Understanding Acrodysostosis type 1 and 2 through a pluripotent stem cell-disease model.

Acrodysostosis is a very rare disease. Symptoms can differ from patient to patient but always affect the skeleton. Acrodysostosis patients are shorter than average and have some skeletal abnormalities including small fingers and toes, this is because their bones do not grow properly. Bone growth is controlled by how a developing tissue called 'growth plate cartilage' responds to hormones which control the formation and growth of the skeleton. Acrodysostosis patients carry genetic mutations in the genes that control how growth plate cartilage responds to these hormones. In our lab we have developed a way to grow human growth plate-like cartilage from stem cells in a test tube. We have also generated human stem cell lines and methods to make controlled and specific changes in genes. In this project we will create the Acrodysostosis genetic mutations in stem cells and then generate growth plate cartilage tissue that will carry the genetic defects associated with Acrodysostosis. We will then compare this tissue to that generated from unaffected stem cells (without the genetic alteration) to determine why Acrodysostosis-affected growth plate cartilage responds differently to the growth-inducing hormones. By looking globally at the RNA molecules made in the cell which carry the codes for all the different proteins, we can identify what has gone wrong in cartilage formation in Acrodysostosis at the level of molecules. From this research we expect to identify several molecules which are not formed, formed at the wrong time or in excess and likely cause disease. These could be corrected in future work using drugs. Thus, this research is expected to point to potential drugs which can be evaluated for the treatment of Acrodysostosis.

Acrodysostosis is a rare disease characterised by short stature and skeletal abnormalities that affects the axial and appendicular skeleton, characteristically presenting with abnormal shortening of the long bones, small fingers and toes and an underdeveloped upper jaw. Acrodysostosis type 1 and 2 are caused by mutations in the type 1 regulatory subunit of cAMP-dependent protein kinase alpha (PRKAR1A) and cAMP-specific phosphodiesterase 4D (PDE4D) respectively. These mutations interfere with response of chondrocytes in the growth plate to growth controlling hormones such as PTHrP. Human induced pluripotent stem cells (hiPSCs) carrying disease causing mutations can be differentiated into growth plate-like cartilage, the template for bone growth, making them ideal for modelling genetic diseases affecting skeleton formation. We will generate mutations in PRKAR1A and in PDE4D, known to cause Acrodysostosis type 1 and 2, in hiPSCs already present in the lab using CRISPR gene editing. Then we will employ our novel hiPSC differentiation protocol with these syngeneic pairs of mutant and non mutant hiPSC lines, to identify pathways driving the pathogenic mechanisms of Acrodysostosis. Using RNAseq and single nuclear RNAseq as well as conventional immuno- and histo-chemistry and appropriate cAMP signaling (CRE) reporter lines produced in this project, we will identify molecular differences between the lines including in response to hormones (e.g. PTHrP). Specifically affected cell cohorts and potential molecular targets for future drug targeting will be identified through our in house bioinformatic pipelines, which may lead to an effective treatment in future.