Identification And Characterization Of Genetic Susceptibility Gene EMB In Hirschsprung’s Disease

Part OneIdentification of candidate pathogenic genes for Hirschsprung’s disease by whole exome sequencing Objective: The peripheral blood of children with Hirschsprung’s disease(HSCR)and normal children were collected for whole exome sequencing,and new candidate pathogenic genes were obtained through bioinformatics analysis.Methods: Peripheral blood of 107 children with HSCR and 133 healthy children were collected.DNA extraction kit was used to extract peripheral blood genomic DNA.Whole exome sequencing was performed using the Illumina second-generation sequencing platform.and finally new HSCR candidate pathogenic genes were obtained through bioinformatics analysis software and public database analysis.Results: The DNA quality and concentration were qualified from the peripheral blood of 107 children with Hirschsprung’s disease and 133 normal children,and the second-generation whole-exome sequencing was performed.Case-control analysis was carried out through the three algorithms of b.collapse test,VT and SKAT-O,and 62 candidate pathogenic genes were initially obtained.Through the GEO database mouse enteric neural crest cell data set GSE34208,the candidate genes were further fucused to 11 genes.Conclusion: The quality of WES sequencing is qualified.In addition to the RET gene,10 new unreported candidate pathogenic genes GPSM1,MAST1,PSD3,AGBL5,ASPH,GNPTAB,HIRIP3,VPS16,SGSM1,and EMB were identified.Part Two Preliminary verification of candidate pathogenic genes PSD3,VPS16 and EMB Objective: The expression levels of Psd3,Vps16 and Emb in the intestinal tract at different stages in mouse development were detected.Candidate pathogenic genes were knocked down and the development of enteric nervous system(ENS)were observed in zebrafish.Methods: Intestinal tissues of C57BL/6J mice at different developmental stages were collected,and the expression of Psd3,Vps16 and Emb was detected by RT-q PCR.Morpholinos injection were performed to knock down gene expression of psd3,vps16 and emb in zebrafish.5dpf zebrafish embryos were usd for immunofluorescence detection.The number of zebrafish intestinal neurons were observed.Results: Psd3,Vps16 and Emb were expressed in mouse embryos at all stages,and the expression level was higher after birth,then gradually decreased,and stabilized at 6w.After knockdown of psd3 and vps16 in zebrafish,the number of enteric neurons did not change significantly,while after knockdown of emb,the number of zebrafish intestinal neurons decreased.Eight children in the HSCR group carried EMB mutations,and the results of Sanger sequencing confirmed seven types of mutations in HSCR group.Six of the mutation types were predicted as pathogenic mutations by a variety of prediction software.Conclusion: Loss of emb causes developmental disorders of the zebrafish ENS and the loss of enteric neuron.Part Three Construction of zebrafish emb gene knockout model and its effect on the development of ENS Objective: We established a zebrafish emb gene knockout model using CRISPR/Cas9 gene editing technology,and performed a series of detailed study on the development of ENS,in order to further explore the emb function on ENS.Methods: CRISPR/Cas9 technology was used to obtain emb zebrafish knockout model.Immunofluorescence was used to detect the expression of Hu C/D at the 5dpf and 7dpf stage of zebrafish embryos,and the changes in the number of enteric neurons were observed.The 7dpf zebrafish were fed fluorescent feed and photographed at 1h,3h and 6h after eating to observe the intestinal peristalsis function.In situ hybridization detects the neural crest cell marker crestin in mutant and WT zebrafish embryos at different stages.The expression level of 5-HT was detected by immunofluorescence.TUNEL assay was used to detect the apoptosis signals in embryonic.Result: A zebrafish model with emb gene knockout was obtained through CRISPR/Cas9 technology.The expression level of emb is higher in the early stage of zebrafish embryos,and then gradually decreases and stabilizes.emb is mainly expressed in zebrafish brain and intestinal tissues.Compared with WT and heterozygous,emb-/-mutants have significantly higher mortality during 7-14 dpf.Staining of enteric neurons revealed that the number of enteric neurons in emb-/-mutants was significantly less than that of heterozygous and WT,and the distal neurons were missing.Intestinal transit function experiments showed that the intestinal function of the emb-/-mutant was significantly weakened,and the heterozygous mutant had no obvious change.In situ hybridization staining revealed that the crestin expression of emb-/-mutant was reduced,and immunofluorescence staining revealed that the expression of 5-HT in the intestinal tract of emb-/-mutant was significantly reduced.The proliferation level detection found that the proliferation signal in the emb-/-mutant was significantly weakened,while the TUNERL assay detected the level of apoptosis,and the result showed that there was no significant difference between the emb-/-mutant and the WT embryo.Conclusion: emb gene involved in the regulation of the development of zebrafish ENS,and the loss of emb can lead to the reduction of zebrafish intestinal neurons and abnormal intestinal motility.Part Four The mechanism research of emb in the development of zebrafish enteric nervous system Objective: To explore the signal pathways that affect the development of zebrafish ENS after emb gene knockout Methods: Mutants and WT zebrafish embryos in 5dpf were collected,and were used to perform RNA transcriptome sequencing.Differentially expressed genes and GO and KEGG enrichment analysis were performed.Western blot was used to detect the changes in the PI3K/AKT signaling pathway between WT and emb-/-mutant.PI3 K and AKT agonists were added into the culture of WT and emb-/-mutant embryo separately,and the changes in the expression of signal pathways were detected.At the same time,embryos were collected for enteric neuron staining and proliferation signal detection.Results: Transcriptome analysis showed that PI3K/AKT signaling pathway was significantly inhibited after emb knockout.Western blot revealed that the phosphorylated protein levels of both PI3 K and AKT significantly decreased.After the stimulation of PI3 K and AKT agonist,the levels of PI3 K and AKT phosphorylated protein were up-regulated,and the number of enteric neurons and proliferation signals were restored to a certain extent.Conclusion: emb affects the development of zebrafish ENS through PI3K/AKT signaling pathway.