|Year : 2018 | Volume
| Issue : 6 | Page : 474-479
Evaluation of collagenase-3 matrix metalloproteinase-13 gene-associated polymorphisms 11A/12A and −77A/G and its associated alleles with and without periodontitis
Jammula Surya Prasanna1, Mutyap Divya Aishwarya1, Parupalli Karunakar2, Koduganti Rekharani1, Bodiga Vijayalakshmi3, Puppala Jharna3
1 Department of Periodontics, Panineeya Mahavidyalaya Institute of Dental Sciences and Research Centre, Hyderabad, Telangana, India
2 Department of Conservative and Endodontics, Panineeya Mahavidyalaya Institute of Dental Sciences and Research Centre, Hyderabad, Telangana, India
3 Department of Genetics, Institute of Genetics and Hospital for Genetic Diseases, Hyderabad, Telangana, India
|Date of Submission||14-May-2018|
|Date of Acceptance||26-Jul-2018|
|Date of Web Publication||1-Nov-2018|
Dr. Jammula Surya Prasanna
Department of Periodontics, Panineeya Mahavidyalaya Institute of Dental Sciences and Research Centre, Kamala Nagar, Road No. 5, Dilsuck Nagar, Hyderabad - 500 060, Telangana
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Connective tissue devastation in periodontitis and other chronic inflammatory diseases is a major concern. There are several inflammatory mediators associated with this process among which matrix metalloproteinases (MMPs) play a predominant role. Collagen degradation is primarily mediated by the collagenases. MMP-13 is familiar as collagenase-3, which has the aptitude to humiliate fibrillar collagen. Aims: This study aims to evaluate MMP-13 promoter polymorphism, 11A/12A, and −77A/G and associated alleles in patients with and without chronic periodontitis (CP). Settings and Design: This was an observational case–control study. Materials and Methods: Of the total 100 patients, 50 with CP (test group) and 50 without CP (Control group), blood was collected for deoxyribonucleic acid isolation. The 11A/12A and −77A/G polymorphisms of the MMP-13 gene were picked out by polymerase chain reaction (PCR)- single-strand conformation polymorphism analysis method and PCR-restriction fragment length polymorphism by BseNI restriction enzyme, respectively. Statistical Analysis Used: Association between MMP-13 genotype (GTs) (11A/12A, 11A/11A, 12A/12A) and (AA, AG, GG) was assessed by Chi-square and Student's t-test for intergroup comparison. Results: 11A/12A GT was seen in 24% and 20%, 11A/11A 64% and 72%, 12A/12A 12% and 8% in test and control groups, respectively. However, the association was not statistically significant. −77A/G polymorphism associated GT s AA was 56% and 62%, AG 24% and 28%, GG 20% and 10% in test and controls, respectively. An association of GG was statistically significant. Conclusion: The present study results indicated that MMP-13 −77A/G gene polymorphisms, GG GT may be predicted intensive ability for CP. On the other hand, there was no significant association between MMP-13 11A/12A gene polymorphisms with CP.
Keywords: Collagen (s), deoxyribonucleic acid microarrays, gene expression, genomics, matrix metalloproteinases, periodontitis
|How to cite this article:|
Prasanna JS, Aishwarya MD, Karunakar P, Rekharani K, Vijayalakshmi B, Jharna P. Evaluation of collagenase-3 matrix metalloproteinase-13 gene-associated polymorphisms 11A/12A and −77A/G and its associated alleles with and without periodontitis. J Indian Soc Periodontol 2018;22:474-9
|How to cite this URL:|
Prasanna JS, Aishwarya MD, Karunakar P, Rekharani K, Vijayalakshmi B, Jharna P. Evaluation of collagenase-3 matrix metalloproteinase-13 gene-associated polymorphisms 11A/12A and −77A/G and its associated alleles with and without periodontitis. J Indian Soc Periodontol [serial online] 2018 [cited 2018 Dec 19];22:474-9. Available from: http://www.jisponline.com/text.asp?2018/22/6/474/244566
| Introduction|| |
Chronic periodontitis (CP) has been defined as an infectious disease culminating to inflammation within the upholding tissues of the teeth, leads to constant attachment loss, and bone loss. Annihilation of the supporting bone persuade mobility and ultimately leads to tooth loss. Periodontal disease, however, encompasses an expanded spectrum of etiology. Identification of these etiological risks is an interesting challenge.
Despite the fact that the predominant etiologic agents of the periodontal disease are bacteria, the host response to this attack causes a major destruction of the periodontal tissues. Susceptibility to several diseases and conditions, including periodontitis is influenced by the genetic background of an individual. The influence of the host genetic makeup along with environmental factors in the etiology of periodontal disease is a well-established fact. Periodontal disease, susceptibility to immunity was “probably inherited,” this term was determined way back in 1930.,,
Degradation of extracellular matrix in periodontitis is arbitrated by entangled cascades of events comprehend by both host and microbial-derived proteinases. Matrix metalloproteinases (MMPs) are a group of proteinases which plays a major role in the destruction. Among these proteinases, MMP-13 (Collagenase-3) expression is considered as an enzyme bound to bone resorption and cartilage destruction in rheumatoid arthritis and osteoarthritis. Evidence suggests that MMP-13 could further contribute to alveolar bone destruction in periodontitis., Genetic polymorphisms positioned in the promoter region of the MMP genes, the protagonist to a predisposition of various diseases considering the conversion in protein expression, structure, and function.,, MMP-13 gene is found to exhibit certain variants such as 11A/12A and −77A/G with 11A, 12A, A and G as allele frequencies that qualified them as polymorphisms. Antecedently, numerous investigations speculated that MMP gene polymorphisms in CP patients of specific populations yielded harmonious determination. However, reciprocal results were found in relation with MMP-13 and CP., There is no clear-cut literature review pertaining to periodontitis and MMP-13 gene polymorphisms (−77A/G and 11A/12A) and MMP-13 genotypes (GTs) (11A/12A, 11A/11A, and 12A/12A) and (AA, AG, GG). In consequence, the intent of the present study was to investigate MMP-13 gene polymorphisms (−77A/G and 11A/12A) and further to determine the association between MMP-13 GTs (11A/12A, 11A/11A, 12A/12A) and (AA, AG, and GG) along with associated alleles (11A, 12A, A, and G) in whole blood of the individuals with CP and without periodontitis.
| Materials and Methods|| |
This case–control study constituted of 200 patients who attended the Department of Periodontics at Hyderabad and consequentially enrolled from 2015 to 2016. Initially, a total of 200 patients were examined, but finally, only 100 individuals were taken into consideration as they met the inclusion criteria [Figure 1]. The sample size was estimated by setting α value at 0.05 and power to 80% (1− β = 0.8). All these 100 individuals were categorized into two groups, test group with CP and control group without CP, with 50 in each group. Prior approval was taken from the Institutional Ethical Committee and was also registered under https://clinicaltrials.gov (NCT02423304). A prior authored endorsement was acquired from all the individuals and recruited by following the Helsinki's Protocol. All the laboratory analysis was done by one person.
|Figure 1: Showing selection of patients and study design. n = No of patients|
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Both males and females aged between 30 and 55 years were included in this study. The American Academy of Periodontology 1999 classification, was taken into consideration to categorize CP patients into a test group. Control group consisted of individuals without even milder form of gingivitis.
Current and former Smokers, other oral diseases with the exception of periodontitis, previous history of dental treatment within the past 6 months, patients suffering from any systemic diseases or sickness even in the form of simple cold and using any sort of medications such as simple paracetamol, which hampers periodontal conditions were excluded. Any other diagnosed genetic disorder with family history, pregnant and lactating women were excluded from the study.
Deoxyribonucleic acid separation and matrix metalloproteinase-13 genotyping
A volume of 2 ml of blood was collected from brachial plexus by vein puncture method and transferred to Ethylenediaminetetraacetic Acid vacutainers (Pammvi Exports Private Limited, Andheri, Mumbai). Stored at −20°C until use. Samples were brought to the room temperature before Deoxyribonucleic Acid (DNA) isolation.
DNA was isolated by a rapid extraction method called “Worden lab sucrose method” including rupture of red blood cells and rupturing of the nucleus of white blood cells to release a large quantity of DNA which was then precipitated with a saturated sodium chloride solution.
Deoxyribonucleic acid quantitation
- By spectrophotometer: DNA was quantified by spectrophotometer (advance research instruments company, Okhla Industrial Area, New Delhi, India). 5 ml of isolated genomic DNA was diluted to 1 ml with autoclaved distilled water, and optical density (O.D) was measured at 260 nm and 280 nm. The ratio of readings at 260 nm and 280 nm (O.D 260/280) provides an estimate of the purity of the DNA. The 260/280 nm values were between 1.8 and 2 and indicated good qualities of DNA
- By agarose gel electrophoresis: 5 ml of isolated genomic DNA was electrophoresed on 0.8% agarose gel along with the DNA of known amount. The ethidium bromide stained gel was visualized under ultraviolet gel documentation system (Bio Technologies Incorporation, Madhuban Chowk, Vikas Marg, New Delhi, India). Comparison between the band intensity of known amounts of DNA to that of unknown provides the concentration of DNA present in the unknown sample.
Detection of matrix metalloproteinase-13 gene variants
A specific region of a DNA strand (the DNA target) expounded using PCR. The 11A/12A polymorphisms 11A/11A, 11A/12A, 12A/12A and 11A, 12Aalleles of the MMP-13 gene was identified by polymerase chain reaction- single strand conformation polymorphism analysis and −77A/G polymorphisms AA, AG, GG, and A, G alleles gene was identified by PCR-restriction fragment length polymorphism (PCR-RFLP) by BseNI restriction enzyme.
Open Epi software (Bill and Melinda Gates Foundation to Emory University, Rollins School of Public Health) was used for the analyses. The comparison between two groups was done by Student's t-test. Allele frequencies and allocation of GT were correlated with Chi-square test. The results were particularized as P values, odds ratio (OR), at 95% confidence intervals (95% CI).
| Results|| |
Identification of novel matrix metalloproteinase-13 promoter variants
MMP-13 gene variants, two were differentiated, 11A/12A insertion/deletion at nucleotide (nt) –291 and A to G transition of −77 G/A at nt. The two derivatives had allele frequencies that qualified them as polymorphisms. The anticipated capacity of the PCR products was 123 or 124 bp for 11A/12A as shown in gel picture [Figure 2]. PCR products were disassociated by electrophoresis on 10% polyacrylamide gels containing formamide to resolve the one-base differences between different alleles [Figure 3]. The GT distributions of 11A/12A and allele frequencies are given in [Table 1] and [Table 2]. Indicative distinctness was not found in the GT dissemination of 11A/12A polymorphism among the two groups.
|Figure 2: Amplification of matrix metalloproteinase-13 gene polymorphism region. Lane M: 100 bp deoxyribonucleic acid ladder. Lanes 1–7: samples|
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|Figure 3: Single-stranded conformational polymorphism-polyacrylamide gel electrophoresis gel picture showing. 11A/12A matrix metalloproteinase-13 polymorphism|
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|Table 1: Distribution of 11A/12A genotypes and allele frequencies in the study population|
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|Table 2: Analysis of 11A/12A genotypes and alleles among chronic periodontitis patients and controls|
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11A/11A GT was present in 64%, 11A/12A GT in 24% and 12A/12AGT in 12% of CP cases. Similarly, 11A/11AGT was present in 36%, 11A/12A in 10%, and 12A/12A in 8% of controls. 11A allele was present in 76% and 12A in 24% of CP cases. Similarly, 11A allele was present in 82% and 12A in 24% of controls. Both groups were not in consonance with Hardy–Weinberg Equilibrium (P < 0.05) [Table 1] and [Graph 1]. The 11A/12A polymorphism (insertion/deletion at nt 291) was also resolved by the direct DNA sequencing method in some randomly selected cases for confirmation.
PCR-RFLP-based method was carried out to analyze -77 A/G polymorphism of MMP-13 gene [Figure 4] and [Figure 5]. The frequency of “GG” GT of single nt polymorphisms (SNP) A −77G was remarkably abundant in test group when compared to controls. Further, the allelic frequency of the SNP was analyzed to understand the risk allele for the study phenotype. However, the “G” allele of A −77G polymorphism was prevailed significantly diagnosticate with CP (P = 0.008) [Table 3] and [Table 4]. AA GT was present in 56%, AG in 24%, and GG in 20% of PC cases. Similarly, AA GT was present in 62%, AG in 28%, and GG in 10% of controls. Allele “A” was present in 68% and “G” in 32% of cases of CP. Similarly, “A” allele was present in 76% and “G” in 24% controls [Table 3] and [Graph 2].
|Figure 4: Genotype frequencies of 11A/12A polymorphism in patient and control groups|
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|Figure 5: Amplification of matrix metalloproteinase-13 gene −77A/G polymorphism region. Lane M: 100 bp deoxyribonucleic acid ladder. Lanes 1–7: samples|
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|Table 3: Distribution of matrix metalloproteinases 13 (-77A/G) genotypes and allele frequencies in the study population|
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|Table 4: Analysis of -77A/G matrix metalloproteinases 13 genotypes and alleles among chronic periodontitis patients and controls|
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The GG GT was found to be hooked up with an elevated risk of CP when compared to AA and AG. AA versus GG adjusted OR = 0.29; 95% CI, 0.08–1.03; χ2 = 3.84; P = 0.04 (significant). Similarly, the G allele was identified with elevated risk of CP related with A. G versus A adjusted OR = 2.47; 95% CI, 1.25–4.87; χ2 = 6.98; P = 0.008 (significant), respectively. Dominant AA versus AG + GG adjusted OR = 0.40; 95% CI, 0.17–0.94; χ2 = 4.41; P = 0.03 (significant) codominant AG v/s AA + GG adjusted OR = 1.65; 95% CI, 0.61–4.49; χ2 = 0.99; P = 0.31 (not significant). Recessive GG versus AA + AG adjusted OR = 2.87; 95% CI, (0.83–9.88); χ2 = 2.96; P = 0.08 (not significant). Nevertheless, there was a suggestive dissimilarity in the GT and allele frequencies of −77A/G polymorphisms between the two groups [Table 4]. Dominant status shows gene expressiveness, codominant variant gene expressed along with wild gene, recessive if both the chromosomes are having variant gene then only it is expressed, i.e., expressed when it is associated with another recessive variant.
| Discussion|| |
In the search for better comprehension for the causes of CP, researchers have focused on two essential aspects: compassionate the character of the bacteria that provokes gingival inflammation, and the genetic and physiologic substructure that can arbitrate the body's repercussion to that provocation. The meticulous research in the last 10 years has seen an epidemic optimization of the information, claiming the interface for genetic polymorphisms with a diversified medical disease, principally chronic immune, and inflammatory diseases. This growth area also upsurges in the field of periodontal research. This recent analysis has harmonized with marked up discerning of the genome functional linkage of gene products and environmental agents. By virtue of flare-up of awareness, it is incontestable that there is a genetic basis for a majority of the diseases, in addition to CP. With the present exponential knowledge, number of investigations related to gene association, genetic polymorphism, and CP progression were opened to view.,
MMP-13 is a component of the MMP group, structurally and functionally similar enzymes authoritative for the proteolytic degradation of extracellular matrix elements along with collagen fibrils in the bone matrix usually expressed by osteoblasts. MMP-13 is predominantly up-regulated by the parathyroid hormone, a bone resorbing factor. On the contrary, very negligible to no expression of MMP-13 has been seen in normal adult tissue.,
This precise action of MMP-13, mutually with its capability to subdue both Type I and II collagen theorizes it to be an essential agonist of bone resorption and a substantial objective in inflammatory bone diseases. The expanded declension action of MMP-13 and its marked up appearance in inflammatory bone diseases necessitates a better understanding of MMP-13 expression and regulation which may lead to remedial approaches to restrict bone destruction.
The current study has estimated the potentiality of MMP-13 gene polymorphisms 11A/12A and −77A/G in CP and healthy individuals. There was no agreeable variation in the GT distribution of 11A/12A polymorphism among both groups. However, there was a remarkable variation in the GT and allele frequencies of −77A/G polymorphisms among the groups. GG GT was significantly elevated in CP. Interestingly, the findings of this research are partially in agreement with the results of a previous study done by Pirhan et al. In their study, they did not find any associated difference of 11A/12A in periodontitis and healthy individuals, which is similar to our study. However, in our study, we found significantly elevated GG GT among the test group compared to controls which differs from the findings of Pirhan et al. Even after periodontal treatment, they found no significant change. Hernandez et al. observed 100% association of MMP-13 in gingival crevicular fluid (GCF) samples of CP with significantly higher proportions associated with active sites than inactive sites. Hernández Ríos et al. found that MMP-13 could influence the humiliation of soft and hard tissue during the progression of CP. There are very few studies explaining MMP-13 polymorphisms role in periodontitis; and hence, it limits the comparison of this study results.
Other MMPs polymorphisms and periodontitis risk were analyzed by many investigators, but the concluding results were enigmatic.,,, MMP-13 is a part of collagenases group known as collagenase-3. A primary detachment of interstitial collagens by collagenases (MMPs 1, 8, and 13) is considered to serve as a key step in the periodontal lesion amelioration. The appearance of MMP-13 in inflamed gingival tissue and expanded levels in GCF from CP compared to healthy sites has been authenticated.,,, MMP-13 upregulation has been implicated in periodontitis evolution, especially in the bone loss.,,
In this study, only GG GT of −77A/G polymorphism of MMP-13 gene was associated with CP. MMP activity accounts for the speed of matrix turnover or destruction, and also the modulation of the immunologic response in more direct action., There is a small amount or nonexistence of MMP-13 in healthy tissues, representing a very limited dispersion pattern. The transpiration of disease activity perchance better ascribed to the disproportion among MMP-13 and its inhibitors, emerging in expanded enzyme activity. The presence of MMP-13 reflection in alveolar bone loss has been proposed as the trademark of periodontitis advancement., With this result, we could conclude that MMP-13 omnipresence has been consolidated with periodontal inflammation. Despite the fact that the existing mechanisms of MMPs in vivo are still ambiguous for the annoyance tissue and disease type, but based on preliminary evidence, it is comprehensible that they might play an important character in periodontal annihilation.
Sometimes, manual errors such as freezing, processing might affect the quality of outcome. And also, assuming that these divergent results might be because of the antibodies applied within the immunoassays for the detection of MMPs and their regulators might have an effect on the measuring outcomes.
| Conclusion|| |
In defiance of exhaustive studies intensifying the relation of gene polymorphisms with the perceptivity and/or austerity of periodontitis, there exists a high degree of inconsistency, and the results are inconclusive. In contempt of considerable progress in the appreciation of genetic risk factors for periodontal disease, we are still in the way of deciding the genetic support of both aggressive and CP. Wherefore, determining allelic variants of genes which are used to gauge periodontal disease risk has gained interest, but till now, only a few genetic polymorphisms were reported. Within the limitations of this study, we can conclude that GG GT of −77A/G polymorphism of MMP-13 gene can be associated with CP. However, more wide-ranged studies with preponderant sample size, extemporize techniques, and alternative ethnical dwellers should be embarked on, that one may throw light on the presumptive concernment of the MMP-13 −77A/G and 11A/12A gene polymorphisms in the pathogenesis of periodontitis.
We would like to thank the Institute of genetics and hospital for genetic diseases. Vijaya Lakshmi Bodiga and Jharna Puppala for help in the genetic procedure involved in this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]