Mutation Detection And Functional Research About Hereditary Aortic Diseases Based On Next-Generation Sequencing

Hereditary aortic disease is a type of cardiovascular emergency with extremely high mortality,which seriously threatens people’s lives.Patients often die due to massive blood loss after aortic rupture.The clinical manifestations of hereditary aortic disease are heterogeneous and it is difficult to identify the type of disease based on the symptom alone.Therefore,finding an effective diagnostic method is essential for the prevention and treatment of hereditary aortic diseases.In recent years,many researches have found that genes involved in extracellular matrix formation,smooth muscle cell contraction and metabolism,and transforming growth factor signaling pathways are closely related to the occurrence of hereditary aortic diseases,so using next-generation sequencing to screen genes can achieve the precise diagnosis,which is fast and efficient.In addition,the abnormal splicing of RNA can disrupt the normal structure and physiological functions of proteins,which is an important cause of hereditary aortic diseases.Therefore,it is significant to clarify whether mutations affect RNA splicing for molecular diagnosis of aortic disease.However,predicting the results of splicing through software is not completely reliable,and the laboratory research still plays an important role.This study intends to use next-generation sequencing to detect the relevant pathogenic genes of hereditary aortic diseases to analyze the causes of patients.We investigate splicing mutations of unknown clinical significance in this test result and hope to clarify their molecular pathogenesis and provide the laboratory evidence for clinical diagnosis.1.Method: We detected the major hereditary aortic disease-related genes,including all exons and their flanks of ACTA2、COL3A1、FBN1、FBN2、MYH11、MYLK、NOTCH1、PRKG1、SKI、SLC2A10、SMAD3、SMAD4、TGFB2、TGFBR1 and TGFBR2.We extracted the genomicDNA from the patient’s blood,performed sequencing through the Ion torrent PGM and verified the results with Sanger sequencing.We used the bioinformatics software,guidelines,databases and literatures to analyze the pathogenicity of the mutations.Results: We conducted genetic testing on 205 subjects and identified a total of147 mutations.64 of these 147 mutations were positive mutations(PV+LPV),of which 22 mutations were first discovered;Patients with positive mutations have a high risk of disease and significant disease performance;Compared with missense mutations,patients with truncated or spliced mutations of the FBN1 have earlier onset.2.Method: Minigene verified the splicing mutation: we constructed the wild and mutant vectors of FBN1 c.2418A>G,which were transfected into Hela cells,and detected the gene expression after 48 hours in vitro culture,and analyzed the splicing results by electrophoresis and sequencing.RT-PCR verified the splicing mutation: we extracted RNA from lymphocytes for RT-PCR and analyzed the PCR products by electrophoresis and sequencing.The gene expression was quantitatively analyzed by qPCR.Results: The results of Minigene: the expression of the mutant type was significantly lower than that of wild type(P<0.01).The results of electrophoresis and sequencing showed that the amplification products of both vectors were 266 bp in size and the nucleic acid sequences were completely the same.The amplification products of mutant vectors did not exist.c.2418A>G mutation.The results of RT-PCR: The size and nucleic acid sequence of the cDNA amplification product of the patient is the same as that of the normal person,but the FBN1 expression in the patient is reduced by about 50% compared with the normal person(P<0.01;P<0.05;P<0.01).3.Method: We extracted RNA from lymphocytes for RT-PCR and analyzed the PCR products by electrophoresis and sequencing.Results: Electrophoresis results showed that the size of cDNA amplification products in normal subjects was 288 bp,while there were one 288 bp normal product and one 162 bp truncated product in the patient.Sanger sequencing confirmed that exon 35 was missing from the truncated product in the patient,resulting the truncation of 126 bp.Conclusion: Gene targeting detection based on next-generation sequencing technology can screen for mutations efficiently and quickly,providing an effective method for the precise diagnosis of hereditary aortic diseases.We identified 22 mutations for the first time,enriching the genetic mutation spectrum of hereditary aortic diseases.Using Mnigene or PCR technology for the first time verified that FBN1 c.2418A>G and c.4336G>A mutations can lead to abnormal RNA splicing.The c.2418A>G causes the degradation of abnormal m RNA,while the c.4336G>A causes the deletion of exon 35.Both mutations disturb the normal structure and function of proteins.This study provides the laboratory evidence for the diagnosis of hereditary aortic disease and is greatly significant for the molecular research of hereditary aortic diseases.