Functional Polymorphisms In The Promoter Region Of MMP-2, MMP-9 And TIMP-2 Gene And Generalized Aggressive Periodontitis In A Chinese Population

Periodontal diseases, involving microbial challenge and host responses, are one group of the most common disorders. Although bacteria are an initial factor for human periodontitis, their impact may be modified by an individual's predisposition. Generalized aggressive periodontitis (G-AgP), as a subgroup of periodontitis, is characterized by rapid degradation and destruction of periodontal supporting tissue in otherwise clinically healthy the juvenile or early adult subjects. AgP is less common than chronic periodontitis (CP) and differ in many respects with regard to their etiology and pathogenesis. It has been suggested that there is a genetic basis and a predisposition for individuals to suffer from AgP.Most of the association studies to date have utilized the candidate gene polymorphisms approach to identify the gene variation related to periodontitis. Previous studies have focused on the relationship between genetic variation in the candidate genes of the immune and pro-inflammatory systems and periodontitis. These genes include interleukin-1, human leukocyte antigen, vitamine D receptor, and Fc gamma receptor et al.. Actually, these pro-inflammatory cytokines stimulate cells of the host to produce a number of matrix metalloproteinases (MMPs), which are eventually responsible for degradation of periodontal connective tissues in the pathogenesis of periodontitis. MMPs are a family consisting of over 20 related zinc-dependent endopeptidases, most of which have been demonstrated to be associated significantly with many biological or pathological processes, such as embryogenesis, inflammation, and cancer metastasis et al.. Furthermore, MMPs perform multiple roles in the host response to the procession of infection, facilitating leucocyte recruitment, cytokine and chemokine processing and matrix remodeling. Both resident cells and infiltrating inflammatory cells produce excessive MMPs that are thought to be mediators in pathogeneses of periodontitis. The important members of MMPs exampled by MMP-2 and MMP-9 (named as gelatinase A and gelatinase B), mainly cleaving type IV collagen and gelatins, are also believed to play important roles in tissue destruction in periodontitis. MMP-2 is synthesized by various fibroblasts, endothelial cells and osteoblasts, while MMP-9 is expressed by polymorphonuclear leukocytes (PMN), macrophages, and epithelial cells. Besides transcriptional regulation and substrate specificity, the activity of MMPs is also regulated by endogenous factors, including a family of anti-proteinases known as tissue inhibitors of metalloproteinases (TIMPs). Elevated MMP-2 and MMP-9 levels of tissue or gingival crevicular fluid (GCF) have been observed in inflammatory sites in periodontitis.Several studies so far focus on the association between MMP1 polymorphism and chronic periodontitis (CP) or AgP among MMPs. These results demonstrate that a single nucleotide polymorphism in the MMP1 promoter region of -1607bp is associated with generalized aggressive periodontitis (G-AgP) in the Chinese population, with severe CP in the Brazilian population and slightly with CP in the Czech population. As non-inflammatory mediators, MMPs are highlightened in the candidate gene polymorphisms related to aggressive periodontitis. Several functional single nucleotide polymorphisms in the MMP-2 and MMP-9 promoter region have distinct effects on MMP expression, which might enhance susceptibility to periodontitis. A cytosine (C) to thymine (T) substitution at position -1306bp of MMP-2 promoter region changes cis-regulatory elements by disrupting a Sp1-type promoter site (CCACC box). This functional polymorphism displays a strikingly different promoter activity between the C and T allele. Other studies indicate that the position -1562bp in the MMP-9 promoter region also has a C to T substitution, which has an allele-specific effect on MMP-9 transcription. In vitro experiments using the reporter assay technique have showed that the -1562T allele has higher promoter activity in driving gene expression than the -1562 C allele. Of the four members in the TIMP family, TIMP-2 is particularly interesting because of its dual functions in terms of regulating MMP-2 activity. A guanine (G) to C transition located at -418bp has also been identified in the consensus sequence for the Sp1-binding site in the promoter region of TIMP-2. It is reasonable to postulate that the polymorphism may down-regulate TIMP-2 expression and consequently cause an imbalance between the activities of TIMP-2 and MMP-2, which is believed to have a significant impact on periodontitis development and progression. Further research is therefore needed on the relationship between MMP-2, -9, TIMP-2 polymorphisms and AgP.Compute-based Patients Record (CPR), which is used for department of periodontology, is the perfect tool for the statistical analysis of periodontal indices in the research of clinical samples. It is indispensable for CPR to develop the research of clinical periodontology. Due to the development of periodontology and the digital management, the utilization and criteria of CPR need to be further completed and improved. In the present study, we established the new model of patients'record based on computer in stead of traditional periodontal record.Additionally, healthy and diseased gingival sample were collected from the patients with AgP or the the controls. Immunohistochemical method was used to analysis the localization and semi-quantitive expression of MMP-9 and TIMP-2. We aimed for understanding the level of expression in gingival specimen with different genotypes by the result of Part two.As mentioned above, the goal of the present study was to evaluate the genotype distribution and allele frequencies of the MMP-2,9 and TIMP-2 of G-AgP patients in a Chinese population and also to investigate whether genetic variations of MMP-2, 9 and TIMP-2 are gene biomarkers for susceptibility to G-AgP.Part one Establishment of Compute-based Periodontal case RecordIn the present study, active server page and access of internet information server were used to process the Compute-based Patient Record of periodontal indices importing. In sequence, the whole image of periodontal condition of individual can be formed in the screen of the computer in clinical practice. The designed CPR could facilitate the daily clinincal data collection and processing at department of periodontology. To the best of our knowledge, this is the first software to record periodontal indices in China.Part two Analysis of the MMP-2, MMP-9 and TIMP-2 gene promoter polymorphisms in patients with generalized aggressive periodontitis in a Chinese populationA total of 207 unrelated Chinese individuals were selected for the study population, consisting of 79 G-AgP patients and 128 periodontally healthy volunteers serving as the control group. All participants were ethnically homogeneous Han Nationality. The power calculations performed for this study show that the power of sample size was larger than 75%, which was enough to detect association with an acceptable level of confidence. Genomic DNA was extracted from oral mucosa swab sample of the study subject by Chelex-100 method. MMP-2-1306bpC/T genotypes were determined by PCR-based denaturing high performance liquid chromatography analysis while MMP-9-1562 C/T and TIMP-2-418G/C genotypes were identified by a PCR-based restriction fragment length polymorphism. The genotypes distributions and allele frequencies of three candidate genes were evaluated by Chi-square test, so were a potential composite MMP-2 and TIMP-2 halpotypes.Results The extraction of DNA from swab sample using Chelex-100 is more efficient and rapid than conventional phenol-chloroform extraction for studying genetic polymorphism. Two groups were comparable in terms of gender and age (p = 0.157 and 0.124, respectively). The mean ages of the patients and the controls were 27.3 and 28.5 years old respectively. The distribution of MMP-2, MMP-9 and TIMP-2 genotype in the G-AgP patients and the controls was in accordance with Hardy-Weinberg equilibrium by Chi-square test (χ2 = 0.001 to 0.83, P >0.05). Chi-square test after Yates'correction was used to investigate the possible association of the genotypes with the G-AgP. Corrected p values were calculated for multiple testing by the Bonferroni method. If 3 separate comparisons had been made, then the corrected significance level would be 0.017. In the patients and the controls, the most common genotype of MMP-2 and MMP-9 gene was CC, while the TT homozygote patients were very rare. No significant difference was observed in the distributions of the genotypes and alleles in MMP-2 and MMP-9 between the patients and the controls (p>0.05). The homozygotes and heterozygote of TIMP-2 appeared to be distributed evenly in the two groups. Yates'correction showed a marked difference of the distribution of TIMP-2 GC and CC genotypes in the groups (p=0.030; OR: 1.97, 95% CI: [1.11; 3.50]), which would be no significant after adjusted p-values level for multiple comparison. The frequencies of the alleles TIMP-2-418G and -418C were 71.5%and 28.5% in the patients and 82.4% and 17.6% in the controls, respectively. A significant increase in the allele C of the patients compared to the controls occurred (p =0.013; OR: 1.87, 95% CI: [1.17; 2.99]). Because the MMP2 -1306 TT and TIMP-2-418 CC homozygotes were rare in the present study, these genotypes were respectively combined with the MMP2 -1306 CT and TIMP-2 -418 GC heterozygotes for estimation of susceptibility to G-AgP. Using the most common genotype MMP-2CC and TIMP-2GG as a reference, however, no significant synergistic effect on the occurrence of G-AgP was observed in subjects carrying the composite genotypes of MMP-2 and TIMP-2 variants (p >0.05).Conclusion Although gene polymorphisms for MMP-2 and MMP-9 did not show any association with the G-AgP, the analysis of the TIMP-2 -418G to C gene polymorphism revealed significant differences between the patients and controls. Compared with controls, a significant increasing trend of TIMP-2 -418C carrier in the G-AgP patients occurred (p=0.013). The carriers of TIMP-2 -418C polymorphism in the Chinese subjects may be more at risk of suffering from G-AgP.Part three Expressions of MMP-9 and TIMP-2 in the gingival tissues of AgP patients with different genotypes.Six healthy gingival tissues and nine AgP gingival samples were obtained in the procedure of periodontal surgery. By using immunohistochemistry and computerized image analysis, the expression level of MMP-9 and TIMP-2 was assessed on the different groups in according to MMP-9-1562bpC/T and TIMP-2-418G/C genotypes.Results Rare expression of MMP-9 and TIMP-2 was observed in the healthy gingival tissues. The distribution of MMP-9 was trended to the connective tissue nearby inflammatory cell. The TIMP-2 was often observed in epithelium layer. As to the amount and the gray value of the MMP-9 positive cells, there was no significant difference between both the groups, with or without MMP-9-1562T alleles. Similarly, no significant difference was found in the amount and the gray value of TIMP-2 positive cells between both the groups, with or without TIMP-2 -418C alleles.Conclusion MMP-9 and TIMP-2 are involved in the pathological gingiva of AgP. Aciassaton between MMP-9/TIMP-2 expression level and their respective genotypes in gingival sample can not be demonstrated.