The Analysis Of Copy Number Fetus In Patients With Congenital Cleft Lip And/or Palates And Study On The Mechanism Of Gene MEIS2 In Congenital Cleft Lip And Palate

Congenital cleft lip and palate(CL/P)is the fourth largest birth defect disease in humans.The pathological basis of CL/P formation is thought to be caused by the insufficient mass or number of mesenchymal cells formed by neural crest cell transformation,with the inability to fill and connect the gaps between the synaptic plates.The causes of CL/P are complex,and genetic causes are an important part.With the improvement of prenatal diagnostic genetic testing techniques,more and more studies have found that chromosomal submicrodeletion/microduplication-related genomic diseases are closely related to the occurrence of cleft lip and palate.However,the CNVs excavation related to cleft lip and palate is still limited,and further testing found the pathogenic mechanism of cleft lip and palate related CNVs,found in previous study,and further study of the pathogenic mechanism of cleft lip and palate needs to be further studied.This topic was selected in the Prenatal Diagnosis Center of Guangzhou Women and Children's Medical Center from January 2014 to January 2020 for consultation,and after ultrasound and/or MRI examination,the fetus was diagnosed as cases of cleft lip and palate or the consultant was cleft lip or palate.Using the fluff/amniotic fluid/umbilical blood fetal sample,we analyzed the relationship between whole genome copy number variation(CNVs)in cleft lip and palate and cleft lip and palate.A 15 q14 fragment of 3.83 Mb microdeletion was found in 2 cases of cleft palate in the Decipher database,containing the pathogenic gene MEIS2,OMIM database that MEIS2 was associated with cleft lip and palate.Sanger sequencing screened 100 cleft lip and palate with one fetal MEIS2 exon c.1066G>A:p.G356R mutation,which was not found in parents of cleft lip and palate and 100 normal controls.This mutation can cause the coon GGA to become the amino acid encoded by AGA,to change from glycine(G)to R),which is detrimental to protein production predictions.The effect of this point mutation on MEIS2 mRNA generation was detected by the qPCR method.MEIS2 is known to be expressed in a variety of stem cells,Wnt/?-catenin signaling is also expressed in human embryonic stem cells,and in some cases of malignant tumors,MEIS2 can work by affecting the Wnt/?-catenin signaling pathway.cyclin D1,c-Myc,c-Jun these three channel signal molecules,are located downstream of Wnt/?-catenin,and are an important link in the execution function of Wnt signaling path.MEIS2,as a HOX cofactor,can bind DNA with PBX and/or HOX proteins,enhancing the affinity and specificity of DNA binding activity.Guess whether MEIS2 can work on the Wnt/?-catenin pathway by affecting cyclin Dl,c-Myc,c-Jun early in embryonic development.MEIS2 interference slow viral vector plasmid was selected in H9 cells for MEIS2 gene interference/overexpression.The amount of MEIS2,c-jun;c-myc;cyclin D1 protein in the normal/negative plasmid/MEIS2 interference/MEIS2 overexpression group was detected by Western Blot.Experimental results show that:1.A total of 165 fetuses with cleft lip/palate detected a total pathogenic CNVs rate of 9.7%of CNVs,;7 fetuses,4.2%,and 142 with 86.1%.2.Pregnational diabetes(GDM)has a higher proportion in the older pregnancy group.Increasing risk of cleft lip and palate in older+GDM.3.The expression of MEIS2 mRNA in the c.1066G>A:p.G356R mutation group decreased significantly compared with the unchanged group.4.The expression of MEIS2;?-catenin;c-jun;c-myc;cyclin D1 in MEIS2 silence group decreased significantly compared with other three groups,while MEIS2;?-catenin;c-jun;c-myc;cyclin D1 expression in MEIS2 overexpression group increased,and single-factor variance analysis showed statistical differences.This experiment confirmed that CMA examination can increase the genetic etiology of cleft lip and palate and use CMA as a CL/P first-line examination technique.Higher risk of cleft lip and palate,early pregnancy(11 weeks 0 days-6 days 13 weeks)ultrasound found NT thickening and/or cleft lip and palate.Verify that 15q14 microdeletion caused insufficient plodose of MEIS2 resulting in cleft lip and palate.Additional evidence support for prenatal diagnosis and genetic counseling in fetuses with cleft lip and palate.In vitro cell experiments show that MEIS2 can influence WNT/?-catenin downstream molecular cyclin D1,c-Myc,c-Jun.in vitroThe understanding of the action mechanism of MEIS2 is further improved.