Effect Of Compound Heterozygous ASXL3 Gene Mutation In A Mouse Model By Integrated Bioinformatic Analysis And Its Interaction With SOCS3 And PSD95

Objective1.To explore the application value of chromosome microarrays and high-throughput sequencing technology in ultrasonic abnormal structure in a family.2.To explore the application value of RNA-seq sequencing in the ASXL3 compound heterozygous mutant mouse model.3.Explore the possible mechanism of ASXL3 gene and the relationship between SOCS3 and PSD95.Methods1.A family of three consecutive children with congenital heart disease was selected at the prenatal diagnosis center of Guangzhou women and children's medical center.Meanwhile,100 healthy controls and 122 children with congenital heart disease were collected and expanded.2.The genomic DNA of fetus(children)was extracted according to the standard operation procedure,and the concentration and purity of DNA were measured by spectrophotometer.After that,cell culture and routine G banding karyotype analysis were performed.3.The DNA samples of fetuses were run on CytoScanTM HD Array using the manufacturer's protocol.Array is scanned and generates intensity(CEL)file data.Then the probe level analysis on CEL file data is performed in ChAS software(Affymetrix,California,USA).The CNVs detected were further aligned with known CNVs listed in database at home and databases publically available online,such as the database of DECIPHER,OMIM,DGV,UCSC and ISCA.4.Application of all exons of high-throughput sequencing in the 5 samples were detected by the American Illumina company Hiseq 2500 high-throughput experimental operation platform,carries on corresponding processing to the sequencing data in accordance with the operation manual,screening and analysis of pathogenic gene family,the sequencing data were analyzed by ClinVar,Exac,HGMD,Mutation Taster,SIFT and PloyPhen database analysis.5.A generation of sequencing(Sanger sequencing)method was used to verify the screening mutation and the control group.6.The mutant gene was constructed in a mouse model.7.The mouse brain,cerebellum and heart tissue were selected after the construction,and the RNA-seq sequencing was carried out according to the operation process.The selected differentially expressed genes were analyzed by GO analysis,KEGG analysis and co expression network analysis.8.The neuroblastoma cells(SK-N-SH)to verify the function of the candidate gene,according to the standard operation process experiment,SK-N-SH cells were cultured in vitro,the lentiviral vector(Lv-control)and expression of ASXL3 recombinant lentivirus(Lv-siASXL3)respectively,SK-N-SH cells were treated as control group and experimental group.The cell proliferation was measured by CCK-8,flow cell apoptosis was observed,Western-blot and real-time fluorescence quantitative PCR(qPCR)to detect the expression of SOCS3 and PSD95 proteins in the cells and mRNA.Results1.In congenital heart disease,abnormal fetal karyotype was not detected,chromosomal microarray analysis(CMA)were not detected in clear pathogenic microdeletions or micro repeat.2.Through the whole exome sequencing,the results showed that patients with ASXL3 gene were present in c.2168 C > G(p.P723R)and c.5449 C>G(p.P1817A)compound heterozygous,and found in the analysis of the parents,the father of c.2168 C >G(p.P723R),heterozygous carriers,mother c.5449C>G(p.P1817A)complex composite carriers.The known gene mutation associated with congenital heart disease was not detected.3.In 222 cases(fetus)extended validation was also found in the ASXL3 gene in a patient with c.3256C>T(p.R1176W)and c.4643A>G(p.D1548G)compound heterozygous mutations,including children of father c.3256C>T(p.R1176W)heterozygous carriers,the mother was c.4643 A > G(p.D1548G)carriers.4.Through RNA-seq sequencing we found compound heterozygous mutation expression of ASXL3 in mice was significantly lower than that in wild group,detected in 56917 lncRNAs and 16567 m RNAs,according to the co expression network analysis,we found 3 lncRNAs(NONMMUT041409.2,NONMMUT047824.2,and,NONMMUT135643.1)3 mRNAs and associated(Fos SOCS3,and Slc6a4).5.In vitro cell experiments,CCK-8 test results showed that after ASXL3 interference,the proliferation ability of the experimental group(Lv-siASXL3)was not significantly enhanced compared with that of the control group(Lv-control).The results of flow cytometry also showed that there was no significant difference in apoptosis between the experimental group(Lv-siASXL3)and the control group(Lv-control)after ASXL3 interference.The results of Western blot and qPCR showed that ASXL3 interference could promote the expression of SOCS3 and inhibit the expression of PSD95.Conclusions1.Chromosome microarray and high throughput sequencing technology are of great value in the analysis of fetal structural abnormalities.2.RNA-seq sequencing technology can enrich the diagnosis of disease and study the association of candidate genes.3.ASXL3 can regulate the expression of SOCS3 and PSD95 in SK-N-SH cells.