An estimated 400,000 children in Australia are living with rare diseases, the majority resulting from genetic factors. They present a significant contribution to child mortality, accounting for 35% of deaths in children under 1 year old, and 10% in children aged 1-15 years. While total disease burden is significant, individual diseases within this category may affect only a small number of children, and often involve novel gene mutations. Thus, obtaining a definitive diagnosis for these children can be a lengthy and difficult process, on average 5-7 years, that leaves ≤50% of children with no diagnosis. This creates additional burden for affected children and their families, and delays implementation of treatment. More rapid methods for patient diagnosis are required.
This study aims to fill the gap between gene identification and diagnosis using CRISPR gene editing in patient-specific inducible pluripotent stem cell (iPSC) models. CRISPR/Cas 9 gene editing technology is used to introduce patient genetic variants into iPSC lines. Alternatively, patient derived iPSCs are reverted to wildtype. Amplicon sequencing is used to isolate successfully gene edited iPSC clones, which are then stimulated to mature into neural progenitors to elucidate effects on neuronal cell development and function. RNA sequencing and functional genomics are used to identify changes in neuronal development relevant to disease phenotype, with the goal of obtaining a molecular diagnosis. Characterisation of the genetic pathways relating to a patient’s specific variant may also allow identification of existing drugs that can be re-purposed for patient treatment.
The novel pipeline proposed aims to reduce the time for patient diagnosis from years to months, provide insight into disease mechanism and enable identification of new treatment strategies for patients. Furthermore, the pipeline can be readily adapted for the assessment of other neural genetic variants, and for genetic variants relevant to other iPSC cell model systems.