E-Poster Presentation 30th Australian and New Zealand Bone and Mineral Society Annual Scientific Meeting 2020

Generating a pipeline for screening genetic variants associated with bone fragility disorders (#74)

Alexandra O'Donohue 1 2 , Genevieve Dammery 1 3 , Craig F Munns 2 4 , Andrew Biggin 2 4 , Aaron Schindeler 1 2
  1. Bioengineering & Molecular Medicine Laboratory, The Children's Hospital at Westmead, Sydney, New South Wales, Australia
  2. Discipline of Child & Adolescent Health, The University of Sydney, Sydney, New South Wales, Australia
  3. Applied Medical Science, The University of Sydney, Sydney, New South Wales, Australia
  4. Institute of Endocrinology and Diabetes, The Children's Hospital at Westmead, Sydney, New South Wales, Australia

Background: The expansion of genetic screening is increasingly identifying new variants of unknown significance (VUSs), and there is currently a major gap in terms of functional genomics. Osteogenesis imperfecta (OI) classically features collagen mutations, however the list of causative genes has expanded over the past two decades. Our team is developing streamlined methods for CRISPR-based gene editing to reproduce OI patient VUSs in cell lines, and then perform functional assays to evaluate their pathogenicity.

Aims: Prior to testing patient VUSs, it is critical to evaluate the editing efficacy of our system and confirm the phenotypic changes in functional assays. Thus our aims are (1) To create a bank of human osteoblast lines with knockout, pathogenic and benign variants; (2) To functionally assess differentiated gene-edited cells.

Methods: Patient VUSs were screened using the PolyPhen2 database to identify likely pathogenic mutations and create a priority list. CRISPR guides were generated using crispr.mit.edu and ligated into Cas9 plasmids. Guides were screened using the T7E1 assay in HEK293 and ASC52telo cells to confirm editing efficiency. Subsequently, individual cells were picked for clonal expansion. Differentiation of ASC52telos was assessed after 14 days using alkaline phosphatase and alizarin red S staining. 

Results: Assessment of VUSs from a cohort of 120 bone fragility disorder patients identified COL1A1, LRP5, BMP1, and FKBP10 as high priority genes. CRISPR guides were screened in a 2-step process, initially in HEK293 and again in ASC52telos to confirm mutational efficiency. Following validation, clonal populations were generated and are being expanded. Initial assays have demonstrated that differentiated ASC52telo cells have a high level of alkaline phosphatase activity and matrix mineralisation, ideal for a functional pipeline.

Discussion: This approach is currently being expanded to trial additional functional assays, with the goal to streamline the process for use in a NATA-accredited hospital laboratory within 2 years.