Methods: Samples from 58 patients with aggressive periodontitis have so far been partially examined. DNA from blood samples has been extracted and 5 primer pairs have used to amplify a 2,2 kb region of the MMP–9 promoter. A three-tiered strategy is employed for detecting all polymorphisms: denaturing high performance liquid chromatography (DHPLC) for initial screening of nucleotide variants using Transgenomic Wave analysis, followed by pooled multiplex analysis and finally confirmatory sequencing analysis using BigDye v2. MMP-9 protein levels in GCF samples are determined by ELISA and used to test if specific polymorphisms and commonly occurring haplotypes are associated with overall increased production of MMP-9 in the GCF.
Results: Examination of MMP-9 –1670 to –2170 has demonstrated the presence of three known polymorphisms at sites –1951, -1832 and –1913. In addition we have detected a novel SNP polymorphism at position -2043 in this region. Use of transgenomic Wave analysis has allowed us to rapidly genotype samples at all polymorphic sites without the need for direct sequencing of all samples. Preliminary ELISA data shows the detection of GCF MMP-9 concentrations of up to 12.3 ng/ml in diseased sites.
Conclusion: Application of these methods allows us to investigate systematically all polymorphic sites within a gene and to test their association with protein production and disease susceptibility. We have already identified 1 new MMP-9 polymorphism and expect to discover further polymorphisms along the rest of the promoter region.