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

Lead Research Organisation: The University of Manchester
Department Name: School of Biological Sciences

Abstract

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.

Technical Summary

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.
 
Description George Baillie 
Organisation University of Glasgow
Department Institute of Cardiovascular and Medical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We are developing an invitro human model for Acrodysostosis type 2 caused by mutation in PDE
Collaborator Contribution Advice and information on PDE enzymes. Potential inhibitors and agonists in due course.
Impact Too early
Start Year 2022
 
Description Sarah Teichman 
Organisation The Wellcome Trust Sanger Institute
Department Human Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution We collaborate with the Sanger over single cell RNAseq comparison between our protocols for chondrogenesis and fetal limb cells
Collaborator Contribution Singel cell RNAseq and bioinformatic expertise. Single cell fetal limb dataa
Impact Paper under review
Start Year 2020
 
Description Knutsford Sci bar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Presentation and discussion on developments an uses in Pluripotent stem cell research. Discussed therpeutic uses in e,g, drug development anf testing and cell based therapy.
Year(s) Of Engagement Activity 2023
 
Description Scibar Disbury 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talk about the uses and developments in pluripotent stem cell biology anf the potential for drug testing and cell therapy
Year(s) Of Engagement Activity 2023