RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / NAME OF THE CANDIDATE
ADDRESS / DR.PREETHI SOMASHEKHAR
DEPARTMENT OF ORAL AND MAXILLOFACIAL PATHOLOGY,
Dr. SYAMALA REDDY DENTAL COLLEGE,HOSPITAL & RESEARCH CENTRE,
111/1 SGR COLLEGE MAIN ROAD,
MUNNEKOLALA, MARATHALLI,
BANGALORE-560037.
2. / NAME OF THE INSTITUTION / Dr. SYAMALA REDDY DENTAL COLLEGE,HOSPITAL & RESEARCH CENTRE,
BANGALORE-560037.
3. / COURSE OF STUDY AND SUBJECT / MASTER OF DENTAL SURGERY IN DEPARTMENT OF ORAL AND MAXILLOFACIAL PATHOLOGY.
4. / DATE OF ADMISSION TO THE COURSE / 5-8-2013.
5. / TITLE OF THE TOPIC / “DETECTION OF POLYMORPHISMS IN TRANSFORMING GROWTH FACTOR β GENE IN ORAL SUBMUCOUS FIBROSIS ”.
6.BRIEF RESUME OF THE INTENDED WORK
6.1:NEED FOR THE STUDY
Oral submucous fibrosis(OSF), is a potentially malignant disease predominantly seen in people of Asian decent. Recent epidemiological data indicate rapid increase in number of OSF cases in India. The reported reason is due to upsurge in the popularity of commercial areca nut preparations (pan masala/gutka) and an increased uptake of this habit by young people due to easy access and effective price changes.
The most important histopathological feature of oral submucous fibrosis is the increased deposition of collagen in the sub epithelial tissue. Collagen synthesis is influenced by a variety of mediators, such as growth factors, hormones, cytokines, and lymphokines. Transforming Growth Factor beta (TGFβ) a prominent mediator stimulates the production of collagens and other matrix components.
PCR is the enzymatic amplification of a specific DNA sequence invitro to obtain a large number of DNA copies .This technique amplifies DNA without involving any living organism. It is used in the identification of chromosomal disorders and heriditary diseases.
The present study aims to detect polymorphisms in TGF β gene in Oral Submucous Fibrosis (OSF) if any in experimental rats subjected to application of pan masala/ arecanut extracts/ gutka and in Oral Submucous fibrosis patients by Real time PCR method.
6.2 REVIEW OF LITERATURE
1)In this study seven polymorphisms (SNPs) of transforming growth factor β-1 gene in patients with oral submucous fibrosis, belonging to South Indian ethnic extraction was focussed. DNA samples from 50 subjects of the same ethnic group and comparable demographic features who have had practiced the habit of arecanut chewing of almost equal duration, but remained free of disease constituted controls. All DNA samples were collected progressively and purified from the peripheral blood employing standard protocols and tested for SNPs. Two polymorphisms in the promoter region(C-509T and G-800A), three polymorphisms in exon-1(Arg25Pro(G915 C),Leu10 pro(T869C),Glu 47 Gly(A979G) and two in 5 UTR regions(C→T(rs13306708) an G→A(rs9282871). The extracted DNA samples along with the primers underwent PCR amplification and genotypic and allelic frequencies were calculated using the SPSS software.The PCR products were purified and subsequently sequenced using Flour STM multi-imager system (Biorad).The sequenced data were analysed using the BioEdit sequence analysis software.The polymorphism in 5 UTR C-T in TGF β1 gene had a significant association with the pathophysiology of Oral Submucous Fibrosis.
2) To study the molecular pathogenesis of oral submucous fibrosis (OSF), a chronic inflammatory disease, gene expression profiling was performed in 10 OSF tissues against 8 pooled normal tissues using oligonucleotide arrays. Microarray results revealed differential expression of 5288 genes. Among them, 2404 were down regulated and 2884 were up regulated. Quantitative real-time PCR and immunohistochemistry confirmed up regulation of transforming growth factor-β1 (TGF- β1) and down regulation of bone morphogenic protein 7 (BMP7) in OSF tissues. Further activation of TGF-β pathway was also evident in OSF. Treatment of keratinocytes and oral fibroblasts by TGF-β confirmed the regulation of few genes identified in microarray including up regulation of connective tissue growth factor, and down regulation of BMP7, which is a known negative modulator of fibrosis. Taken together, the data suggested activation of TGF-β signaling and suppression of BMP7 expression in the manifestation of OSF7.
3) This study compared the association of oral submucous fibrosis (OSF) and polymorphisms of six collagen-related genes which included collagen 1A1 and 1A2 (COL1A1 and COL1A2), collagenase-1(COLase), transforming growth factor β1 (TGF-β1), lysyl oxidase (LYOXase), and cystatin C (CST3), between patients with high and low exposure to betel quid’s. 166 patients with OSF from a medical center and 284 betel quid chewers who were free of OSF and oral cancer, from the same hospital and five townships, were recruited. PCR-based restriction fragment length polymorphism assays were used to determine the genotypes of the six collagen related genes situated on different chromosomes. It was found that the genotypes associated with the highest OSF risk for collagen 1A1, collagen 1A2, collagenase-1, transforming growth factor β1, 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. A trend was observed for an increased risk of OSF with increasing number of high-risk alleles for those with both high and low exposures for betel quid. The cell selection mechanism of oral fibroblasts explains the effect of the modification of cumulative betel quid exposure on the risk profiles of collagen-related genes. The results implied that susceptibility to OSF could involve multigenic mechanisms modified by the betel quid-exposure dose6.
4) This study investigates the significance of transforming growth factor beta 1 (TGF β 1) in the pathogenesis of oral submucous fibrosis (OSF). In situ hybridization technique was used to determine TGF β 1 mRNAs in keratinocytes of the paraffin embedded tissues of 25 OSF cases, 5 normal’s (NOR) and 10 oral lichen planus(OLP) . The result revealed that there was an expression of TGF β 1 mRNA in keratinocytes of 15 OSFs (60%). The positive expression was mainly seen in keratinocytes of early and middle stage OSF. There was no expression in that of 5 NORs and 10 OLPs. The study suggests that keratinocytes of OSF tissue may synthesize and release TGF β1 which may play an important role in the pathogenesis of OSF and participate as a mediator in the pathogenic process of OSF5.
5)This study aimed at establishing the animal model of oral submucous fibrosis in the buccal mucosa of S D (Sprague and dawley) rats by injecting aqueous arecanut extracts submucosally followed by surface painting of the same. The animals in the different groups were randomly killed in the 12th, 16th, 22nd and 28th week. The collected buccal mucosae were studied under light microscope and transmission electron microscope. The results showed OSF (Oral Submucous Fibrosis) like lesions appearing in different degrees among some animals of the model groups. Tissue changes included atrophic epithelium with partial or complete loss of retepegs, accumulation of collagen fibers in the subepithelial region, inflammation along with muscle degeneration. Animals in the control group did not show any histomorphologic changes. The results implied that the animal model for Oral Submucous Fibrosis could be induced rapidly by submucosal injection and surface painting of aqueous arecanut extracts1.
6) In this study oral cavity of 21 albino rats was painted with a paste of an instant betel nut preparation on alternate days for a period of six months. At the beginning of the study and every two months thereafter biopsies were taken from the oral mucosa and compared histopathologically with those obtained from a control group of 14 albino rats. Changes observed when compared to the control group included mild to moderate loss of nuclear polarity and increase in keratoses, parakeratosis, inflammatory cell infiltration and vascularity. The increase in mitotic figures was statistically insignificant and no definite changes in pigmentation or atypical cells were seen. These findings suggest the possibility of mild leukoplakia. Submucosal collagen increased steeply and steadily throughout the study period and at the end of six months, 88.23 per cent of biopsies showed thickened and condensed sub-mucosal collagen, indicating submucous fibrosis2.
OBJECTIVES OF THE STUDY:
To detect polymorphisms in the transforming growth factor β gene, if any, causing tissue changes subsequent to application of pan masala/arecanut extracts/gutka over a set period of time in the experimental rats and Oral Submucous Fibrosis patients tissues by Real time-PCR method.

7. MATERIALS AND METHODS

7.1 SOURCES OF DATA
Tissues obtained from experimental rats subjected to application of pan masala/arecanut extracts/gutka over a specified period of time and Oral Submucous Fibrosis patients. Clearances from the respective ethical clearance committee will be ensured.
7.2 METHOD OF COLLECTION OF DATA
1) Experimental rats subjected to treatment with pan masala–10.
2) Experimental rats subjected to treatment with arecanut extract -10.
3) Experimental rats subjected to treatment with gutka- 10.
4) Untreated buccal mucosa -10 each ( human and rats).
5) Human biopsied OSF tissues -10 .
METHODOLOGY:
1.  The experimental rats will be treated with pan masala/arecanut/gutka extracts over a set period of time, sufficient to assess tissue changes after application.
2.  The rats will be sacrificed at the time period of 30 weeks when the fibrosis in the tissue reaches its peak.
3.  The tissues obtained from the buccal mucosa of the control and test rats and collected from patients with different grades of OSF will be subjected to genetic analysis for detecting polymorphisms in the TGF β gene.
4.  Real time PCR method will be used to assess the polymorphisms in the TGF β gene.
7.3 Does the study require any investigation or interventions to be conducted on patients or other human or animals? If so, please describe briefly.
Yes, biopsies from human subjects and tissues from experimental rats to be used.
7.4 Has the ethical clearance obtained from your institution in case of 7.3?
Yes.

8. LIST OF REFERENCES

1)Rajendran R,Harish RK, Sukumaran A,Vidyadharan R,Banerjee M .Transforming growth factor β-1 polymorphism are infrequent but exist at selected loci in oral submucous fibrosis.Indian J Dent Res.2010;21(3):413-19.
2) Khan I, Agarwal P, Thangjam GS, Radhesh R, Rao SG, Kondaiah P. Role of TGF-β and BMP7 in the pathogenesis of oral submucous fibrosis. Growth Factors 2011 ; 29(4):119-27.
3) Chiu CJ, Chang ML, Chiang CP, Hahn LJ, Hsieh LL, Chen CJ.Interaction of Collagen-related Genes and Susceptibility to Betel Quid-induced Oral Submucous Fibrosis. Cancer Epidemioligical Biomarkers . 2002; 11(7):646-53.
4) Gao Y, Ling T, Wu H. Expression of transforming growth factor beta 1 in keratinocytes of oral submucous fibrosis tissue. Chinese journal of stomatology 1997; 32(4):239-41.
5) Huang S, Ling T, Wu H. Experimental study on aqueous areca nut extracts inducing oral submucous fibrosis in rats. I. Observation of histomorphology. West China journal of stomatology 1997; 15(2): 91-3, 96.
6) Khrime RD, Mehra YN, Mann SB, Mehta SK, Chakraborti RN. Effect of instant preparation of betel nut (pan masala) on the oral mucosa of albino rats. Indian J Med Res 1991 ; 94:119-24.
9. SIGNATURE OF THE CANDIDATE
10. REMARKS OF THE GUIDE / SATISFACTORY
11. NAME AND DESIGNATION OF (IN BLOCK LETTERS):
11.1 GUIDE:
11.2 SIGNATURE: / Dr. V.V. KAMATH,
PROFESSOR AND HEAD,
DEPT. OF ORAL AND MAXILLOFACIAL PATHOLOGY
Dr SYAMALA REDDY DENTAL COLLEGE, HOSPITAL & RESEARCH CENTRE,
BANGALORE-560037.
11.3 CO-GUIDE, IF ANY:
11.4 SIGNATURE: / 1.Dr. KRISHNANAND. P. SATELUR
READER,
DEPT. OF ORAL AND MAXILLOFACIAL PATHOLOGY,
Dr SYAMALA REDDY DENTAL COLLEGE, HOSPITAL & RESEARCH CENTRE,
BANGALORE - 560037.
2.Dr. KISHORE.G.BHAT
PROFESSOR AND HEAD,
DEPT. OF MOLECULAR BIOLOGY AND IMMUNOLOGY,
MARATHA MANDAL’S NGH DENTAL INSTITUTE OF DENTAL SCIENCES AND RESEARCH CENTRE,
BELGAUM - 590010.
1.
2.
11.5 HEAD OF THE DEPARTMENT
11.6 SIGNATURE / Dr. V.V. KAMATH,
PROFESSOR AND HEAD,
DEPT. OF ORAL AND MAXILLOFACIAL PATHOLOGY,
Dr SYAMALA REDDY DENTAL COLLEGE, HOSPITAL & RESEARCH CENTRE,
BANGALORE - 560037.
12.1 REMARKS OF THE CHAIRMAN AND PRINCIPAL
12.2 SIGNATURE

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