| Objectives We aim to screen high-risk pathogenic genes for patients with otomandibular dysostosis syndrome in a five-generation family,construct a zebrafish mutation model of candidate gene,verify the pathogenicity of candidate genes by observing the phenotype of zebrafish embryos mandibular development,explore the pathogenesis.Methods Whole exome sequencing was performed on the proband and family members of the hemifacial microsomia pedigree.The sequencing results were analyzed by bioinformatics analysis such as frequency comparison,functional annotation and pathogenicity prediction to screen out possible high-risk pathogenic mutations.RTPCR and in situ hybridization were used to establish the spatiotemporal expression pattern of candidate genes during zebrafish embryonic development.The CRISPR/Cas9 rapid phenotypic screening system was used to knock out the homologous genes in zebrafish,zebrafish mandibular cartilage developmental malformations can be observed.Alcian blue cartilage staining and wheat germ agglutinin(WGA)staining were performed on zebrafish embryos to visually observe the development of zebrafish maxillofacial cartilage.The proliferation and apoptosis of zebrafish embryonic neural crest cells and the expression of specific antibodies were investigated by immunofluorescence staining of whole embryo antibodies.The in situ hybridization probes of several important marker genes during the development of neural crest cells of zebrafish were synthesized in vitro,and the in situ hybridization was performed on zebrafish embryos at the corresponding developmental stage to explore the mechanism of the candidate genes in the development of maxillofacial chondrogenesis of zebrafish.Result:By WES,FBLN2 was identified as a high-risk pathogenic gene for this pedigree.In situ hybridization suggested that the zebrafish homologous gene fbln2 was continuously expressed during early zebrafish embryo development,and was specifically highly expressed in the mandibular cartilage region.Using CRISPR/Cas9 rapid phenotype screening system to knock out fbln2,mandibular cartilage developmental deformity of zebrafish were observed.Alcian blue cartilage staining results suggested that fbln2 knocking-out caused mandibular cartilage morphology.In situ hybridization results of fgf3 and nkx2.3 suggested that pharyngeal sac development process was not affected in fbln2 mutants.The pharyngeal arch development of fbln2 mutant embryos under.Tg(fli1a:EGFP)transgenic background was observed dynamically,and no significant abnormality was found in the formation and structure of the mutant pharyngeal arch.In situ hybridization results of crestin,foxd3 and barx1 suggested that the formation and aggregation of cranial neural crest cells in fbln2 mutants were not seriously affected.In situ hybridization of sox9a and col2a1a suggest that fibulin-2 functional deficiency affected the differentiation of mandibular cartilage.TUNEL staining suggested that fibulin-2 loss of function(LOF)led to increased apoptosis of neural crest cells in early embryonic development of zebrafish.WGA chondrocyte membrane staining suggested that fbln2 knockout caused morphological changes of zebrafish mandibular chondrocytes and disordered arrangement of chondrocytes.PHH3 antibody staining results suggested that fibulin-2 functional deficiency might affect the process of early embryonic neural crest cell proliferation,while p-smad 1/5/8 antibody staining results suggest that fibulin-2 functional deficiency caused abnormal BMP signal pathway in zebrafish embryos.Conclusion:FBLN2 is the high-risk pathogenic gene of this hemifacial microsomia pedigree.The fibulin-2 functional deficiency in zebrafish embryos may cause the increase of apoptosis and decrease of proliferation of neural crest cells in early embryonic development by affecting the BMP signaling pathway,the differentiation of neural crest cells into chondrocytes was blocked and the arrangement of mandibular chondrocytes was affected,and eventually lead to mandibular chondrogenesis malformation. |
