| BackgroundIn the China Cardiovascular Disease Report 2017,cardiovascular disease deaths occupy the first place in the death composition of Chinese residents,accounting for more than 40%.As a state between stable angina pectoris and acute myocardial infarction,unstable angina pectoris is one of the common diseases of cardiovascular system.As early as 2013,the concept of atherosclerotic cardiovascular disease(ASCVD)has been defined in the ACC/AHA blood lipid therapy guidelines,and the related risk assessment methods have been given.In the current growing trend of ASCVD research,one of the central types,coronary artery disease,and its subordinate unstable angina pectoris both attracting more and more attentions.Traditional markers for clinical diagnosis and prognosis assessment of patients with unstable angina pectoris have limited efficacy,and new markers need to be developed.In recent years,gene-related research has become a hotspot.There were no explorations of whole blood messenger RNAs and exons with high-throughput sequencing technology in the reports of unstable angina pectoris genes.While,this field is full of infinite possibilities.The author is making an effort to find something might be applied as possible new markers by employing the new technology in detecting the expression level of unstable angina pectoris patients' mRNAs,and screening families of unstable angina pectoris patients with a family history of sudden cardiac death of coronary artery disease.In the analysing process,genetics and biomformatics methods were performed at the same time.Objectives1.To provide new bases for clinical diagnosis,individualized treatment and prognosis judgment for unstable angina pectoris patients by screening the expression profiles of peripheral blood mRNAs in UAP patients with the existing data.2.To find out the related gene loci by full exon analysis of a sudden death pedigree of coronary artery disease.Methods1.1)According to hospitalization,15 patients with unstable angina pectoris and 15 healthy controls were selected without malignant tumors and acute infectious diseases,and 2.5 ml of venous blood was collected by PAXgene test tube,respectively.2)Extraction and purification of whole blood mRNA.3)High-throughput sequencing of peripheral blood mRNA.4)Data analysis and comparison were carried out by R software and DESeq2.Genes that might be used as markers were screened by correcting p value and difference multiple.5)Gene functions,gene associations and signaling pathways were analyzed and classified according to the relevant databases of screened genes such as NCBI.6)Find out the target gene range that may be used for clinical diagnosis or prognosis analysis in patients with unstable angina pectoris2.1)According to the clinical inpatients,the patients with unstable angina pectoris were definitely diagnosed,and the family history was questioned.One family with a family history of sudden death of coronary artery disease(more than 3 sudden cardiac death)was selected and the family diagram was drawn.2)In the 3rd and 4th generations,5 ml venous blood was extracted by EDTA tube on an empty stomach.3)DNA was extracted and purified separately.4)High-throughput sequencing of all exons was performed in the third generation of patients with unstable angina pectoris.5)Gene correlation analvsis was carried out by bioinformatics method,and pathogenic genes with strong pathogenicity were screened out.6)In the third and fourth generations of human specimens,one-generation sequencing was performed.7)According to the results of validation combined with relevant bioinformatics analysis,we can get the possible gene loci that may be suseeptible to sudden death of eoronary artery disease.Results1.1)565 genes were screened according to the difference multiples,of which 80 were up-regulated and 485 were down-regulated.2)Screened the functional enrichment of differentially expressed genes on GO is mainly expressed in structural and molecular activity,extracellular matrix receptor interaction,neuroactive ligand-receptor interaction,membrane-enclosed lumen,organelle lumen and innate immunity.3)Screened differential gene function in KEGG signaling pathway is associated with systemic lupus erythematosus and cancer,and all of them are related to focal adhesion.2.1)In this family,ACMG screened 3 pathogenic mutations,1 on chromosome 4 CEP 135(POS 56865814),1 on chromosome 5 LARS(POS145508538),and the other 1 on chromosome 16 UTP4(POS 69199289).The five possible mutation sites were located on chromosome 4 TLL1(POS 166996084),chromosome 5 DMGDH(POS 78338181)and WDR36(POS110462528),chromosome 6 COL12A1(POS 75836118),and chromosome 16 DX19A(POS 70380905).2)Gene mutations screened by other methods:564 remaining SNP mutation sites,202 remaining insertion and deletion mutation sites9 0 remaining CNV mutation sites.18 candidate genes corresponding to SNP and 1 candidate gene corresponding to InDel were detected by dominant genetic model.1 candidate gene corresponding to SNP and 0 candidate genes corresponding to InDel,and 2 candidate genes corresponding to the compound heterozygous mutation sites were detected by recessive genetic model.3)GO function MF is mainly enriched in protein complex binding,collagen binding,electron carrier activity,and motor activity.CC is mainly enriched in cell parts,intracellular organelles,cytoplasm and cytoskeleton.BP is mainly enriched in cell component,cell component parts,biogenetics,cell differentiation and cell development process.KEGG pathway analysis hints the functional pathways of the genes in this sudden death family are mainly enriched in the biogenesis of eukaryotic ribosomes.4)1541 different associations among the selected candidate genes were detected by GeneMania.5)Gene-disease phenotype association was not detected in DisGetNet database.6)The first generation sequencing confirmed that the three pathogenic loci obtained by high throughput sequencing analysis were identical both in mutation point and genotype.7)The first generation sequencing also found carriers of mutant gene loci in members of the 4th generation family.Conclusions1.1)There was a significant difference in the expression of peripheral blood mRN A between patients with unstable angina pectoris and healthy controls.2)The down-regulated gene expression in peripheral blood of patients with unstable angina pectoris of coronary artery disease is predominant,which may be related to the pathophysiological state of the disease.3)Peripheral blood mRNAs in terms of structural and molecular activity,extracellular matrix receptor interaction,neuroactive ligand-receptor interaction,membrane-enclosed lumen,intracellular organelles and innate immunity may be new markers for the diagnosis,treatment and prognosis of the disease in patients with unstable angina pectoris of coronary artery disease.4)Patients with unstable angina pectoris of coronary artery disease may be associated with systemic lupus erythematosus and cancer,in which there may be the involvement of focal adhesion.These diseases may share common mechanisms in the occurrence and development of the disease.2.1)In this sudden death pedigree of unstable angina pectoris patients,there are indeed pathogenic gene mutations:CEP 135,LARS,UTP4.These three gene mutation loci have not been reported and may be related to sudden death of coronary artery disease.These should be included in the screening scope of the vulnerable population of sudden death of unstable angina pectoris.2)The carriers of mutation loci in 4th generation families were discovered by Generation 1 sequencing which provided valuable information for clinical follow-up and follow-up. |
PDF Full Text Request