Our objective is to identify genes that regulate development of bone density, quality and strength in childhood. Childhood is a critical window for lifelong musculoskeletal health. Failure to achieve optimal bone accrual during childhood results in suboptimal peak bone mass and bone fragility later in life. In excess of 50 million older US adults have osteoporosis or low bone mass. Osteoporosis has a strong heritable component, yet only 20% of adult bone mineral density (BMD) variability is explained by genetic variants discovered to date. Pediatric studies should be highly effective in distilling the genetics of this complex phenotype, given (a) the duration of environmental influences is shorter, and (b) the genetic determinants of growth, body composition and maturation also influence bone accrual. Uncovering the genetic architecture of childhood bone accrual is critical for understanding lifelong skeletal health and identifying targets for preventing and treating bone fragility. Dual energy x-ray absorptiometry (DXA) measures of areal BMD are widely used in genetic studies. With DXA software advances, elements of bone quality and structural strength can be extracted, along with body composition parameters known to influence bone accrual. These deeper DXA-derived phenotypes have great potential to shed further important, novel insights into genetic determinants of the developing skeleton. We have genome-wide genotyped the NICHD Bone Mineral Density in Childhood Study (BMDCS) cohort which is unique for its large size, broad age range, high data quality, diversity and longitudinal design. We will derive new phenotypes from existing DXA and radiograph images, and apply advanced multidimensional phenotyping and multivariate GWAS methods to identify new loci. GWAS only reports genomic signals associated with a given trait and not necessarily the precise location of culprit genes. Therefore, we will use high-resolution `variant to gene mapping' techniques established in our `Center for Spatial and Functional Genomics' to investigate both previously reported pediatric novel loci and our anticipated new loci. Our approach first prioritizes putative causal SNPs using open chromatin and enhancer epigenetic signatures, and then identifies 3D genomic contacts between these prioritized SNPs and their target gene promoters, using a high-resolution promoter-based chromatin conformation capture technique. To validate these target genes, we will use CRISPR/Cas9 to edit the putative regulatory SNPs and use siRNA to target genes and show an effect on bone-relevant phenotypes. We will apply our techniques in primary pediatric human mesenchymal progenitor cell (MSC)-derived osteoblasts, a very relevant bone cellular model for understanding pediatric bone mass accrual. Thus, our proposal is an unparalleled opportunity to interrogate novel phenotypes and functionally characterize the actual effector genes using high resolution chromatin conformation capture approaches at these new, as well as previously known bone-related loci.
SUMMARY Osteoporosis is a major disease of aging that has origins in childhood since failure to achieve optimal bone accretion during growth results in suboptimal peak bone mass and bone fragility in older adulthood. Osteoporosis has a strong heritable component, and pediatric studies may be more effective in distilling the genetic component in this complex disease, because duration of environmental exposure has been less. Utilizing existing data from the NICHD Bone Mineral Density in Childhood Study, we will use advanced techniques to extract new information about bone quality and structural strength, and bone-related body composition, and apply high resolution `3D Genomics' and CRISPR based techniques to pinpoint causal genetic variants and corresponding effector gene(s) to deepen our understanding of bone accretion during this critical phase of the life cycle.
