Understanding the molecular mechanisms causing osteogenesis imperfecta type XIV due to loss-of-function in TMEM38B

Osteogenesis imperfecta (OI) type XIV is a recessive brittle bone disorder characterized by bone fragility and multiple fractures without other organ involvement. Other typical OI features such as blue sclerae, dentinogenesis imperfecta and hearing loss are not consistently present. Homozygous mutations in TMEM38B gene encoding for a potassium channel (TRIC-B) are responsible for the disease. TRIC-B is present in the endoplasmic reticulum membrane of non excitable cells and allows the potassium ions to move from the cytosol to the ensoplasmic reticulum (ER), thus acting as positive charge exchanger required for the exit of positive calcium ions from the ER without altering the potential membrane. Calcium homeostasis is very important for cellular function. The exact molecular mechanism linking the loss of TRIC-B function to the skeletal phenotype in OI type XIV is still unknown. The aim of our research will be to develop and use in vitro and in vivo models to test the hypothesis that altered proliferation and differentiation caused by altered intracellular calcium concentration could be causing OI type XIV bone fragility. The TMEM38B gene will be knocked out by CRISPR/Cas9 genomic engineering tool in an immortalized human osteoblast line widely used as model for osteoblastogenesis investigation. Proliferation and differentiation will be evaluated in control and mutated cells. To confirm our in vitro data we plan to generate a zebrafish model for OI type XIV using the CRISPR/Cas9 technology to knock out tmem38b gene. The success of the targeting will be demonstrated at the gene and protein level and mutant zebrafish bone phenotype will be investigated to validate it as model for OI type XIV. If successful the project will allow the identification of some of the molecular mechanism responsible for OI type XIV and will provide useful in vitro and in vivo tools for future investigation of the disease mechanism