- Email: [email protected]
Association study for the role of Matrix metalloproteinases 2 and 3 gene polymorphisms in dental caries susceptibility
Accepted Manuscript Title: Association study for the role of Matrix metalloproteinases 2 and 3 gene polymorphisms in dental caries susceptibility Author: Dobrina Karayasheva Maria Glushkova Ekaterina Boteva Vanyo Mitev Tanya Kadiyska PII: DOI: Reference:
S0003-9969(16)30071-1 http://dx.doi.org/doi:10.1016/j.archoralbio.2016.03.007 AOB 3570
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
30-12-2014 22-11-2015 17-3-2016
Please cite this article as: Karayasheva Dobrina, Glushkova Maria, Boteva Ekaterina, Mitev Vanyo, Kadiyska Tanya.Association study for the role of Matrix metalloproteinases 2 and 3 gene polymorphisms in dental caries susceptibility.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2016.03.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Association study for the role of Matrix metalloproteinases 2 and 3 gene polymorphisms in dental caries susceptibility
Dobrina Karayashevaa, Maria Glushkovab, Ekaterina Botevaa, Vanyo Mitevc, Tanya Kadiyskab,c
a
Department of Conservative Dentistry, Faculty of Dental Medicine, Sofia Medical University, Sofia-
1431, 1 Sv. G. Sofiiski Blvd., Sofia, Bulgaria b
Genetic Medico-Diagnostic Laboratory Genica, 90 Tsar Asen str., Sofia-1643, Bulgaria
c
Department of Medical Chemistry and Biochemistry, Medical Faculty, Sofia Medical University, 2
Zdrave str., Sofia-1431, Bulgaria
Running Title: MMP2 and MMP3 SNPs and dental caries
Correspondence to: Tanya Kadiyska, Department of Medical Chemistry and Biochemistry, Sofia Medical University, Sofia-1431, 2 Zdrave str., Sofia-1431, Bulgaria Tel/Fax: +359 2 9530715; E-mail: [email protected]
1
Highlights
MMP2 genotype frequencies differ between CF and LCE groups MMP2 genotype AA increase the risk for dental caries development about 3,5 times MMP3 allele and genotype frequencies differ between CF and caries experience groups MMP3 genotype frequencies differ between LCE and HCE groups MMP3 genotype GG decrease the risk of HCE about 4 times
2
ABSTRACT Objective: Various exogenous and endogenous risk factors have been described as contributing to dental caries susceptibility. In the last decade it has been established that both pro and active forms of host derived Matrix metalloproteinases (MMPs) are present in the oral cavity. MMPs role in caries development has been hypothesized. The aim of this study was to analyse MMP2 (rs2287074) and MMP3 (rs679620) single nucleotide polymorphisms (SNPs) and their role in caries susceptibility. Design: The two SNPs were analysed by PCR- restriction fragment length polymorphism (RFLP) in a sample of 102 ethnic Bulgarian volunteers (42 males and 60 females), all students in Sofia Medical University. Results: Statistical analysis of the MMP2 SNP showed significant differences for the genotype frequencies between the caries free (CF, DMFT=0) and low caries experience (LCE, DMFT≤5) groups. Analysis for the non-synonymous MMP3 SNP found significant differences between both CF vs caries experience groups (LCE+ high caries experience (HCE, DMFT≥5)) and LCE vs HCE groups. The presence of allele G decreased the risk of HCE about 4 times. Conclusions: MMP2 and MMP3 genes are likely to be involved in caries susceptibility in our population. However, as dental caries is a multifactorial disorder and several genes are likely to have influence on it, it is reasonable to expect that SNPs, even those proven to be functional like rs679620, potentially play a significant, but not major role in the disease outcome.
Key words: candidate genes, dental caries, Matrix metalloproteinases, MMP2, MMP3
3
INTRODUCTION
Dental caries is a complex disease characterized by demineralization of inorganic portion and destruction of organic portion of teeth, eventually resulting in cavitation. This process is caused by certain cariogenic bacteria of the oral cavity, and various exogenous and endogenous risk factors have been described as contributing to dental caries development. In addition, the impact of individual genetic variation on the development of dental caries, including its severity and extent has been demonstrated in twin studies (1). In the last decade it has been established that both pro and active forms of host derived MMPs are present in the oral cavity. They are activated on-site by the lower pH, caused by the presence of the cariogenic bacteria, suggesting their importance in dental lesion process (2). MMPs are a family of zincdependent endopeptidases that degrade extracellular matrix proteins. Twenty five members of the MMP family have been identified in humans. On the basis of substrate specificity and homology, MMPs can be divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs (MT-MMPs), and other MMPs (3,4,5). MMPs contribute to various physiological processes, e.g. embryonic development, tissue turnover, and wound healing, as well as to pathological processes, e.g. cancer, cardiovascular diseases, arthritis, periodontitis, and fibrosis (6,7,8,9). Several MMPs are found to have a role in tooth development. It was suggested that they may regulate mineralization by controlling the proteoglycan turnover (10). Although most MMPs are expressed during normal dentin-pulp complex formation and maintenance, MMP-20 was identified as the first proteinase secreted into the developing enamel matrix (11). The important role of genetics in the multifactorial etiology of dental caries is well established. However, Genome wide association studies (GWAS) on caries risk are limited in the literature. A recent published paper by Wang et al. in 2013 identified 53 potential susceptibility genes for dental caries and their protein-protein interactions. Functional analyses of these 53 genes revealed three major clusters: cytokine network relevant genes, matrix MMPs family, and transforming growth factor-beta (TGF-β) family (12). Therefore the aim of the present study was to analyse single nucleotide polymorphisms (SNPs), located into two MMP genes- MMP2 (rs2287074, p.Thr460Thr, c.1380G>A) and MMP3 (rs679620, p.Lys45Glu, c.133A>G) and to evaluate their role in dental caries susceptibility in a clinically selected group of 102 Bulgarian students.
4
MATERIALS AND METHODS
More than 140 subjects, all of them students in Sofia Medical University completed a questionnaire about their ethnicity, parent’s education and social environment, oral hygiene habits, dietary habits, snacking between meals, previous preventive fluoride treatments, orthodontic treatment, frequency of preventive dental examinations, self-evaluation of salivary flow, medical history. Of them, 102 fulfilled the initial requirements for being clinically healthy, without systemic diseases or generalized periodontal inflammations, perfect oral hygiene habits and at least annual dental examinations. The selected participants were ethnic Bulgarians, aged from 20 to 32 years (42 males and 60 females). Subsequently all 102 subjects were clinically examined. Clinical examinations were performed with dental mirrors, explorers, artificial light and photo-polymerizing lamps. Radiographs were not performed. The study was conducted according to the World Medical Association Declaration of Helsinki and additionally approved by the Ethics Committee of Sofia Medical University. Written informed consent was obtained from all participants prior to genetic testing. The studied subjects were classified according to the caries experience level using the DMFT indexes. According to recently published papers, the different genetic loci and SNPs could lead to either increased or decreased activities and thus contribute differently to caries experience. In this context, the genetic factors have been divided into caries protective genes (associated with CF phenotype) and caries susceptibility genes (associated with LCE or HCE phenotypes) (13,14). The subjects were divided into three groups: CF (with DMFT=0, n=20), LCE (with DMFT≤5, n=41) and HCE (with DMFT≥5, n=41). Epithelial buccal cells were collected in sterile, Еpendorf-type plastic containers of 2 ml. Each container was labeled indicating the name and individual number. The samples were collected after normal hygiene and breakfast individual habits of the participants, between 9 and 11.30 a.m. DNA was extracted from buccal cells by the Chelex® 100 (Bio-Rad Laboratories, Hercules, CA) extraction technique. The vials with buccal cells were centrifuged at 7,500 rpm for 15 min, and the supernatant was removed. Then the pellet was resuspended thorough mixing in 150 µl of 5% Chelex 100 resin and Proteinase K solution, followed by incubation at 56°C for 2 hours in a dry heat block. The mixture was boiled for 10 min and then chilled on ice for 5 min. It was then centrifuged at 12,000 rpm for 10 min. The supernatant was carefully removed, with the Chelex avoided. The DNA was stored at 20°C prior to analysis. 5
For MMP2 SNP genotyping, DNA fragments were amplified by the use of primer pairs MMP2F 5´GTCCAGGCATCTTCTTGTTA-3´ and MMP2R 5´-GAGGACAAGAAGCAAGCTCC-3´ (322 bp). PCR amplification was performed in 25 µl volume containing 100 ng of genomic DNA. The thermal cycles were initiated for 5 minutes at 95 C, followed by 30 cycles of 40 seconds at 95 C, 30 seconds at 58 C, and 30 seconds at 72°C, and a final extension at 72°C for 10 minutes. The PCR products were subjected to restriction fragment length polymorphism (RFLP) with 5 U of BseYI (NEB, Germany) at 37°C overnight, and the products were separated on a 3% agarose gel and stained with Ethidium bromide in order to yield G (122 and 200bp) and A (322 bp) alleles, allowing, therefore, the determination of the GG, GA, and AA genotypes. For MMP3 SNP genotyping, DNA fragments were amplified by the use of primer pairs MMP3F 5´GATTAAGAAGTGAGCAACTGCA-3´ and MMP3R 5´-CCTCCAATCCAAGGAACTTC-3´ (212 bp). PCR amplification was performed using the conditions for the MMP2 SNP. The PCR products were then subjected to RFLP with 5 U of TaqI (NEB, Germany) and incubated overnight at 65°C. The products were separated on a 3% agarose gel and stained with Ethidium bromide in order to yield A (212bp) and G (120 and 92 bp) alleles, allowing, therefore, the determination of the AA, AG, and GG genotypes. Genetic data was entered and processed with SPSS 16.0 software (SPSS, Chicago, IL). Frequencies of alleles and genotypes were compared using χ2 test. OR and their corresponding 95% CI were determined as per standard statistical methods. The Hardy-Weinberg equilibrium among the CF volunteers (used as control group) was also tested using standard χ2 statistics. P values of <0.05 are considered statistically significant.
RESULTS
All 102 volunteers were successfully genotyped for the MMP2 and MMP3 SNPs. The distribution of the allele and genotype frequencies is shown in Table 1. There are no differences in age and gender distribution between the groups. Considering the two SNPs independently, genotype distributions do not deviate from Hardy-Weinberg equilibrium among the groups (p>0,05). There were no significant differences of the allele and genotype frequencies between the subjects from our study and the reported in dbSNP database for other European populations. For MMP2 (rs2287074), the reported minor allele frequency (MAF) is 35%, while in our study is 39%. For MMP3 (rs679620), the reported MAF is 37%, while in our study is 47%. The statistical analysis for the MMP2 gene showed significant differences for the genotype frequencies between the CF and LCE groups. We found that the presence of genotype AA increased the risk for 6
dental caries development about 3,5 times. The comparison between the other investigated groups of CF vs HCE and CF vs LCE+HCE showed differences close the borderline of p=0.05 (Table 1). However, after applying the Yates correction for the small sample sizes, the p-values showed no statistical differences between the three groups. Our study for the non-synonymous MMP3 SNP found significant differences between both CF vs LCE+ HCE and LCE vs HCE groups. The presence of genotype GG decreased the risk of HCE about 4 times (Table 1). When the three groups were divided according to their sex (data not shown), no significant differences were found between the groups and MMP2 allele and genotype frequencies. After statistical analysis of MMP3, it was noted that the significant difference between the groups of CF or LCE and HCE cases was due to the high impact of female, but not male cases.
DISCUSSION
Although caries prevalence has decreased over the past few decades in the North West of Europe, South and Eastern European populations are increasingly affected. In the last years the research was focused to investigate the susceptibility genes for dental caries. Special interest was carried out in the study of genetic variations of genes involved in the tooth formation and differentiation. It has been shown that variations of genes which alter enamel and dentin organization may increase the individual caries susceptibility. Several authors verified the relationship between enamel defects and dental caries (15,16). Genes involved in amelogenesis alone or in combination with high Mutans streptococci levels, were shown to result in increased caries rates while genes involved in dentin formation as MMP2 and 3 might contribute to faster carious lesion progression in dentin and periapical pathologies (17,18,19) Another group of genes, involved in ingestive behaviour has been widely studied. Some examples are the TAS1R2 gene (Taste receptor, Type 1, Member 2), found to influence taste perception (20) and GLUT2 gene (Glucose transporter type 2), involved in the energy homeostatic pathways (21). Functional polymorphisms of DEFB1 (beta defensin 1) and Lactotransferrin genes have also been shown to be associated with caries susceptibility (22,23). We hypothesized that MMP genetic variations may be one of several contributing factors to the susceptibility of dental caries. MMP-2 and MMP-3 genes were chosen as representative candidates for dental caries susceptibility and progression, described also from Chaussain-Miller in 2006 (24).
7
MMP2, also known as gelatinase A, is a membrane-bound protein that is important for extracellular matrix turnover. It has proteolytic activity against components of the basement membrane, preferentially cleaving collagen types IV, V, VII, and XI and gelatin (25). MMP-3, also known as stromelysin 1 is the main metalloproteinase that is secreted by fibroblasts, synovial cells and chondrocytes. It has proteolytic activity against proteoglycans, fibronectin, laminin and type IV collagens (26). The transcriptional level of most MMPs is regulated by growth factors and cytokines, but also singlenucleotide polymorphisms (SNPs) of several MMP genes have been shown to be transcriptional regulators (27,28). The two SNPs analysed in the recent study have been previously associated with host susceptibility of developing periapical lesions in individuals with untreated carious lesions (19). Furthermore, it has been shown that hormones, especially oestrogen, do upregulate MMP2 levels (29). There is no sufficient data about MMP3 gene expression and oestrogen. Some authors demonstrated MMP3 up-regulation in oestrogen-deficient mouse osteoblasts, while others did not show any significant effect of oestrogen on MMP3 (30,31). Following our results of stronger association between MMP3 allele G female carriers and dental caries resistance, we can speculate that there is a negative regulation of MMP3 expression by oestrogen. Despite the relatively small number of participants (102 people in total) in this pilot study for Bulgaria, the selected group of volunteers was characterized by a very high homogeneity with regard to: ethnicity, age (20-32 year-olds), social status and education (students), parents' education (higher), nutritional and hygiene habits, health culture, awareness of and motivation for their general and dental health care. The evidence that individuals exposed to the same levels of environmental risk factors do present differences in the DMFT index suggests even stronger influence of genetic factors in the aetiology of the disease. We emphasize the hypothesis that MMP inhibitors may have role in preventing caries progression and functional SNP detection may guide therapeutic decision making. Based on our results, it could be concluded that MMP2 and MMP3 genes are likely to be involved in caries susceptibility in our population. However, as dental caries is multifactorial disorder and several genes are likely to have influence on it, it is reasonable to expect that SNPs, even those proven to be functional like rs679620, potentially play a significant, but not major role in the disease outcome. Although these results look promising, additional studies exploring larger sample sizes and extended genetic and haplotype data are needed to clarify the association of dental caries with MMP genetic polymorphisms.
8
ACKNOWLEDGEMENTS
The authors are grateful to the volunteers for participating in this study. This study was partially supported by research grant No. 26/2014, Committee of Medical Sciences, Sofia Medical University.
CONFLICT OF INTEREST
Herewith we declare that we have no conflict of interest.
9
REFERENCES: 1. Rintakoski K, Kaprio J, Murtomaa H. Genetic and environmental factors in oral health among twins. J Dent Res 2010; 89(7): 700-4. 2. Tjäderhane L, Larjava H, Sorsa T, Uitto VJ, Larmas M, Salo T. The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions. J Dent Res 1998; 77(8): 1622-9. 3. Murphy G, Knäuper V. Relating matrix metalloproteinase structure to function: why the "hemopexin" domain? Matrix Biol 1997; 15(8-9): 511-8. 4. Murphy G, Nagase H. Progress in matrix metalloproteinase research. Mol Aspects Med 2008; 29: 290–308. 5. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003; 92(8): 827-39. 6. Hadler-Olsen E, Winberg JO, Uhlin-Hansen L. Matrix metalloproteinases in cancer: their value as diagnostic and prognostic markers and therapeutic targets. Tumour Biol 2013; 34(4): 2041-51. 7. Agewall S. Matrix metalloproteinases and cardiovascular disease. Eur Heart J 2006; 27(2): 1212. 8. Malemud CJ. Matrix metalloproteinases (MMPs) in health and disease: an overview. Front Biosci 2006; 11: 1696-701. 9. Sorsa T, Tjäderhane L, Salo T. Matrix metalloproteinases (MMPs) in oral diseases. Oral Dis 2004; 10(6): 311-8. 10. Hannas AR, Pereira JC, Granjeiro JM, Tjäderhane L. The role of matrix metalloproteinases in the oral environment. Acta Odontol Scand 2007; 65(1): 1-13. 11. Bartlett JD. Dental enamel development: proteinases and their enamel matrix substrates. ISRN Dent Sep 2013; 16: 684607. 12. Wang Q, Jia P, Cuenco KT, et al. Multi-dimensional prioritization of dental caries candidate genes and its enriched dense network modules. PLoS One 2013; 8(10): e76666. 13. Tannure PN, Küchler EC, Falagan-Lotsch P, Amorim LM, Raggio Luiz R, Costa MC, Vieira AR, Granjeiro JM. MMP13 polymorphism decreases risk for dental caries. Caries Res. 2012;46(4):401-7. 14. Vieira AR, Modesto A, Marazita ML. Caries: review of human genetics research. Caries Res. 2014;48(5):491-506.
10
15. Milgrom P, Riedy CA, Weinstein P, Tanner AC, Manibusan L, Bruss J. Dental caries and its relationship to bacterial infection, hypoplasia, diet, and oral hygiene in 6- to 36-month-old children. Community Dent Oral Epidemiol 2000; 28(4): 295-306. 16. Targino AG, Rosenblatt A, Oliveira AF, Chaves AM, Santos VE. The relationship of enamel defects and caries: a cohort study. Oral Dis 2011; 17(4): 420-6. 17. Patir A, Seymen F, Yildirim M, Deeley K, Cooper ME, Marazita ML, et al. Enamel formation genes are associated with high caries experience in Turkish children. Caries Res 2008; 42(5): 394-400. 18. Slayton RL, Cooper ME, Marazita ML. Tuftelin, mutans streptococci, and dental caries susceptibility. J Dent Res 2005; 84(8): 711-4. 19. Menezes-Silva R, Khaliq S, Deeley K, Letra A, Vieira AR. Genetic susceptibility to periapical disease: conditional contribution of MMP2 and MMP3 genes to the development of periapical lesions and healing response. J Endod 2012; 38(5): 604-7. 20. Nelson G, Hoon MA, Chandrashekar J, Zhang Y, Ryba NJ, Zuker CS. Mammalian sweet taste receptors. Cell 2001; 106(3): 381-90. 21. Brown GK. Glucose transporters: structure, function and consequences of deficiency. J Inherit Metab Dis 2000; 23(3): 237-46. 22. Ozturk A, Famili P, Vieira AR. The antimicrobial peptide DEFB1 is associated with caries. J Dent Res 2010; 89(6): 631-6. 23. Brancher JA, Pecharki GD, Doetzer AD, et al. Analysis of polymorphisms in the lactotransferrin gene promoter and dental caries. Int J Dent 2011; 2011: 571726. 24. Chaussain-Miller C, Fioretti F, Goldberg M, Menashi S. The role of matrix metalloproteinases (MMPs) in human caries. J Dent Res 2006; 85(1): 22-32. 25. Steffensen B, Wallon UM, Overall CM. Extracellular matrix binding properties of recombinant fibronectin type II-like modules of human 72-kDa gelatinase/type IV collagenase. High affinity binding to native type I collagen but not native type IV collagen. J Biol Chem 1995; 270: 11555–11566. 26. Kähäri VM, Saarialho-Kere U. Matrix metalloproteinases and their inhibitors in tumour growth and invasion. Ann Med 1999; 31(1): 34-45. 27. Warner RL, Bhagavathula N, Nerusu KC, Lateef H, Younkin E, Johnson KJ,, et al. Matrix metalloproteinases in acute inflammation: induction of MMP-3 and MMP-9 in fibroblasts and epithelial cells following exposure to pro-inflammatory mediators in vitro. Exp Mol Pathol 2004; 76(3): 89-95.
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28. Yan C, Boyd DD. Regulation of matrix metalloproteinase gene expression. J Cell Physiol 2007; 211(1): 19-26. 29. Marin-Castaño ME, Elliot SJ, Potier M, Karl M, Striker LJ, Striker GE, et al. Regulation of estrogen receptors and MMP-2 expression by estrogens in human retinal pigment epithelium. Invest Ophthalmol Vis Sci 2003; 44(1):50-9. 30. Breckon JJ, Papaioannou S, Kon LW, Tumber A, Hembry RM, Murphy G, et al. Stromelysin (MMP-3) synthesis is up-regulated in estrogen-deficient mouse osteoblasts in vivo and in vitro. J Bone Miner Res 1999; 14(11):1880-90. 31. Lee YJ, Lee EB, Kwon YE, Lee JJ, Cho WS, Kim HA, et al. Effect of estrogen on the expression of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 and tissue inhibitor of metalloproternase-1 in osteoarthritis chondrocytes. Rheumatol Int 2003; 23(6):282-8.
12
Table 1. Allele and genotype distribution of the MMP2 and MMP3 polymorphisms among the three groups of volunteers (n=102); Comparison of the allele and genotype frequencies between the different groups CF
LCE
HCE
CF/ LCE
CF+LCF+LCE/HCE CF+LLCE/HCE
n=20
n=41
n=41
p-value
p-value
p-value
rs2287074
n (%)
n (%)
n (%)
allele G
19 (48)
26 (32)
34 (41)
allele A
21 (52)
56 (68)
48 (59) p>0.05
p>0.05
p>0.05
genotype GG
3 (15)
4 (10)
8 (19)
genotype GA
13 (65)
18 (44)
18 (44) р=0.13
p>0.05
p>0.05
genotype AA
4 (20)
19 (46)
15 (37) р=0.04
p>0.05
p>0.05
rs679620
n (%)
n (%)
n (%)
allele A
23 (58)
32 (39)
41 (50)
allele G
17 (42)
50 (61)
41 (50) p=0.05
p>0.05
p>0.05
genotype AA
7 (35)
9 (22)
7 (17)
genotype AG
9 (45)
14 (34)
27 (66) p>0.05
p=0.01
р= 0.004
genotype GG
4 (20)
18 (44)
7 (17)
p=0.03
р=0.008
MMP2 gene
MMP3 gene
p=0.06
13
S0003-9969(16)30071-1 http://dx.doi.org/doi:10.1016/j.archoralbio.2016.03.007 AOB 3570
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
30-12-2014 22-11-2015 17-3-2016
Please cite this article as: Karayasheva Dobrina, Glushkova Maria, Boteva Ekaterina, Mitev Vanyo, Kadiyska Tanya.Association study for the role of Matrix metalloproteinases 2 and 3 gene polymorphisms in dental caries susceptibility.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2016.03.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Association study for the role of Matrix metalloproteinases 2 and 3 gene polymorphisms in dental caries susceptibility
Dobrina Karayashevaa, Maria Glushkovab, Ekaterina Botevaa, Vanyo Mitevc, Tanya Kadiyskab,c
a
Department of Conservative Dentistry, Faculty of Dental Medicine, Sofia Medical University, Sofia-
1431, 1 Sv. G. Sofiiski Blvd., Sofia, Bulgaria b
Genetic Medico-Diagnostic Laboratory Genica, 90 Tsar Asen str., Sofia-1643, Bulgaria
c
Department of Medical Chemistry and Biochemistry, Medical Faculty, Sofia Medical University, 2
Zdrave str., Sofia-1431, Bulgaria
Running Title: MMP2 and MMP3 SNPs and dental caries
Correspondence to: Tanya Kadiyska, Department of Medical Chemistry and Biochemistry, Sofia Medical University, Sofia-1431, 2 Zdrave str., Sofia-1431, Bulgaria Tel/Fax: +359 2 9530715; E-mail: [email protected]
1
Highlights
MMP2 genotype frequencies differ between CF and LCE groups MMP2 genotype AA increase the risk for dental caries development about 3,5 times MMP3 allele and genotype frequencies differ between CF and caries experience groups MMP3 genotype frequencies differ between LCE and HCE groups MMP3 genotype GG decrease the risk of HCE about 4 times
2
ABSTRACT Objective: Various exogenous and endogenous risk factors have been described as contributing to dental caries susceptibility. In the last decade it has been established that both pro and active forms of host derived Matrix metalloproteinases (MMPs) are present in the oral cavity. MMPs role in caries development has been hypothesized. The aim of this study was to analyse MMP2 (rs2287074) and MMP3 (rs679620) single nucleotide polymorphisms (SNPs) and their role in caries susceptibility. Design: The two SNPs were analysed by PCR- restriction fragment length polymorphism (RFLP) in a sample of 102 ethnic Bulgarian volunteers (42 males and 60 females), all students in Sofia Medical University. Results: Statistical analysis of the MMP2 SNP showed significant differences for the genotype frequencies between the caries free (CF, DMFT=0) and low caries experience (LCE, DMFT≤5) groups. Analysis for the non-synonymous MMP3 SNP found significant differences between both CF vs caries experience groups (LCE+ high caries experience (HCE, DMFT≥5)) and LCE vs HCE groups. The presence of allele G decreased the risk of HCE about 4 times. Conclusions: MMP2 and MMP3 genes are likely to be involved in caries susceptibility in our population. However, as dental caries is a multifactorial disorder and several genes are likely to have influence on it, it is reasonable to expect that SNPs, even those proven to be functional like rs679620, potentially play a significant, but not major role in the disease outcome.
Key words: candidate genes, dental caries, Matrix metalloproteinases, MMP2, MMP3
3
INTRODUCTION
Dental caries is a complex disease characterized by demineralization of inorganic portion and destruction of organic portion of teeth, eventually resulting in cavitation. This process is caused by certain cariogenic bacteria of the oral cavity, and various exogenous and endogenous risk factors have been described as contributing to dental caries development. In addition, the impact of individual genetic variation on the development of dental caries, including its severity and extent has been demonstrated in twin studies (1). In the last decade it has been established that both pro and active forms of host derived MMPs are present in the oral cavity. They are activated on-site by the lower pH, caused by the presence of the cariogenic bacteria, suggesting their importance in dental lesion process (2). MMPs are a family of zincdependent endopeptidases that degrade extracellular matrix proteins. Twenty five members of the MMP family have been identified in humans. On the basis of substrate specificity and homology, MMPs can be divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs (MT-MMPs), and other MMPs (3,4,5). MMPs contribute to various physiological processes, e.g. embryonic development, tissue turnover, and wound healing, as well as to pathological processes, e.g. cancer, cardiovascular diseases, arthritis, periodontitis, and fibrosis (6,7,8,9). Several MMPs are found to have a role in tooth development. It was suggested that they may regulate mineralization by controlling the proteoglycan turnover (10). Although most MMPs are expressed during normal dentin-pulp complex formation and maintenance, MMP-20 was identified as the first proteinase secreted into the developing enamel matrix (11). The important role of genetics in the multifactorial etiology of dental caries is well established. However, Genome wide association studies (GWAS) on caries risk are limited in the literature. A recent published paper by Wang et al. in 2013 identified 53 potential susceptibility genes for dental caries and their protein-protein interactions. Functional analyses of these 53 genes revealed three major clusters: cytokine network relevant genes, matrix MMPs family, and transforming growth factor-beta (TGF-β) family (12). Therefore the aim of the present study was to analyse single nucleotide polymorphisms (SNPs), located into two MMP genes- MMP2 (rs2287074, p.Thr460Thr, c.1380G>A) and MMP3 (rs679620, p.Lys45Glu, c.133A>G) and to evaluate their role in dental caries susceptibility in a clinically selected group of 102 Bulgarian students.
4
MATERIALS AND METHODS
More than 140 subjects, all of them students in Sofia Medical University completed a questionnaire about their ethnicity, parent’s education and social environment, oral hygiene habits, dietary habits, snacking between meals, previous preventive fluoride treatments, orthodontic treatment, frequency of preventive dental examinations, self-evaluation of salivary flow, medical history. Of them, 102 fulfilled the initial requirements for being clinically healthy, without systemic diseases or generalized periodontal inflammations, perfect oral hygiene habits and at least annual dental examinations. The selected participants were ethnic Bulgarians, aged from 20 to 32 years (42 males and 60 females). Subsequently all 102 subjects were clinically examined. Clinical examinations were performed with dental mirrors, explorers, artificial light and photo-polymerizing lamps. Radiographs were not performed. The study was conducted according to the World Medical Association Declaration of Helsinki and additionally approved by the Ethics Committee of Sofia Medical University. Written informed consent was obtained from all participants prior to genetic testing. The studied subjects were classified according to the caries experience level using the DMFT indexes. According to recently published papers, the different genetic loci and SNPs could lead to either increased or decreased activities and thus contribute differently to caries experience. In this context, the genetic factors have been divided into caries protective genes (associated with CF phenotype) and caries susceptibility genes (associated with LCE or HCE phenotypes) (13,14). The subjects were divided into three groups: CF (with DMFT=0, n=20), LCE (with DMFT≤5, n=41) and HCE (with DMFT≥5, n=41). Epithelial buccal cells were collected in sterile, Еpendorf-type plastic containers of 2 ml. Each container was labeled indicating the name and individual number. The samples were collected after normal hygiene and breakfast individual habits of the participants, between 9 and 11.30 a.m. DNA was extracted from buccal cells by the Chelex® 100 (Bio-Rad Laboratories, Hercules, CA) extraction technique. The vials with buccal cells were centrifuged at 7,500 rpm for 15 min, and the supernatant was removed. Then the pellet was resuspended thorough mixing in 150 µl of 5% Chelex 100 resin and Proteinase K solution, followed by incubation at 56°C for 2 hours in a dry heat block. The mixture was boiled for 10 min and then chilled on ice for 5 min. It was then centrifuged at 12,000 rpm for 10 min. The supernatant was carefully removed, with the Chelex avoided. The DNA was stored at 20°C prior to analysis. 5
For MMP2 SNP genotyping, DNA fragments were amplified by the use of primer pairs MMP2F 5´GTCCAGGCATCTTCTTGTTA-3´ and MMP2R 5´-GAGGACAAGAAGCAAGCTCC-3´ (322 bp). PCR amplification was performed in 25 µl volume containing 100 ng of genomic DNA. The thermal cycles were initiated for 5 minutes at 95 C, followed by 30 cycles of 40 seconds at 95 C, 30 seconds at 58 C, and 30 seconds at 72°C, and a final extension at 72°C for 10 minutes. The PCR products were subjected to restriction fragment length polymorphism (RFLP) with 5 U of BseYI (NEB, Germany) at 37°C overnight, and the products were separated on a 3% agarose gel and stained with Ethidium bromide in order to yield G (122 and 200bp) and A (322 bp) alleles, allowing, therefore, the determination of the GG, GA, and AA genotypes. For MMP3 SNP genotyping, DNA fragments were amplified by the use of primer pairs MMP3F 5´GATTAAGAAGTGAGCAACTGCA-3´ and MMP3R 5´-CCTCCAATCCAAGGAACTTC-3´ (212 bp). PCR amplification was performed using the conditions for the MMP2 SNP. The PCR products were then subjected to RFLP with 5 U of TaqI (NEB, Germany) and incubated overnight at 65°C. The products were separated on a 3% agarose gel and stained with Ethidium bromide in order to yield A (212bp) and G (120 and 92 bp) alleles, allowing, therefore, the determination of the AA, AG, and GG genotypes. Genetic data was entered and processed with SPSS 16.0 software (SPSS, Chicago, IL). Frequencies of alleles and genotypes were compared using χ2 test. OR and their corresponding 95% CI were determined as per standard statistical methods. The Hardy-Weinberg equilibrium among the CF volunteers (used as control group) was also tested using standard χ2 statistics. P values of <0.05 are considered statistically significant.
RESULTS
All 102 volunteers were successfully genotyped for the MMP2 and MMP3 SNPs. The distribution of the allele and genotype frequencies is shown in Table 1. There are no differences in age and gender distribution between the groups. Considering the two SNPs independently, genotype distributions do not deviate from Hardy-Weinberg equilibrium among the groups (p>0,05). There were no significant differences of the allele and genotype frequencies between the subjects from our study and the reported in dbSNP database for other European populations. For MMP2 (rs2287074), the reported minor allele frequency (MAF) is 35%, while in our study is 39%. For MMP3 (rs679620), the reported MAF is 37%, while in our study is 47%. The statistical analysis for the MMP2 gene showed significant differences for the genotype frequencies between the CF and LCE groups. We found that the presence of genotype AA increased the risk for 6
dental caries development about 3,5 times. The comparison between the other investigated groups of CF vs HCE and CF vs LCE+HCE showed differences close the borderline of p=0.05 (Table 1). However, after applying the Yates correction for the small sample sizes, the p-values showed no statistical differences between the three groups. Our study for the non-synonymous MMP3 SNP found significant differences between both CF vs LCE+ HCE and LCE vs HCE groups. The presence of genotype GG decreased the risk of HCE about 4 times (Table 1). When the three groups were divided according to their sex (data not shown), no significant differences were found between the groups and MMP2 allele and genotype frequencies. After statistical analysis of MMP3, it was noted that the significant difference between the groups of CF or LCE and HCE cases was due to the high impact of female, but not male cases.
DISCUSSION
Although caries prevalence has decreased over the past few decades in the North West of Europe, South and Eastern European populations are increasingly affected. In the last years the research was focused to investigate the susceptibility genes for dental caries. Special interest was carried out in the study of genetic variations of genes involved in the tooth formation and differentiation. It has been shown that variations of genes which alter enamel and dentin organization may increase the individual caries susceptibility. Several authors verified the relationship between enamel defects and dental caries (15,16). Genes involved in amelogenesis alone or in combination with high Mutans streptococci levels, were shown to result in increased caries rates while genes involved in dentin formation as MMP2 and 3 might contribute to faster carious lesion progression in dentin and periapical pathologies (17,18,19) Another group of genes, involved in ingestive behaviour has been widely studied. Some examples are the TAS1R2 gene (Taste receptor, Type 1, Member 2), found to influence taste perception (20) and GLUT2 gene (Glucose transporter type 2), involved in the energy homeostatic pathways (21). Functional polymorphisms of DEFB1 (beta defensin 1) and Lactotransferrin genes have also been shown to be associated with caries susceptibility (22,23). We hypothesized that MMP genetic variations may be one of several contributing factors to the susceptibility of dental caries. MMP-2 and MMP-3 genes were chosen as representative candidates for dental caries susceptibility and progression, described also from Chaussain-Miller in 2006 (24).
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MMP2, also known as gelatinase A, is a membrane-bound protein that is important for extracellular matrix turnover. It has proteolytic activity against components of the basement membrane, preferentially cleaving collagen types IV, V, VII, and XI and gelatin (25). MMP-3, also known as stromelysin 1 is the main metalloproteinase that is secreted by fibroblasts, synovial cells and chondrocytes. It has proteolytic activity against proteoglycans, fibronectin, laminin and type IV collagens (26). The transcriptional level of most MMPs is regulated by growth factors and cytokines, but also singlenucleotide polymorphisms (SNPs) of several MMP genes have been shown to be transcriptional regulators (27,28). The two SNPs analysed in the recent study have been previously associated with host susceptibility of developing periapical lesions in individuals with untreated carious lesions (19). Furthermore, it has been shown that hormones, especially oestrogen, do upregulate MMP2 levels (29). There is no sufficient data about MMP3 gene expression and oestrogen. Some authors demonstrated MMP3 up-regulation in oestrogen-deficient mouse osteoblasts, while others did not show any significant effect of oestrogen on MMP3 (30,31). Following our results of stronger association between MMP3 allele G female carriers and dental caries resistance, we can speculate that there is a negative regulation of MMP3 expression by oestrogen. Despite the relatively small number of participants (102 people in total) in this pilot study for Bulgaria, the selected group of volunteers was characterized by a very high homogeneity with regard to: ethnicity, age (20-32 year-olds), social status and education (students), parents' education (higher), nutritional and hygiene habits, health culture, awareness of and motivation for their general and dental health care. The evidence that individuals exposed to the same levels of environmental risk factors do present differences in the DMFT index suggests even stronger influence of genetic factors in the aetiology of the disease. We emphasize the hypothesis that MMP inhibitors may have role in preventing caries progression and functional SNP detection may guide therapeutic decision making. Based on our results, it could be concluded that MMP2 and MMP3 genes are likely to be involved in caries susceptibility in our population. However, as dental caries is multifactorial disorder and several genes are likely to have influence on it, it is reasonable to expect that SNPs, even those proven to be functional like rs679620, potentially play a significant, but not major role in the disease outcome. Although these results look promising, additional studies exploring larger sample sizes and extended genetic and haplotype data are needed to clarify the association of dental caries with MMP genetic polymorphisms.
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ACKNOWLEDGEMENTS
The authors are grateful to the volunteers for participating in this study. This study was partially supported by research grant No. 26/2014, Committee of Medical Sciences, Sofia Medical University.
CONFLICT OF INTEREST
Herewith we declare that we have no conflict of interest.
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Table 1. Allele and genotype distribution of the MMP2 and MMP3 polymorphisms among the three groups of volunteers (n=102); Comparison of the allele and genotype frequencies between the different groups CF
LCE
HCE
CF/ LCE
CF+LCF+LCE/HCE CF+LLCE/HCE
n=20
n=41
n=41
p-value
p-value
p-value
rs2287074
n (%)
n (%)
n (%)
allele G
19 (48)
26 (32)
34 (41)
allele A
21 (52)
56 (68)
48 (59) p>0.05
p>0.05
p>0.05
genotype GG
3 (15)
4 (10)
8 (19)
genotype GA
13 (65)
18 (44)
18 (44) р=0.13
p>0.05
p>0.05
genotype AA
4 (20)
19 (46)
15 (37) р=0.04
p>0.05
p>0.05
rs679620
n (%)
n (%)
n (%)
allele A
23 (58)
32 (39)
41 (50)
allele G
17 (42)
50 (61)
41 (50) p=0.05
p>0.05
p>0.05
genotype AA
7 (35)
9 (22)
7 (17)
genotype AG
9 (45)
14 (34)
27 (66) p>0.05
p=0.01
р= 0.004
genotype GG
4 (20)
18 (44)
7 (17)
p=0.03
р=0.008
MMP2 gene
MMP3 gene
p=0.06
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