Skeletal dysplasias are disorders of skeletal development and occur at a rate of approximately 1 in 5000 births. DNA sequencing is capable of identifying causative genes for these disorders, but mechanistic studies are needed to understand how identified mutations cause abnormal bone growth. This proposal addresses the mechanistic gap in knowledge for spondyloepimetaphyseal dysplasia with joint laxity- leptodactylic type (SEMDJL2), a skeletal dysplasia characterized by defects in endochondral bone formation. Causative dominant mutations in the gene KIF22 have been identified in SEMDJL2 patients. However, the function of KIF22, a kinesin motor protein, in bone development and the consequences of single amino acid changes in KIF22 on this process are not known. Endochondral bone growth requires the differentiation of mesenchymal stem cells to chondrocytes and osteoblasts. Chondrocytes proliferate and produce a calcified cartilage template for bone growth. Upon chondrocyte hypertrophy, osteoblasts are recruited to the calcified cartilage and deposit bone matrix. We hypothesize that mutations in KIF22 result in disruption of this process via defects in cell division and proliferation, defects in the differentiation of progenitors to chondroctyes and osteoblasts, or defects in both division and differentiation. To test the function of KIF22 in division and proliferation of progenitor cells at the cellular level, we will use a combination of fixed and live cell imaging to assess the ability of KIF22 with SEMDJL2- derived mutations to generate forces for the movement of chromosome arms in mitotic cells. Consequent cell cycle arrest or reduction in proliferation will be assessed. To determine whether mutations in KIF22 result in SEMDJL2 pathology due to defects in pluripotent mesenchymal stem cell differentiation, we will induce differentiation of cells to chondrocytes and osteoblasts in vitro. Differentiation and maturation will be assessed in these systems by histological staining for matrix production and mineralization, as well as expression levels of markers indicative of chondrocytes, osteoblasts, or osteocytes. Together, these assays will allow us to determine the mechanism by which mutations in KIF22 affect endochondral bone growth by combining expertise in cell division with expertise in the control of bone development.
Treatment of skeletal developmental disorders requires an understanding of the mechanistic causes of abnormal bone growth. While individual disorders may be caused by different genetic mutations, there are common pathways related to membranous and endochondral bone formation that are defective in multiple disorders. As such, studying the molecular mechanism of one skeletal dysplasia, SEMDJL2, is expected to reveal not only the cellular abnormalities that contribute to the dysplasia, but also provide insight into novel basic mechanisms of bone physiology.
