Screening And Functional Study Of Pathogenic Genes In A Small Family With Bilateral Microtia Based On Whole Genome Sequencin

OBJECTIVETo screen out the suspected pathogenic gene variants based on the whole genome sequencing analysis of proband-parents small pedigrees,and to validate the function of the target gene variants using zebrafish model animal platform.METHODSEleven children with bilateral microtia and their biological parents with normal auricle phenotypes were recruited for whole genome sequencing analysis.The original data were subjected to quality control,sequence alignment and gene annotation,using the strategy of family co-segregation analysis,the suspected pathogenic gene variants were screened.The temporal and spatial expression profiles of target gene were determined by referring to the spatial transcriptome data of zebrafish embryos and in situ hybridization.Using CRISPR/Cas9 gene editing system,the target gene was knocked out in zebrafish embryos,and the developmental phenotypes of the first and second pharyngeal arches of zebrafish embryos were observed.The development of zebrafish maxillofacial cartilage was investigated by Alcian blue staining and immunofluorescence staining of chondrocyte membrane.The proliferation and apoptosis of zebrafish embryos were evaluated by PHH3 and TUNEL immunofluorescent staining.Using canonical marker genes as probes,in situ hybridization was carried out in wild-type zebrafish and mutant zebrafish to identify the effect of gene dysfunction on important nodes in the development and differentiation of maxillofacial cartilage.RESULTSIn 11 families,9 probands were male and 2 probands were female.High-quality raw data with a total size of 5.92 terabytes was obtained through whole genome sequencing.The number of de novo rare variants in each family was 299-346,and the number of cosegregated variant genes was 25-44.After excluding synonymous and intronic variants,three genes were repeated in at least two pedigrees.The tubulin folding cofactor E(TBCE)gene,which was mutated in families 3,5 and 6,was selected as the target gene for further functional study.The zebrafish homologous gene tbce was continuously expressed in the early stage of zebrafish embryos and expressed in the pharyngeal arch region.After knockout of tbce using CRISPR/Cas9 gene editing techniques,Alcian blue cartilage staining,and chondrocyte membrane immunofluorescence staining showed abnormal cartilage development and disordered cell arrangement in the region of the pharyngeal arch in mutant embryos.Loss of tbce inhibited the proliferation and promoted the apoptosis of neural crest cells in the pharyngeal arch.In zebrafish embryos,knock-out of tbce did not affect the formation of pharyngeal sac,nor did it affect the formation and migration of neural crest cells,but it did hinder the cartilage differentiation of neural crest cells.CONCLUSIONTBCE gene mutation may be one of the causes of bilateral microtia.TBCE dysfunction in zebrafish early embryos results in developmental deformities of maxillofacial cartilage by interfering with the proliferation-apoptosis balance of neural spines and hindering cartilage differentiation.