MON-714 CRISPR / Cas9 Genomic Edition of the CDH2 Gene in Zebrafish Leads to Eye and Cardiac Malformation

Abstract

Introduction: The CDH2 gene encodes a transmembrane protein responsible for calcium-dependent cell-cell adhesion that participates in the process of embrionic development. Neural development is achieved by neuronal neurulation, migration and differentiation, as well as axon growth and synapse formation. During pituitary development, CDH2 was associated to the epithelium-to-mesenchymal transition, with cell migration from the marginal zone to the anterior pituitary gland, followed by terminal differentiation. Thus, a dysfunction in n-cadherins disrupts the architecture of the neural tube, cortical architecture of the embryonic brain and pituitary development. In a previous study in our laboratory, a patient was diagnosed with a homozygous variant located in the N-terminal region of CDH2 (p.Val289IIe) that culminated in congenital hypopituitarism and pituitary hypoplasia. Together, these observations indicate that cadherins, especially N-cadherin, play an indispensable role in the organization of neuroepithelial layers. Zebrafish has been widely used as a model for studies of gene functionality, as it has 70% genetic homology to humans, besides being a small animal with rapid development. Our goal was to generate a zebrafish knockout for the CDH2 gene, using CRISPR Cas9 genomic edition to study its importance during development and analyze this gene in patients with characteristics similar to those observed in zebrafish. Material and methods: Three guides were drawn for the CDH2 gene using crispor program. sgRNA, produced by in vitro transcription, and Cas9 protein were injected into one cell stage. All developmental parameters were observed under a microscope up to 96 hours post fertilization (hpf). Mortality rate were calculated at 24, 48, 72 and 96 hpf. The embryos were genotyped to confirm the deleterious allelic variant. CDH2 coding region was evaluated in 3 female siblings, born from consanguineous parents, presenting micro/anophtalmia and short stature. Exons 2 to 16 were sequenced by the Sanger method. Results: 352 eggs were injected and several deformities such as absence of somites, cardiac edema, spinal curvature, cranial malformation and microphthalmia or total absence of eyes were observed. The mortality rates were 26%, 31%, 40% and 62% at 24, 48, 72 and 96 hpf, respectively. The Sanger sequencing from DNA extracted from the whole animal presented deleterious effect classified as insertions, deletions and missense changes. No deleterious allelic variant was observed in the 15 analyzed exons in the 3 patients. Conclusion: The CDH2 gene is important for neurodevelopment and eyes formation in the zebrafish although pathogenic allelic variants in this gene was not found in the studied patients with short stature and eye abnormalities.