RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA BANGALORE
ANNEXURE II
PROFORMA FOR THE REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / NAME OF THE CANDIDATE AND ADDRESS / : / Dr SAMRIDDHI JAINDEPARTMENT OF ORAL MEDICINE AND RADIOLOGY,
DAPMRV DENTAL COLLEGE
CA 37, 24th MAIN,
J.P.NAGAR IST PHASE
BANGALORE – 560078.
2. / NAME OF THE INSTITUTION / : / DAPMRV DENTAL COLLEGE BANGALORE.
3. / COURSE OF THE STUDY AND SUBJECT / : / MASTER OF DENTAL SURGERY (M.D.S)
ORAL MEDICINE AND RADIOLOGY
4. / DATE OF ADMISSION TO THE COURSE / : / 04/06/11
5. / TITLE OF THE TOPIC / : / DETERMINATION OF LYSYL OXIDASE POLYMORPHISM IN ORAL SUBMUCOUS FIBROSIS
6. BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study:
Oral submucous fibrosis (OSMF) is a chronic debilitating disease of the oral cavity characterized by inflammation and progressive fibrosis of the submucosal tissues (lamina propria and deeper connective tissues). It results in marked rigidity and an eventual inability to open the mouth. javascript:showrefcontent('refrenceslayer');The buccal mucosa is the most commonly involved site, but any part of the oral cavity may be involved.
An ethnic basis is indicated because OSMF is found mostly in Asians or Asians settled in other countries. OSMF is most commonly found in India, although cases have been reported in China, Malaysia, South Africa, Papua-New Guinea, Sri Lanka, Myanmar, United Kingdom and Canada.1 Worldwide, estimates of OSMF indicate that 2.5 million people are affected, with most cases concentrated in the Indian subcontinent (southern states). OSMF has been identified as a potentially malignant condition and its malignant transformation rate has been reported to be 7.6% over a 17 year period.2
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Epidemiological data and intervention studies suggest that areca nut is the main aetiological factor for OSMF. Other etiological factors implicated are chillies, lime, tobacco, nutritional deficiencies such as iron and zinc, and collagen disorders. Areca nut is made up of alkaloid and flavonoid components.3 Four alkaloids arecoline, arecaidine, guvacine and guvacoline have been identified in areca nut, of which arecoline is the most potent agent while flavonoid components like tannins and catechins have a synergistic role. Arecanut consists of high free copper content which is recognised to play a passive role in the pathogenesis of OSMF.2
OSMF is a consequence of disturbances in the equilibrium between synthesis and degradation of extracellular matrix (ECM). Overall increased collagen production and decreased collagen degradation results in increased collagen deposition in the oral tissue leading to fibrosis. Increased soluble copper levels found in oral fluids of chronic chewers may support the fact that copper acts as an initiating factor in OSMF by stimulating fibrosis through up-regulation of lysyl oxidase (LOX) activity. LOX is a copper activated enzyme critical for collagen cross-linking and organization of extracellular matrix. The activity of LOX has been found to increase in fibroblasts cultured from OSMF patients.4
Thus there is a need to assess the up regulation of LOX Arg158Gln polymorphism in OSMF which is a potentially malignant condition. Aim of the present study is to determine LOX Arg158Gln polymorphism in OSMF to assess a probable role in the pathogenesis of OSMF.
6.2 Review of Literature:
Oral submucous fibrosis is a disease due to a chronic, insidious change in fibro-elasticity, characterized by burning sensation in the oral cavity, blanching, and stiffening of the oral mucosa and oropharynx leading to trismus and inability to open the mouth. The overlying epithelium may become the site of dysplastic change and malignant transformation, making it a potentially malignant condition affecting the oral cavity.2
OSMF was initially considered as a precancerous condition. The precancerous nature of OSMF was first postulated by Paymaster in 1956, who described the development of a slow growing squamous cell carcinoma in one third of the cases of OSMF seen among patients in the Tata Memorial Hospital in Bombay. In a 15-year follow-up study a malignant transformation rate of 4.5% has been demonstrated and another study has reported malignant transformation rate of 7.6% over a period of 17years.1
At a workshop coordinated by the WHO Collaborating Centre for Oral Cancer and Precancer in the UK, issues related to terminology, definitions and classification of oral precancer were discussed by an expert group and the term potentially malignant disorders was recommended to refer to precancer, as it conveys that not all disorders described under this term may transform into cancer. Now OSMF is considered as a potentially malignant condition.5
Oral submucous fibrosis (OSMF) is now globally accepted as an Indian disease. The pathogenesis of OSMF is by the dual action of areca nut. It is suggested that arecoline not only stimulates fibroblastic proliferation and collagen synthesis but also decreases its breakdown. Under the influence of areca nut alkaloids ( arecadine and arecoline), fibroblasts differentiate into phenotypes that produce more collagen. There is a dose dependent increase in the production of collagen by fibroblasts under the influence of arecanut alkaloids. Areca nut contains tannin and copper which stabilize collagen by cross-linking it. The authors have concluded that arecoline interferes with the molecular processes of deposition and/or degradation of extracellular matrix (ECM) molecules such as collagen. Due to this interference, phagocytic activity of fibroblasts is reduced, because of up or down regulation of the key enzymes such as lysyl oxidase and alteration in the expression of various ECM molecules. The above mechanisms may explain the progression of fibrosis in OSMF.3
OSMF is a collagen-related disorder induced by betel quid chewing. However, the cumulative exposure to betel quid varies in OSMF patients and there is individual susceptibility to betel quid-induced OSMF. A study compared the association of OSMF and polymorphisms of six collagen-related genes namely, collagen 1A1and 1A2 (COL1A1 and COL1A2), collagenase-1 (COLase), transforming growth factor β1 (TGF-β1), lysyl oxidase (LYOXase), and cystatin C (CST3), between patients with low and high exposure to betel quid using PCR-based restriction fragment length polymorphism assays. It was found that the genotypes associated with the highest OSMF risk for collagen 1A1, collagen 1A2, collagenase-1, transforming growth factorβ1, lysyl oxidase, and cystatin C were CC, AA, TT, CC, AA, and AA, respectively, for the low-exposure group, and TT, BB, AA, CC, GG, and AA, respectively, for the high-exposure group. The authors have reported an increased risk of OSMF with increasing number of high-risk alleles for those with both high and low exposures for betel quid. These results of the study imply that susceptibility to OSMF could involve multigenic mechanisms modified by the betel quid-exposure dose.6
The lysyl oxidase enzyme catalyses the oxidative deamination of lysine residues in elastin and collagens. This converts soluble monomers of collagen and elastin into insoluble fibres in the extracellular matrix. Up-regulation of LOX expression and increase in LOX activity have been demonstrated in several fibrotic disorders, including liver fibrosis, scleroderma and pulmonary fibrosis. Also increase in LOX activity has been demonstrated in fibroblasts cultured from the buccal mucosa of OSMF patients in comparison with normal fibroblasts. The authors have concluded that up-regulation of LOX may therefore be responsible for the formation of the dense fibrotic bands seen in OSMF.7
Increased soluble copper levels present in oral fluids of chronic chewers may support the fact that copper acts as an initiating factor in OSMF by stimulating fibrosis through up-regulation of LOX activity. LOX can be induced by areca nut and copper, and LOX also has transcription activator function, which can activate collagen III and elastin promoters in OSMF. It has been reported that LOX is increased in fibroblasts obtained from OSMF patients and other fibrotic lesions. The authors have investigated the effects of LOX Arg158Gln polymorphism on fibrosis and concluded that approximately 20% areca chewers carried LOX Arg158Gln allelotype and OSMF patients older than 50 years carried much more LOX Arg158Gln polymorphism.4
Lysyl oxidase (LOX) is synthesized in the cells as a preproenzyme. A 50-kDa LOX proenzyme is secreted into the extracellular matrix (ECM) and cleaved between Gly168 and Asp169 by a peptidase to yield a 32-kDa active enzyme and an 18-kDa propeptide. Five LOX family genes have been identified encoding LOX and various LOX-like proteins (LOXL1, LOXL2, LOXL3, and LOXL4. All LOX family members show a highly conserved COOH-terminal region, such as the copper-binding site and other active domains. LOX plays a central role in ECM organization. Among all the single nucleotide polymorphism sites in the LOX coding region, G473A has the highest frequency. The mutation changes the Arg at residue 158 to Gln (LOX Arg158Gln), and the site is near the peptidase cutting sites of LOX. In a study to find out the effects of the functional LOX Arg158Gln polymorphism on oral tumorigenesis the authors have reported that relative to the patients with the wild-type LOX, oral squamous cell carcinoma patients carrying the polymorphic allele have a lower risk of lymph node metastases and tumor progression.8
6.3 Objectives of the study:
1. To determine the LOX Arg158Gln polymorphism in subjects with OSMF.
2. To determine the LOX Arg158Gln polymorphism in subjects with chewing habit (arecanut, pan masala, gutkha, betel quid) but no clinically evident OSMF.
3. To determine the LOX Arg158Gln polymorphism in age and sex matched healthy individuals.
4. To compare the LOX Arg158Gln polymorphism in subjects with OSMF, with chewing habit but no clinically evident OSMF and in age and sex matched healthy individuals.
7. MATERIALS AND METHODS 7.1 Source of data
The subjects for the present study will be selected from the outpatient department of Oral Medicine and Radiology DAPM R.V Dental College, Bangalore. 40 subjects will be selected for the study, which will be divided into the study group and the control group. The study group will be further divided into Group I (OSMF) and Group II (subjects with chewing habit).
Study Group:
· Group I - 15 subjects with OSMF
· Group II - 15 subjects with Chewing habit but no clinically evident OSMF.
Control Group: 10 age and sex matched healthy individuals.
A detailed history will be elicited from the subjects included in the study comprising of medical and habit history following which a thorough clinical examination will be carried out. For the Group I (OSMF) subjects the following criteria will be considered for clinical diagnosis of OSMF:
· Clinically discernible blanching and pallor
· Palpable bands
· Restriction of mouth opening
· Severe burning sensation of mouth, aggravated by use of even moderate spicy food.9
The clinical diagnosis of OSMF in subjects will be confirmed by histopathological examination.
Inclusion criteria:-
Subjects above 18 years of age will be selected for the study.
Exclusion criteria:-
1. Pregnant and lactating women.
2. Subjects with previous treatment for OSMF.
3. Subjects with any collagen diseases or immune mediated disorders.
4. Subjects with history of head and neck tumors, breast cancer and gastric cancer.
5. Subjects with prior neoplastic surgeries and cancer metastases.
6. Other diseases such as myocardial fibrosis, exfoliating glaucoma.
7.2 Method of collection of data
Prior to conducting the study an informed consent (Annexure 1and 2) will be obtained from all the subjects selected for the study. A case history with detailed medical and habit history will be recorded and thorough clinical examination will be carried out for all the subjects included in the study. Habit counselling will be done for cessation of habit for all the subjects included in the study group (Group I and Group II).
2 ml of venous blood will be drawn from the antecubital fossa by venupuncture from all the subjects included in the study and will be collected in vials coated with an anticoagulant namely Ethylenediaminetetraacetic acid (EDTA). Genomic DNA isolation will be done from peripheral leukocytes. The genetic polymorphism, LOX Arg158Gln in the LOX coding region will be determined in peripheral leukocyte DNA using PCR-based restriction fragment length polymorphisms (PCR-RFLP). The results thus obtained will be tabulated and statistical analysis will be done using Analysis of variance test (ANOVA) and Chi square test.
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7.3 Investigations to be conducted on the patient
1. Incisional biopsy of all the subjects included in the Group I (OSMF).
2. Isolation of DNA from leukocytes and performing genotyping using PCR based restriction fragment length polymorphisms for all the subjects included in the study.
7.4 Has Ethical clearance been obtained?
Yes, the Ethical clearance has been obtained.
8. REFERENCES:
1. R. Rajendran. OSMF: etiology, pathogenesis, and future research. Bulletin of the World Health Organization 1994; 72 (6): 985-996
2. Punnya V. Angadi & Sanjay S. Rao. Areca nut in pathogenesis of OSMF: revisited. Oral Maxillofac Surg 2011; 15:1–9
3. M.K. Gupta, Shubhangi Mhaske, Raju Ragavendra , Imtiyaz. OSMF - Current Concepts in Etiopathogenesis. People’s Journal of Scientific Research 2008; 1
4. Shieh TM, Tu FH, Ku TH, Chang SS, Chang KW, Liu CJ. Association between lysyl oxidase polymorphisms and oral submucous fibrosis in older male areca chewers. J Oral Pathol Med 2009; 38:109–113
5. S. Warnakulasuriya, Newell. W. Johnson, I. van der Waal. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J Oral Pathol Med 2007; 36: 575–80.
6. Chung-Jung Chiu, Min-Lee Chang, Chun-Pin Chiang, Liang-Jiunn Hahn, Ling-Ling Hsieh, and Chien-Jen Chen. Interaction of Collagen-related Genes and Susceptibility to Betel Quid-induced OSMF. Cancer Epidemiol Biomarkers Prev 2002; 11: 646-653
7. Trivedy C, Warnakulasuriya KA, Hazarey VK, Tavassoli M, Sommer P, Johnson NW. The up-regulation of lysyl oxidase in OSMF and squamous cell carcinoma. J Oral Pathol Med 1999; 28: 246–51.
8. Shieh TM, Lin SC, Liu CJ, Chang SS, Ku TH, Chang KW. Association of expression aberrances and genetic polymorphisms of lysyl oxidase with areca-associated oral tumorigenesis. Clin Cancer Res 2007; 13: 4378–85.
9. Bailoor DN, Nagesh KS, Reddi Ramechandra. Oral Precancer. In Bailoor DN, Nagesh KS, editors: Fundamentals of Oral Medicine and Radiology.2nd ed. New Delhi: Jaypee Brothers; 2005. p 182-193.
9. SIGNATURE OF THE CANDIDATE:
10. REMARKS OF THE GUIDE: THE STUDY IS FEASIBLE
AND HAS BEEN APPROVED
11. NAME AND DESIGNATION OF:
11.1 GUIDE: DR. DIVYALAKSHMI M.R
READER
DEPARTMENT OF ORAL