Genetic Susceptibility Analysis And Functional Characterization Of A Promoter Polymorphism In MMP3 Gene That Contributes To Sporadic Brain Arteriovenous Malformations Development

Aims:Explore the genetic susceptibility and functional characterization of a promoter polymorphism in MMP3 gene that contributes to sporadic brain arteriovenous malformations (BAVM) developmentMaterials and methods:MMP3 is a key member of the matrix metalloproteinase family, involved in the breakdown of extracellular matrix. MMP3 also plays an important role in angiogenesis and vascular remodeling. We identified five potential MMP3 gene functional single nucleotide polymorphism (SNP) loci in the Chinese population with high-frequency distribution by searching through the online database. And then, we conducted a case-control study, by collecting blood samples from 319 BAVM and 333 healthy control cases, to verify that a SNP within MMP3 gene,-709 A>G (rs522616), is significantly associated with the risk of BAVM. Afterwards, we investigated the molecular mechanisms of the protective effect of G allele for BAVM development. We constructed pGL3-A, pGL3-G and pGL3-mutant luciferase reporter vectors containing-709A,-709G and mutation loci, then transfected them into HEK293 and HUVEC cells. By detecting the luciferase activity, different MMP3 gene transcription activity affected by this SNP was analyzed. The transcription factor binding affinity between A and G allele was compared by adding competitive DNA fragment with-709A or-709G into cells with pGL3-A. Bioinformatics software indicated that transcription factor binding to this MMP3 gene promoter SNP region is c-myb. We constructed the c-myb over-expression luciferase vector to identify its regulatory effect on the transcriptional activity of MMP3 gene. ChIP and EMSA assays were carried out for further confirmation.Results:There is not any statistically significant difference between BAVM and the control group in age and gender (p= 0.062 & 0.945). The association between MMP-3 genotypes and the risk of developing BAVM was assessed using logistic regression analyses. In the five obtained SNP loci, we found that the genotype frequencies were significantly different between patients and controls for the rs522616 A>G variant of MMP-3 (p=0.02). Logistic regression analysis revealed that the variant genotype of this polymorphism was associated with a significantly decreased risk of BAVM (adjusted odds ratio= 0.62,95% confidence interval= 0.44-0.87,p= 0.006 for the AG compared with the AA genotype), we also investigate the mechanism through which the polymorphism rs522616 regulates the expression of MMP3. Our results showed that-709G led to a 2.67-fold lower transcriptional activity compared with the A allele in the HEK293 cells (P< 0.01), as well as 5.89-fold lower in the HUVEC cells (P<0.05). This transcriptional activity can also be depressed by co-transfecting cells with competitive DNA fragments containing-709A (A portion vs G portion= 0.503-fold vs 0.756-fold, P=0.01). Bioinformatics analyses suggested that the transcription factor c-myb might bind to the area with 6 bases TTCAAT around rs522616. Overexpressed c-myb significantly increased the transcriptional activity of-709A compared with-709G or controls that did not overexpress c-myb in HEK293 (1.7-fold) and HUVEC cells(1.2-fold, P< 0.05). ChIP assays indicated that c-myb bound to the SNP region in the two cell lines and three BAVM tissue samples. Together, these data indicated that c-myb can bind to the-709A allele of the MMP3 promoter, activate its transcription and lead to a higher expression of this gene.Conclusions:These findings indicate for the first time that the MMP-3 gene-709A>G (rs522616) polymorphism may contribute to lower risk of sporadic BAVM development.The protective mechanism may be that-709 A>G may compromise the binding affinity of transcription factor c-myb to the MMP3 gene promoter area, then affect the transcriptional activity and reduce the overcxpression of MMP3, confirmed by the functional experiment.