Study On Mutations Of MMP-9 Gene In Patients With Intracranial Aneurysms In Henan Han

Background and Purpose Intracranial aneurysms(IA)is a complex disease caused by the destruction of intracranial blood vessel walls.With the development of aneurysms,IA ruptures,causing subarachnoid hemorrhage(SAH),causing disability or death of patients and bringing serious family and social burdens.The early detection of IA is of great significance to prevent its rupture.The study found that IA is a complex disease affected by the combination of environmental and genetic factors.The risk factors that have been found include smoking,high blood pressure,increasing age,and gender.However,genetic factors play an important role in the occurrence and development of IA.The incidence of first-and second-degree relatives in patients with subarachnoid hemorrhage increases.Compared with sporadic aneurysms,familial aneurysms are more prone.There are also some genetic diseases such as autosomal dominant polycystic kidney disease,neurofibromatosis type I,hereditary hemorrhagic telangiectasia and other related to the formation of intracranial aneurysms.Through methods such as linkage analysis and genome-wide association analysis,it has been found that there are multiple genetic loci related to IA,but only part of them have been verified by different authors,MMP-9 gene is one of them.The MMP-9 gene is located on human chromosome 20,and its translated matrix metalloproteinase 9(matrix metalloproteinase 9,MMP-9)belongs to a class of gelatinases,which can degrade elastin and denatured collagen and collagen fragments.The abnormal expression and function are related to many diseases,but the expression and mutation forms and the pathogenesis of the disease are unclear.This study is mainly to sequence all the exons,some regulatory regions and exon-intron binding regions of gene MMP-9 to find the mutation site.The effects of mutations on protein expression and function were analyzed,and the molecular mechanism associated with the progression of intracranial aneurysms was speculated to lay a foundation for the early diagnosis and early intervention of IA to prevent the occurrence of subarachnoid hemorrhage.Subjects Experimental samples were collected from the Neurointerventional Department of the First Affiliated Hospital of Zhengzhou University.The subjects were 48 patients with intracranial aneurysms who were admitted to hospital from March 2018 to December 2019.Methods 1.Blood DNA was extracted with the 2 ml blood genomic DNA extraction kit produced by Shanghai Laifeng Biological Company,and the concentration and purity of the extracted DNA were determined.2.MMP-9 gene information was searched in NCBI,and the sequence of the MMP-9 gene was obtained to find out the sequence to be amplified.Then,Primer3 online Primer design was used.The amplification range included all exons,the binding part of exon-intron,part of 5’UTR and 3’UTR.Then online Primer-BLAST was performed for specific validation,and gave it to the company to synthesize primers.3.PCR amplification,PCR products are verified by agarose gel electrophoresis,and then recovered by cutting the gel.4.The sequencing results were analyzed using Chromas and Snap Gene software to find bimodal peaks and mutations.5.SPSS 21.0 software was used for statistical analysis of the data to compare whether the difference between the patients and the control group was statistically significant;Prediction information and mutation effect fraction of gene mutation sites were analyzed using Mutation Taster software to predict whether the protein function changed or not.Results 1.Bidirectional sequencing was performed on the exon and partial intron junctions of MMP-9 gene in 48 patients,and a total of 15 point mutations were detected,12 of which were known mutations and 3 of which were newly discovered mutations.Among them,5 are synonymous mutations,3 mutations at introns,5 missense mutations,and 2 mutations at 3’UTR.2.One of the three newly discovered mutations is a missense mutation(chr20:44640324,c DNA.954G>A,Arg312His),which exists in one individual and causes a change in the function of the protein.The other two are synonymous mutations Chr20:44640217(c.847 C>A,Leu276Leu),Chr20:44640367(c.967C>T,Thr316Thr),which exist in one individual.3.At the exon 6 chr20: 44640225 site,the allele frequency of the diseased group and the control group was analyzed in Fisher’s exact probability method,P=0.0142<0.05,statistically significant,indicating that the mutation site was related to the occurrence and development of intracranial aneurysms in Henan Han population.The dominant model(GG+AG: AA)of chr20: 44640225,P=0.0354<0.05,with statistical significance,indicating that the dominant model of mutations GG and AG were correlated with the progression of intracranial aneurysms in Henan Han population.4.Mutation Taster software predicted that 4 of the 5 missense mutations might cause changes in protein functions,chr20:44640225(c DNA.855A>G,Gln279 Arg,mutation effect score 0.98793),chr20:44642406(c DNA.1740G>C,Arg574 Pro,mutation effect score 0.9999),chr20:44643111(c DNA.2022 G>A,Arg668 Gln,mutation effect score 0.99999),chr20:44640324(c DNA.954G>A,Arg312 His,mutation effect score 0.99999).However,among the above 4 gene mutations,only the mutation at chr20:44640225,the allele frequency and genotype frequency were statistically different,while the gene mutation at chr20:44640324(c DNA.954G>A)was a rare mutation,the prediction information of mutation effect showed that these two mutations might cause intracranial aneurysm disease by causing amino acid changes and then protein function changes.Conclusions 1.The chr20:44640324(c DNA.954G>A,Arg312His)on exon 6 of MMP-9 gene is a rare variation,which may be related to intracranial aneurysms and may be a pathogenic mutation.2.The chr20:44640225(c DNA.855A>G,Gln279Arg)locus on exon 6 of MMP-9 gene is related to the occurrence and development of intracranial aneurysms in Henan Han population,the G allele is the risk gene.