Studies On NOTCH1 Point Mutation Mouse Model Of Familian Tetralogy Of Fallot And Its Cardiac Phenotype

The heart is the earliest functional organ formed during the embryonic development of vertebrate,and its development process is regulated by a series of genes.The gene mutation or abnormal expression in its development process will lead to a variety of congenital heart disease.For example,Tetralogy of Fallot(TOF)is a common congenital heart malformation,ranked first among children with cyanotic heart malformations.The basic symptoms are ventricular septal defect,right ventricular outflow tract/pulmonary stenosis,overriding aorta,and right ventricular hypertrophy.The mortality rate of patients with TOF is 25%within one year,40% within three years,and more than 70% within 10 years,which seriously affects human physical and mental health,and also brings heavy economic burden to patients,families and society.Therefore,the studies on the pathological molecular mechanism of TOF have very important theoretical and clinical significance.It is known that NOTCH1 mutations occur at the highest frequency(>10%)in patients with TOF,but NOTCH1 knockout mice did not mimic the symptoms of TOF,which raises the key scientific question of how a NOTCH1 mutation causes TOF.We previously identified a new heterozygous mutation NOTCH1-p.C387 Y of the NOTCH1 gene in a familiar TOF.The heterozygous mutation was co-inherited with the disease,suggesting that this point mutation may be the real molecular etiology of TOF.At present,there is no report on NOTCH1-p.C387 Y point mutation and its animal models.Therefore,this paper established a mouse model of NOTCH1-p.C387 Y point mutation by simulating the point mutation of human patients,and explored the relationship between the point mutation and the occurrence of TOF.A mouse company used CRISPR/Cas9 technology to obtain NOTCH1-p.C387 Y point mutant heterozygote mice.In this paper,a heritable mouse strain was obtained by further identification and reproduction,and the abnormal changes of NOTCH1 point mutant protein from membrane protein to nuclear protein were studied at the cellular level.The cardiac development phenotype similar to TOF caused by this point mutation was analyzed at the body level.The results obtained are as follows:(1)Characteristics of NOTCH1-p.C387 Y heterozygous mutant mice:the genomic DNA was extracted from the mice and the genotypes were identified by PCR and gene sequencing.The genotypes of a total of 216 mice were identified and counted.It was found that no homozygous newborn mice were born,which proved that the homozygous mice with the point mutation were lethal;By dissecting E9.5,E11.5,E14.5,E18.5embryos,it was found that E14.5 was homozygous lethal.Based on the genotype of each generation,the heterozygotes of 3-month-old newborn mice were mated with wild types to obtain heritable heterozygous offspring,and the fifth generation heterozygous mice have been obtained.(2)To explore the abnormal changes of NOTCH1 point mutant protein from membrane protein to nuclear protein at the cellular level:firstly,the whild type plasmid of NOTCH1 gene and the point mutation plasmid of NOTCH1-p.C387 Y were constructed.The plasmids were transfected into HEK293 T cells and q RT-PCR analysis showed that the point mutation upregulated the expression of NOTCH1 at the transcriptional level.Co-immunoprecipitation analysis showed that the point mutation did not affect the binding of NOTCH1 to ligands.It is known that the protein encoded by NOTCH1 is digested by furin convertase in the Golgi apparatus and split into notch extracellular domain(NECD)protein and C-terminal intracellular domain(NICD)protein.Western blot showed that the NOTCH1-p.C387 Y point mutation caused a significant decrease in the expression of the C-terminal intracellular structural protein,suggesting that the point mutation decreased the signal activity of NOTCH1.(3)The phenotype similar to TOF caused by NOTCH1 point mutation was analyzed at the body level: in order to determine the effect of NOTCH1 gene on cardiac structural development,the point mutant and wild type mouse embryos were stained with HE.It was found that the heterozygote heart of point mutant mice showed a mutation phenotype similar to TOF,with aortic overriding and ventricular septal defect.However,due to time,whether there is a right ventricular outflow tract/pulmonary artery stenosis phenotype remains to be tested.In short,the NOTCH1 point mutation mouse model was established,and it was found that the point mutation of the NOTCH1 gene did not affect the binding process between the receptor and the ligand,but affected the splicing of the NICD into the nuclear region;The mouse heterozygous heart of the NOTCH1 point mutation showed a mutant phenotype similar to TOF,suggesting that NOTCH1-p.C387 Y heterozygous mutation is the molecular etiology of TOF.The results obtained in this paper are helpful to clarify the pathological molecular mechanism of TOF caused by NOTCH1 point mutation,and provide molecular markers for clinical genetic diagnosis,which has very important medical and clinical significance.