across developmental stages to uncover novel insights into gene functions in bone biology.

The prevalence of childhood obesity is increasing worldwide, along with the associated common comorbidities of type 2 diabetes and cardiovascular disease in later life. Motivated by evidence for a strong genetic component, our prior genome-wide association study (GWAS) efforts for childhood obesity revealed 19 independent signals for the trait; however, the mechanism of action of these loci remains to be elucidated. To molecularly characterize these childhood obesity loci, we sought to determine the underlying causal variants and the corresponding effector genes within diverse cellular contexts. Integrating childhood obesity GWAS summary statistics with our existing 3D genomic datasets for 57 human cell types, consisting of high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq, and RNA-seq, we applied stratified LD score regression and calculated the proportion of genome-wide SNP heritability attributable to cell type-specific features, revealing pancreatic alpha cell enrichment as the most statistically significant. Subsequent chromatin contact-based fine-mapping was carried out for genome-wide significant childhood obesity loci and their linkage disequilibrium proxies to implicate effector genes, yielded the most abundant number of candidate variants and target genes at the BDNF, ADCY3, TMEM18, and FTO loci in skeletal muscle myotubes and the pancreatic beta-cell line, EndoC-BH1. One novel implicated effector gene, ALKAL2 – an inflammation-responsive gene in nerve nociceptors – was observed at the key TMEM18 locus across multiple immune cell types. Interestingly, this observation was also supported through colocalization analysis using expression quantitative trait loci (eQTL) derived from the Genotype-Tissue Expression (GTEx) dataset, supporting an inflammatory and neurologic component to the pathogenesis of childhood obesity. Our comprehensive appraisal of 3D genomic datasets generated in a myriad of different cell types provides genomic insights into pediatric obesity pathogenesis.