Screening For Pathogenic Variants And Functional Studies Of Hemifacial Microsomia Based On Proband-parent Whole Exome Sequencing And Zebrafish Mode

ObjectiveTo investigate the high-risk pathogenic variation of hemifacial microsomia(HFM),and verify the pathogenicity of candidate genes through zebrafish model.MethodsWhole exome sequencing was conducted in 12 probands with HFM and their asymptomatic parents.The sequencing results were screened for high-risk pathogenic genes through frequency comparison,functional annotation and pathogenicity prediction.The temporal and spatial expression profiles of candidate genes in zebrafish were established by in situ hybridization(ISH).CRISPR/cas9 technique was used to knock out the expression of candidate genes to further observe the maxillofacial development of zebrafish.The maxillofacial cartilage of zebrafish embryos was stained by alcian blue staining technique.The proliferation and apoptosis of embryonic cells were observed by immunofluorescence staining.ISH was used to detect the role of candidate genes in the development of maxillofacial cartilage.ResultsCDC27,MUC6,MUC16,FAM136A and XKR3 may be high-risk pathogenic genes of HFM.Knockout of cdc27 by CRISPR/Cas9 in zebrafish can lead to the phenotype of maxillofacial cartilage malformation,small head,small eye,spinal deformity and cardiac edema.The loss of Cdc27 function encoded by cdc27 can reduce the proliferation of neural crest cells and hinder the differentiation of neural crest cells into chondrocytes.ConclusionThe nonsynonymous variation of CDC27 may be the cause of HFM.During zebrafish embryonic development,the Cdc27 function loss leads to the reduction of the proliferation of neural crest cells and hinders their differentiation into chondrocytes,which eventually leads to the developmental deformity of maxillofacial cartilage and spine.