To Analyze The Mechanism Of Formation Of Thoracic Aortic Aneurysm/dissection Caused By Inscag Mutation Of COL5A1c3636-3637insCAGG

Objective:To investigate whether COL5A1（collagen type V alpha 1 chain）frameshift mutation is the causative gene of TAAD by selecting COL5A1 frameshift mutation in TAAD patients and to investigate the pathogenesis of TAAD by COL5A1 frameshift mutation.COL5A1 is also called type V collagen a1 chain.Collagen V,a less abundant fibrillar collagen present in tissues expressing collagen I,plays a critical role in the nucleation of fiber assembly during the embryonic period of animals and is an essential gene for the formation of collagen fibers and skin integrity.COL5A1 mutation can lead to human classical EDS syndrome,but whether COL5A1 mutation can lead to TAAD is still unclear.Therefore,in this paper,we constructed a mouse model of COL5A1insertion of human syntenic gene by CRISPRS/CAS9 gene editing technology,compared the characteristics of human TAAD,explored the pathogenesis of TAAD caused by Col5A1 mutation,so as to provide new targets for further treatment.Methods:1.Discovery of mutated geneA registry study was conducted in 108 patients with sporadic AD in southern Jiangxi.Aortic and blood samples were collected from 150 patients hospitalized in the First Affiliated Hospital of Gannan Medical College from October 2020 to November2021.Two patients with COL5A1 mutation were obtained by collecting peripheral blood from patients and performing whole-exome targeted sequencing with matched healthy controls.Patient related information was also collected,including patient age,gender,smoking,alcohol use,concurrent hypertension history,cardiovascular system surgery history,and other cardiovascular disease conditions.2.Mouse constructionGRNA of mouse Col5a1 gene,donors oligo and Cas9 containing c.3636＿3637 ins CAGG mutation site were injected into mouse fertilized eggs to generate targeted knockout offspring.The F0 animal was identified by PCR and sequence analysis,and it was bred into wild-type mice to test the transmission of species and F1 generation animals.3.Mouse genotype identification.DNA was extracted from the tail of 1-month-old mice,and Sanger sequencing was performed subsequently after observing the presence of a band at 500 bp–750 bp by agarose gel electrophoresis to determine the mouse gene type by Chromas software.Successful mouse model construction was verified by Western Blo.4.Cardiac ultrasound and arterial blood pressure measurement in mice.4.Cardiac ultrasound and arterial blood pressure measurement in miceA total of 40 Col5a1^+/+and Col5a1^+/-mice aged 5-6 months were selected,including 20 mice in Col5a1^+/-group and 20 mice in Col5a1^+/+group,for ultrasound and arterial blood pressure detection.The diameters of ascending aorta,aortic arch and descending aorta were measured by ultrasound.Arterial blood pressure measures heart rate,systolic blood pressure and diastolic blood pressure.The measured values of ultrasound and arterial blood pressure were statistically analyzed.5.TAAD mouse modelA total of 45 3-week-old male mice were selected,including 20 Col5a1^+/+mice in group A and 25 CO5a1^+/-mice in group B.The daily BAPN intake of each mouse was 1 g·kg^-1·d^-1,which was fully dissolved in the direct drinking water of the mice,and the two groups were fed for 28 days each.Both groups had free access to food and water.The experiment was weighed every 3 days and blood pressure and ultrasound were measured weekly.It was found that paraffin sections were made after dissecting the aorta of dead mice for fixation,HE staining,EVG staining and Masson staining were performed to investigate the cause of death in mice at a later stage6.Transmission electron microscopeCol5a1^+/+and Col5a1^+/-mice aged 5-6 months were selected,and the distribution of collagen fibers in the aorta of the two groups was observed under transmission electron microscope,and whether there was collagen fiber deformity or collagen destruction.7.RNA-seqFour Col5a1^+/+and Col5a1^+/-mice aged 5-6 months were selected,and RNA was extracted from aorta as RNA-seq.According to the results,the differential gene expression between the two groups was discussed,and the differential genes related to the pathogenesis of TAAD were found,so as to explore the pathogenesis and preliminary mechanism of TAAD caused by Col5A1 gene mutation.8.Statistical methodsAll experimental data were expressed as mean±standard error（X±SEM）,and mouse survival curves were statistically compared between two groups using the log-rank test（log-rank Mantel-cox text）using the unpaired t-test.SPSS 26.0 was used as statistical analysis software and Graph Pad prism 8.0 was used as mapping software.P<0.05 was considered statistically significant.Results:1.Col5A1 gene mutation is the pathogenic gene of TAAD,but the pathogenicity of TAAD is weak alone.The morbidity of TAAD in Col5a1^+/-mice stimulated by pregnancy and BAPN drugs is significantly higher than that in Col5a1^+/+mice.2.The cause of TAAD in COL5A1^+/-mice is not vasodilation and hypertension,but destruction of the structure and function of collagen fibers in aortic wall.There was no obvious abnormality in cardiac ultrasound and blood pressure statistics of Col5a1^+/-mice and Col5a1^+/+mice aged 5-6 months.Transmission g electron microscope showed that collagen fibers in the aorta wall of Col5a^1+/-mice were sparse and varied in size.3.Col5a1^+/-mice develop TAAD.Pathogenic mechanisms may be associated with upregulation of Col4a5,Loxl1,Mmp3,and Col1a2 genes,and ECM-receptor interaction,focal adhesion,and PI3K-Akt signaling pathways4.Col5a1^-/-mice have lethal effect in embryonic stage,and the lethal period is between E10.5-E11.5 by taking embryos for gene identification.Conclusion:Col5A1 gene mutation is the pathogenic gene of TAAD.Col5a1^-/-mice died in embryonic stage,and the incidence of TAAD in Col5a1^+/-mice was significantly higher than that in Col5a1^+/+mice after being stimulated by pregnancy and BAPN drugs.