Engineering Zebrafish Model to Study the α-tubulin (TUBA1A) Gene Mutation from a child diagnosed with Neurodevelopmental Disorder
Next-generation sequencing (NGS) technology has provided a new opportunity to identify the genetic underpinnings of many undiagnosed rare disorders. However, the genetic diagnosis of most of the rare disorders lacks effective treatment. To understand the biology of genetic mutation and develop novel therapies, we utilize Zebrafish as a model system to study the neurodevelopmental disorders. At TGen’s Center for Rare Childhood Disorders (C4CRD), we have identified a patient with novel variant heterozygous de novo substitution mutation in the TUBA1A gene (c.344 T>C), resulting in amino acid substitution (I155T). We hypothesize this mutation is the causal variant of the phenotype observed. TUBA1A plays a vital role in cell division and cell migration and is very important for proper brain development. While alpha tubulin is produced in most cells, it is highly expressed in the developing brain and is involved in neuronal migration and elongation. Zebrafish have a highly conserved TUBA1A gene compared to that of humans. As such, and due to their similar central nervous system, easy breeding and maintenance in the lab, and ease of performing genetic manipulation at the single cell stage, we utilized this model to study the mutant TUBA1A in brain development. At the single-cell stage, Zebrafish embryos were microinjected with mutant and/or wild-type version of the human TUBA1A mRNA. Microinjection of mRNA carrying the patient mutation lead to tubulin aggregation in the neuronal tracts, which was identified through confocal microscopy analysis. Currently, we are validating the rescue of mutant gene pathology with the microinjection of WT mRNA. We expect that our research will help us to understand and establish the pathogenicity of the mutation and molecular mechanisms involved in disease pathophysiology.
