Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2019.
Osteoporosis is a polygenic, inherited disease of progressive bone loss culminating in debilitating low-trauma fractures (bone failure) and increased mortality risk. Bone mineral density (BMD) is a quantitative measure of bone mass used clinically to assess the risk of fracture, as BMD is inversely correlated with fracture risk. It is well documented that BMD is a highly heritable trait, and it’s estimated that more than half of the variation in peak bone mass is controlled by genetics alone. Thus, identification of genes that
contribute to the development and maintenance of BMD represents an important area of focus.
Genome-wide association studies (GWAS) have aided in the discovery of novel BMD loci; however, functional validation through characterization of genes within these novel loci is lacking yet necessary to advance our understanding of disease phenotypes. GWAS for BMD have repeatedly identified a genome-wide significant locus at a locus on Chromosome 7 which contains the uncharacterized gene Cadherin-like and PCEsterase Domain containing 1 (CPED1, also known as C7ORF58). CPED1 is an
uncharacterized gene and has no defined function. CPED1 putatively contains an Nterminal signal peptide that should direct the protein product to cell surface where it may be secreted and interact with the extracellular matrix, a cadherin-like domain, and a PC esterase domain with predicted enzymatic activity for the modification of cell surface bioplymers. We hypothesize that Cped1 plays a role in ECM formation by the osteoblast and is involved in the regulation of bone mass. We show that transcription of Cped1 results in numerous mRNA isoforms, producing alternative transcripts lacking individual exons (exon 3, exons 16 and 17 combined) and others that may lack the coding region for one or more of the predicted functional domains. Additionally, other expressed transcripts were shown to contain
alternate 5’- and 3’-UTRs or novel promoter regions upstream of exons 3 and 12. Cped1 is widely expressed in mouse tissues and cell lines including bone and bone cells, but is notably absent from RAW264.7 macrophage/monocyte cells and circulating leukocytes from whole blood. To test the function of Cped1, we delivered siRNA against Cped1 to MC3T3-E1 pre-osteoblasts concurrently with osteogenic media to drive osteoblast differentiation. Transient knockdown of Cped1 led to an increase in the formation of mineralized nodules relative to negative control cells. We additionally observed a significant increase in both mRNA expression and protein levels of Sparc (SPARC, osteonectin) and Ibsp
(BSPII, bone sialoprotein), two important bone extracellular matrix molecules produced by the osteoblast. Our in vivo data shows a clear sexual dimorphism between Cped1mut and WT male and female mice and that disruption of Cped1 has effects on bone mass and osteoblast function. This work confirms that CPED1 is an additional candidate at this GWAS locus and requires further investigation.
