ESPE2023 Free Communications Bone, Growth Plate and Mineral Metabolism (6 abstracts)
Department of Endocrinology, Genetics and Metabolism, Fuzhou Children’s Hospital of Fujian Medical University, Fuzhou, China
Key words: KBG syndrome, ANKRD11, zebrafish, CRISPR/CasRx, Wnt signaling pathway
Background: KBG syndrome (KBGS) is a rare condition caused by variant in ANKRD11 gene, which is characterized by intellectual disability, distinctive facial features, macrodontia of the upper central incisors, skeletal anomalies and short stature. Patients carrying loss-of-function ANKRD11 variants exhibit short stature due to defects of bone development. However, the mechanism of which remains unclear.
Objective: This study aimed to generate a zebrafish model of KBGS by using CRISPR/CasRx method and explore the effects of ankrd11 on bone growth.
Methods: The mixture of mRNA of CasRx and gRNA targeting ankrd11 gene were generated according to the 2:1 ratio, whose final concentration was 200 ng/μL and 100 ng/μL, respectively. The mixture or CasRx mRNA alone was injected into zebrafish single-cell stage fertilized eggs by microinjection (experimental group vs. control group). Alizarin red and Alcian blue staining were used to analyze the craniomaxillofacial skeletal growth. The gene expression profiles of experimental group and control group were compared using RNA genome wide analysis (RNA-Seq) and RT-qPCR analysis.
Results: The relative level of ankrd11 gene was 50.9% in experimental group compared to control group (P<0.05). Impairment growth, with different degrees of deformity (small head, spinal curvature, pericardial effusion, monocular, slow degeneration of yolk sac, mandibular malformation and undeveloped swim bladder) was observed in zebrafish after knocking down ankrd11. Alizarin red and Alcian blue staining of the knockdown zebrafish showed craniomaxillofacial cartilage development defect. Further RNA-Seq and RT-qPCR analysis showed the Wnt signaling pathway was down-regulated in the ankrd11 knockdown zebrafish.
Conclusions: A novel genetic model of KBGS was generated by using CRISPR/CasRx method. It is confirmed that ankrd11 gene regulates bone growth in zebrafish through the Wnt signaling pathway.