Abstract
The skeletal system is tightly regulated by multiple molecular signalling pathways that ensure proper bone development and remodelling in response to external stimuli. How exactly disruptions in these pathways lead to skeletal disease is not entirely understood. The study of rare genetic diseases that affect the skeleton provides a unique opportunity to learn more about these pathways, the key molecules that are a part of them, and the underlying pathogenesis of bone disease. This has the potential to have significant outcomes in the clinical care of patients with rare skeletal diseases as well as provide avenues to find treatments for more common conditions, such as osteoporosis.
Frontometaphyseal dysplasia (FMD) is a rare genetic disorder characterised by skeletal, cardiovascular, respiratory, and urogenital anomalies. Three genes have been implicated in FMD: FLNA which encodes the actin-binding protein filamin A (FLNA), MAP3K7, encoding the signalling factor TGFβ-activated kinase (TAK1), and TAB2 which encodes the TAK1 associated binding protein 2 (TAB2). A close functional relationship is shared between TAK1 and TAB2, but their connection with FLNA remains unexplored.
To understand how TAK1 and FLNA are associated and how pathogenic variants in both of them lead to the same disease, a novel mouse model was engineered with a pathogenic variant in Flna, NM_001110556.2 (NP_001104026.1): c.5169T>G, p.(Cys1723Trp). The skeletal phenotype of these mice was analysed via micro-computational tomography of the femur and calvarium, histomorphometry, and serum markers of bone activity. No evidence of skeletal dysplasia was found in the mice that carried the variant of interest.
To further determine the connection between TAK1 and FLNA, a series of transient transfections with HEK293FT cells, co-immunoprecipitation, and western blotting were conducted to identify if TAK1 and FLNA are interaction partners. Despite the addition of the TAK1-associated binding proteins 1 (TAB1) and TAB2, no evidence of any protein-protein interaction between TAK1 or FLNA was identified. This was further supported by the negative findings for TAK1 and FLNA interaction when proteins that contained FMD-causing pathogenic variants were analysed.
The research carried out in this thesis has not uncovered the connection between TAK1, FLNA, and TAB2 and how they result in FMD. These findings have emphasized the difficulties of both in vivo and in vitro research of skeletal disease; however, they have also highlighted what the best avenues are for the future study of FMD, providing hope that one day the pathomechanism of this disease may be discovered.