Association between matrix metalloproteinases-2 and -9 gene polymorphism with basement membrane disruption in oral lichen planus: A case-control pilot study

Abstract

Objectives

This study investigated genetic polymorphism of matrix metalloproteinases (MMP) −2 and −9 in oral lichen planus (OLP) and their association with the basement membrane status.

Study design

This case-control study involved genotyping of peripheral blood sample of 32 OLP patients and 106 ethnically matched controls. Single nucleotide polymorphisms (SNP) that were assessed in the groups were- MMP9 rs3918242, MMP9 rs17576 and MMP2 rs865094. Basement membrane status of the OLP biopsy samples was microscopically assessed and recorded following Periodic acid Schiff staining.

Results

MMP9 rs3918242 showed significant genotypic and allelic associations between OLP subjects and controls. It was also significantly associated with intact basement membranes in OLP cases with increased frequency of ‘TT’ genotype and ‘T’ allele. No association was found with regard to MMP9 rs17576 and MMP2 rs865094.

Conclusion

Biallelic substitution at the promoter region of MMP9 (rs3918242) gene may be associated with increased risk of development of OLP. It may be involved in compromising the integrity of the basement membrane junction.

Graphical abstract

1. Introduction

Oral lichen Planus (OLP) is the mucosal counterpart of lichen planus. It is a chronic immune-mediated disorder whose exact etiology is currently unknown and has a world-wide prevalence rate of 1.01%.¹ Clinically, the lesions are categorized as - reticular, atrophic, erosive, papular, bullous and plaque-like.² The disorder is marked by periods of exacerbation and remission with significant morbidity associated with the recurring lesions.

Several antigen specific and non-antigenic mechanisms are proposed as the etiopathogenetic agents for the basement membrane changes that are pathognomonic of the OLP lesions. Basement membrane changes in OLP comprise of breaks, branches and duplications.³ Disruption of the integrity of the basement membrane in OLP accounts for the basal cell changes as well as for the observed clinical manifestations³ Primarily, T-cells, mast cells, cytokines and matrix metalloproteinases (MMP) have been widely implicated in the immune dysregulation of OLP.

MMPs are a large family of zinc-dependent endopeptidases, which are capable of digesting extracellular matrix and basement membrane components. On the basis of substrate specificity, sequence similarity, and domain organization, vertebrate MMPs are divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs and others. Gelatinases include MMP2 and MMP9 proteins which can degrade type IV, V, VII, X, XI, and XIV denaturated collagens, gelatin, elastin, aggrecan and fibronectin.⁴ Several immunohistochemical studies have reported altered expression of MMP9 (within inflammatory infiltrate) and MMP2 (within epithelium) in OLP tissues.5, 6, 7 Elevated MMP9 levels are reportedly associated with localized tissue destruction in OLP lesions.⁸ Despite the evidence of their involvement in the production and regulation of the phenotype of the OLP clinical lesions, one is not certain whether these alterations in MMPs are due to underlying genetic polymorphisms in these genes. There exists a lacunae regarding investigation of MMP gene polymorphism in OLP. Hence, this study was conducted to understand the significance of functionally relevant genetic variants of the MMP9 and MMP2 genes in OLP subjects. The study also attempted to decipher the association between MMP2 and MMP9 gene polymorphism with basement membrane disruption in OLP.

2. Materials and methods

2.1. Study subjects and controls

This case control study was conducted for a period of one year and six months (May 2013 to October 2014): included a total of 138 subjects, with 32 cases of oral lichen planus and 106 controls. It was conducted in the department of Oral Pathology and Microbiology of a dental college in collaboration with a national tertiary research centre for Biotechnology following approval from the Institutional ethics committee (IEC/M/04/2012/XXX) and written informed consent from the selected subjects.

Subjects reporting to the outpatient department of the dental college were clinically screened for OLP by a single investigator (A.S). Sociodemographic data and the clinical details of the lesion were recorded. Cases which were clinically and histopathologically diagnosed as OLP using modified World health Organization (WHO) diagnostic criteria⁹ were selected for the study. Histological assessment was performed by A.S and was confirmed by a second investigator who was blinded to the clinical details of the participants (H.R). All subjects suffering from systemic diseases such as diabetes mellitus, cardiovascular diseases, stroke; exhibiting lichenoid lesions; immunocompromised patients; having oral mucosal lesions besides OLP and; suffering from periodontal diseases were excluded from the study. Patients on concurrent or with history of treatment with steroids were excluded as well.

2.2. Histological assessment of basement membrane integrity

Basement membrane zone is a highly specialized extracellular matrix which is composed of laminin, type IV collagen, heparan sulfate proteoglycan and other glycoproteins. Demonstration of the basement membrane zone in the histopathological sections of OLP were done via special staining with Periodic Acid Schiff (PAS) by a single investigator after an interim period of 90 days to prevent information bias. The integrity of the stained basement membrane zone was assessed as “Intact” and “Not Intact” via microscopic examination at 400X magnification (Fig. 2). Any break and loss of continuity in the basement membrane zone were recorded as “Not Intact”.

Fig. 2.

Periodic acid Schiff stained histopathological sections (400X) of oral lichen planus displaying (arrows): a. Intact basement membrane and; b. disrupted basement membrane.

2.3. Genotyping

Peripheral blood was collected by venipuncture from the study subjects and subjected to genomic deoxyribonucleic acid (DNA) isolation using modified salting-out method.¹⁰ The quality and the quantity of the sample DNA were assessed via UV spectrophotometer (Bio Spec-1601, Shimadzu) following which all DNA samples were diluted to 50 ng/μl concentration. This study mainly explored the role of regulatory variants of MMP2 and MMP9 gene in OLP. Single nucleotide polymorphism (SNP) rs3918242 (−1562C > T) and rs17576 of MMP9, and rs865094 of MMP2 gene, were selected for this study (Table 1). SNPs were selected mainly on the basis of their functional and tagging status. The selected SNPs were retrieved from the NCBI SNP database. The SNP rs17576 is located in the exon region while SNP rs3918242 is located in the promoter region of the MMP9 gene on chromosome 20. The SNP rs865094 is located in the intron region of the MMP2 gene on chromosome 16.

Table 1.

Primers and sequences of MMP2 and MMP9 genes.^a

Primer	Sequence 5′-3′	Bases
mmp9rs3918242F	5′-ATgCCTggCACATAgTAggC-3′	20
mmp9rs3918242R	5′-TCgggCAgggTCTATATTCA-3′	20
mmp9rs17576F	5′-ACCATCCATgggTCAAAgAA-3′	20
mmp9rs17576R	5′-gggCTgAACCTggTAgACAg-3′	20
mmp2rs865094F	5′- CCTTgACCCATgCATTCTCT-3′	20
mmp2rs865094R	5′-CCATCCCAATgACCTCATCTA-3′	21

^aAbbreviations used: MMP- Matrix metalloproteinase.

Polymerase chain reaction (PCR) was performed using MMP2 and MMP9 primers and cycling conditions were denaturation for 5 min at 95 °C, followed by 30 cycles of 30s at 95 °C, 30 s at 56 °C, and 30s at 72 °C, with a final extension at 72 °C for 10 min. SNPs MMP9- rs3918242 and MMP2- rs865094 were genotyped via sequencing PCR using BigDye® Terminator v3.1. Following sequencing clean up to purify the sequencing product, it was analyzed by Genetic analyzer (Applied Biosystems 3730xl). AB1 PRISM Genetic analyzer software provided the sequence data in the form of a chromatogram wherein each nucleotide represented a peak.

SNP MMP9 rs17576 was analyzed via Restriction Fragment length Polymorphism method (Fig. 1). The PCR products of MMP9 rs17576 were digested with restriction enzyme Sma1. The digested samples were electrophoresed to determine the genotype.

Fig. 1.

Gel picture showing restriction fragment length polymorphism of rs17576. Lane 4,5,6: Homozygous GG; Lane 1,2,3,8: Heterozygous AG; Lane No 7: Homozygous AA.

2.4. Statistical analysis

The data were analyzed via GraphPad Prism version 5.02 (Graph Pad software, San Diego, California, USA). Frequency of the individual alleles as well as the homozygous and heterozygous genotypes of the MMP2 and MMP9 gene polymorphisms were determined in the subjects. Since the data were non-parametric in nature: to draw inferences and elucidate the associations between different parameters, Chi square (χ2) test was used. Multivariate logistic regression analysis was performed to assess the risk factors (Odds ratio) of different factors for each group. For all statistical evaluations, a two-tailed probability of value, p < 0.05 was considered significant. Hardy–Weinberg equilibrium analysis was also carried out in the control population.

3. Results

The demographic details signifying the sex, age, duration of the clinical manifestation, clinical phenotype of OLP in the patient group are shown in Table 2. Majority of the case subjects were females above 40 years of age with a mean age being 49.64 ± 6.37 years. The mean duration the clinical manifestation was 0.57 ± 0.27 years. The most common type of OLP was reticular followed closely by erosive type with least number of cases of atrophic type. The most common site of presentation was buccal mucosa followed by tongue, gingiva, labial mucosa and palate.

Table 2.

Demographic distribution of OLP subjects.^a

Characteristics	Cases (%)
Gender
Males	10 (31)
Females	22 (69)
Type of OLP
Reticular	14 (44)
Erosive	13 (40)
Atrophic	5 (16)
Age
<40 years	5 (16)
40–50 years	9 (28)
>50 years	18 (56)
Duration
< month	9 (28)
1–6 months	11(34)
6 months- 1 year	4 (13)
> year	8 (25)

^aAbbreviations used: OLP- Oral lichen Planus.

3.1. MMP Gene polymorphism

All the patients and controls were genotyped for rs3918242 (−1562C > T) and rs17576 of MMP9 and; rs865094 of MMP2 gene polymorphism. The control population was found to be in the Hardy–Weinberg equilibrium for genotype frequencies of the MMP2 (rs865094) and MMP9 (rs3918242 [−1562C > T] and rs17576) polymorphism.

While comparing the genotype and allele frequencies of the MMP2 and MMP9 polymorphism in OLP patient and control population, we observed that MMP9 rs3918242 (−1562C > T) polymorphism was significantly associated with OLP at both allelic (OR = 0.5063; 95%CI = 0.278 to 0.920; p value = 0.0241) and genotype levels (p value = 0.0065) (Table 3). There was a higher frequency of the genotype TT (25%) and allele T (40%) in OLP patients. None of the other studied SNPs were associated with OLP.

Table 3.

Comparison of genotype and allele frequencies of MMP9 and MMP2 gene variants between total OLP patients and controls.^a.

MMP9		CC	CT	TT	X2	p-value	C	T	OR (95%CI)	p-value
rs3918242	Cases	14	9	8			37	25
		0.452	0.290	0.258	10.06	0.0065	0.597	0.403	0.5063 (0.2786–0.9201)	0.0241
	Controls	56	40	6			152	52
		0.549	0.392	0.059			0.745	0.255
MMP9		AA	AG	GG	X2	p-value	A	G	OR (95%CI)	p-value
rs17576	Cases	3	18	11			24	40
		0.094	0.563	0.344	0.9759	0.6139	0.375	0.625	0.7826 (0.4406–1.390)	0.4023
	Controls	17	58	31			92	120
		0.160	0.547	0.292			0.434	0.566
MMP2		AA	AG	GG	X2	p-value	A	G	OR (95%CI)	p-value
rs865094	Cases	26	6	0			58	6
		0.813	0.188	0.000	2.215	0.3304	0.906	0.094	1.933 (0.7738–4.831)	0.1522
	Controls	72	31	2			175	35
		0.686	0.295	0.019			0.833	0.167

^aAbbreviations used: MMP- Matrix metalloproteinase; OLP- Oral lichen Planus; OR- Odd's ratio; ‘C’, ‘T’, ‘A’ and ‘G’- nucleotide bases.

Multivariate analysis showed an Odds ratio of 0.5778 (95%CI = 0.2751 to 1.214) for the female gender at the genotypic level.

3.2. Basement membrane integrity and MMP gene polymorphism

Following histopathological examination of the PAS-stained sections, 75% (n = 24) of the sections showed compromised/loss of basement membrane integrity (‘not intact’) while 25% (n = 8) showed intact basement membranes. MMP9 rs3918242 genotypic and allelic frequencies showed a significant association with intact basement membranes in OLP cases with increased frequency of ‘TT’ genotype and ‘T’ allele in the OLP subjects (OR = 0.2566; 95%CI = 0.0850 to 0.7744; p value = 0.0103) However, in case of the OLP patients showing disrupted basement membrane status, no association was found suggesting that the C-allele may be associated with a protective effect at the C-1562T polymorphic site. SNP's MMP9 rs17576 and MMP2 rs865094 did not show any significant association with the basement membrane status.

4. Discussion

Genetic polymorphism in OLP has primarily been investigated with regard to cytokines,^11,12 Toll like receptors,¹³ Mannose binding lectin,¹⁴ Vitamin D receptor,¹⁵ p53 tumor suppressor protein^16(p72), cyclooxygenase-2,¹⁷ Cytotoxic T Lymphocyte Antigen-4,¹⁸ serotonin transporter,¹⁹ ß-defensin,²⁰ myeloperoxidase¹⁷ and, hypoxia inducible factor-1.²¹ There exists an absence of investigation into gene polymorphism of MMPs: family of important extracellular matrix remodelling enzymes in OLP.

MMP9, also known as gelatinase B is known to play a role in basement membrane alterations, leukocyte trafficking, chemokine regulation, and apoptosis. The present study revealed a significant association of MMP9 rs3918242 (−1562C > T), at a genotypic as well as the allelic level, between the OLP subjects and the controls. Evaluation of single nucleotide polymorphism at −1562 (rs3918242) revealed that the cytosine (C) to thymidine (T) transition was more common in OLP case. T allelic substitution at C-1562T may lead to increased transcriptional expression of gelatinase B in OLP and that the subjects having this particular nucleotide substitution may demonstrate clinical lesions concurrent with that of OLP.

Promoters are involved in initiating transcription and are therefore among the many important cis-acting elements that regulate gene expression that might harbour functionally relevant polymorphisms.²² Zhang et al. determined the C-1562T polymorphic site to be a binding site for a transcription repressor protein and suggested that a C-to-T substitution at the polymorphic site (−1562) would result in the restriction of DNA-protein interaction, leading to a higher promoter activity of the T-allelic promoter.²³ Various investigators have postulated that the presence of minor allele i.e T allele alters the transcriptional regulation of the MMP9 enzyme.^23,24 SNP rs3918242 has previously been associated with atherosclerosis and coronary artery disease (CHD)²⁴; multiple gingival recessions,²⁵ type 2 diabetes,²⁶ psoriasis,²⁷ etc. The present study excluded patients with other systematic diseases and oral lesions in order to avoid confounding of the study.

MMP9 rs17576 also known as Gln279Arg is likely influences protein conformation, leading to a change in substrate-binding and enzyme activity.²⁸ However, we did not find significant association between MMP9 rs17576 gene polymorphism and OLP in our study. MMP2 molecule has been implicated by several studies in the pathogenesis of OLP.^29,30 However, we did not find significant association between MMP2 rs865094 gene polymorphism and OLP in our study.

Mazzarella et al., has previously reported higher expression of MMP mRNAs in erosive lichen planus in comparison to the reticular OLP.³¹ The same may be explored in the future studies with increased sample size as the limited sample size of the study prevented the present investigators to explore gene polymorphism distribution in various clinical subtypes of OLP.

Basement membrane changes in the OLP lesions are associated with the inflammatory cell infiltrates and clinical manifestations of OLP.³² Breaks and discontinuities of the basal lamina are found more frequently in erosive forms with duplication reported in both reticular and erosive forms of OLP.³³ We found MMP9 rs3918242 genotypic and allelic frequencies to be significantly associated with the OLP cases with intact basement membranes.

The recent World Health Organization 2017 classification on Head and Neck tumors has identified lichen planus as an oral potentially malignant disorder.³⁴ Hence, it is imperative to identify the risk factors affecting the basement membrane continuity in these lesions so as to prevent and monitor progression to malignancy. In this regard, MMP2 promoter polymorphism has been previously identified as a risk factor for oral carcinogenesis in OLP.³⁵

The association between OLP, diabetes mellitus and vascular hypertension is described as the Grinspan syndrome.³⁶ Since MMP9 rs3918242 is altered in CHD²⁴ and diabetes,^26,37 we believe that genetically influenced MMP9 production can influence co-morbid diseases in OLP patients as well.

The clinical significance of this study is underlined by the fact that SNP identification can be used for disease gene mapping, drug development and for understanding of individual drug response.³⁸ Disease gene mapping using SNPs requires additional research consisting of haplotype analysis and linkage disequilibrium. Pharmacogenetics has evolved to develop personalized medication and SNPs can alter the efficacy of the drugs as demonstrated in a CHD study by Xu et al.³⁹ wherein the TT genotype patients of MMP9 rs3918242 displayed a significantly greater reduction of low-density lipoprotein cholesterol than those carrying CC genotype following drug therapy. Future studies investigating the efficacy of the drugs may be planned in the OLP patients especially refractory cases.

Despite the encouraging results, we acknowledge the limitation of the present study: small sample size and the need to include subjects from other regions and ethnicities. We suggest future studies to take these factors into consideration along with investigation of exon and promoter regions of MMP2 gene in OLP. In essence, a larger sample size with adequate representation of each clinical subtype (reticular, erosive and atrophic) would allow for determination of association with MMP gene polymorphism.

5. Conclusion

Apart from causing direct damage to the basement membranes and the epithelium, MMPs can modify several non-matrix proteins such as cytokines and chemokines; suggesting that MMPs may have complex regulatory role over various chemical mediators. MMP9 gene polymorphism: biallelic substitution (C to T) at the position -C1562T may be associated with susceptibility to developing OLP. The association of OLP with the comorbid diseases is not co-incidental, but can be influenced by MMP9 gene polymorphism.

Source of funding

No funding was used.

Conflict of interest/Declarations of interest

None.

Author contributions

A.S, L.M.C, R.H, M.B: Conceptualization; A.S: Investigation and Data acquisition; A.S and S.S: Methodology; A.S: Project administration; A.S, S.S and M.B: Resources; L.M.C, R.H, M.B: Supervision; A.S: Writing - original draft; S.S, M.B, R.H and L.M.C: Writing - review & editing.