Prenatal Genetics Diagnosis Of The Fetuses With Corpus Callosum Abnormality And Pathogenesis Of Fetal Corpus Callosum Abnormality Caused By NOTCH3^c.4738T>A

Background:Epilepsy,mild to severe neurological and mental retardation are commonly associated with corpus callosum abnormality(CCA).The prognosis of CCA is closely related to genetic etiology.The next generation high-throughput sequencing(NGS)is a new method that can analyze multiple genes or even the whole genome at the same time.It promotes the development of new genetic detection,such as disease-specific genes,whole exome sequencing(WES)and whole genome sequencing.However,the application of WES in prenatal genetic diagnosis of CCA is still in the exploratory stage.Due to the low incidence rate of CCA and the difficulty in obtaining clinical samples,the mechanisms of the candidate pathogenic gene has been greatly restricted.Objective:1.To explore the application value of chromosome karyotype analysis,chromosomal microarray analysis(CMA)and WES in prenatal diagnosis of CCA fetus.2.To verified the function of the new candidate pathogenic gene(NOTCH3 c.4738T>A).The mutant plasmid and genetic engineering mouse model were constructed to explore the possible pathogenic mechanism of fetal CCA induced by NOTCH3 c.4738T>A.Methods:Pregnant women with CCA fetuses receiving invasive prenatal diagnosis were recruited for participation in our study.Karyotype analysis,CMA and WES were detected for CCA fetuses and the abnormal results were analyzed to explore new candidate genes.Wild-type and mutant plasmids of NOTCH3c.4738T>A were transfected into mouse neural stem cells respectively to detect the influence of the differentiation of neural stem cells to neuron.The Notch3 mutant mice were constructed.Hematoxylin-eosin staining and scanning electronic microscopy were performed on brain tissues,and the morphological phenotype of corpus callosum was observed between the mutant and wild-type mice.Morris water maze test was used to test the behavioral phenotype of the two group mice.Results:1.Forty-five cases with CCA were detected by karyotype analysis.The overall detection rate of karyotype analysis was 11.11%(5/45).2.Twenty-six cases with negative result of karyotype analysis were detected by CMA/CNV-seq.The detection rate was 11.54%(3/26).3.Thirty cases with negative karyotype analysis and(or)CMA/CNV-seq results were further detected by WES.The overall detection rate was 43.33%(13/30).4.In this study,two pathogenic genes(PTDSS1 and NOTCH3)were found to be new potential candidate genes for fetal CCA by WES.5.The molecular dynamics study of NOTCH3c.4738T>A showed that the conformational fluctuation of mutant protein was increased compared with wild type protein.The wild-type and mutant plasmids of NOTCH3c.473 8T>A were constructed and transfected into mouse neural stem cells respectively.It was found that the proliferation of neural stem cells transfected with the mutant plasmids decreased(P<0.05),the activation product NICD3 of NOTCH3 increased(P<0.05),the expression of Notch signaling pathway target gene Hesl increased(P<0.05),and the differentiation of neural stem cells into neurons decreased compared with wild-type group(P<0.05).The Notch3 mutant mice were constructed.Hematoxylin-eosin staining and scanning electronic microscopy were performed on brain tissues.It was found that the white matter and the fiber arrangement of corpus callosum of mutant mice were loose.Morris water maze test was used to test the behavioral phenotype of adult mutant mice and wild-type mice.It was found that the spatial learning and memory ability of mutant mice decreased(P<0.05).Conclusion:In this study,we conducted a systematic and comprehensive genetic examination of CCA fetuses from cellular genetics to molecular genetics,and verified the application of CMA as a first-tier detection technology in CCA fetuses;We found that WES can significantly improve the detection rate of CCA in fetuses with negative results of karyotype analysis and(or)CMA.The results of this study provide a reference for evaluating the prognosis of CCA fetus;In this study,we constructed NOTCH3c.4738T>A plasmids and mouse model,and explored the pathogenic mechanism of fetal CCA caused by NOTCH3c.4738T>A.