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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION A BRIEF RESUME OF THE INTENDED WORK Incidence rates of OSCC has been increasing globally. It is estimated that 35,310 new cases of OSCC were diagnosed in Need for the study : US during 2008. Head and neck malignancies constitute the sixth most common malignancy, and more than 90 % malignancies are squamous cell carcinoma. Oral and oropharyngeal malignancies constitute 30-40 % of all types of cancer in Indian population making it a leading cause of cancer mortality . In one of the survey done in Karnataka cancer therapy research institute, Navnagar Hubli carcinoma of the oral cavity was the 3rd most prevalent after carcinoma of cervix & oesophagus 2 Neoplasia is a result of imbalance between cell proliferation and apoptosis. This further fecilitates results in tumor growth and is influenced by angiogenesis, cell - cell and cell - extracellular matrix(ECM) interactions. ECM consists of proteins and polysaccharides distributed in many different tissues of the body. ECM environment provides appropriate conditions for cell growth, cell differentiation and survival of tissues. It constitutes fibrous proteins such as collagen and elastin, elongated glycoproteins such as fibronectin and laminin ,which provide cell matrix adhesion. The role of ECM in the tumour microenvironment is not limited to acting as a physical barrier to neoplasia, but it also works as a reservoir for ligand proteins and growth factor 1. Matrix metalloproteinases are a family of zinc dependent endo peptidases that are capable of degrading most components of the extra cellular matrix (ECM). Degeneration of matrix is a key invent in invasion and metastasis of malignant lesions of the head and neck. A polymorphism is a genetic variant observed in atleast 1% of population. It represents natural sequence variants which may occur in more than one form.90% of DNA polymorphisms are single nucleotide polymorphisms (SNPs) due to a single base exchange 1. Common bi-allelic SNPs have been found in the promoter region of several MMPs in patients with HNSCC. Evaluation of such genetic polymorphisms are vital because they can be used as biomarkers for malignant lesions and thus may be involved in early intervention and diagnosis of malignancies. MMP-3 gene has been mapped to the long arm of chromosome 11q22.3 and the level of expression of this gene can be influenced by single nucleotide polymorphisms(SNPs) in the promoter region of their respective gene3.The promoter region of MMP3 is characterized by a 5A/6A promoter polymorphism at position-1171 in which one allele has six adenosine(6A) and the second has five adenosine(5A).A single adenosine insertion/ deletion polymorphism(5A/6A) at position -1171 of the MMP-3 promoter region causes different transcription of MMP-3.Invitro assays of promoter activity showed that the 5A allele had a two fold higher promoter activity than the 6A allele 3. The prevalence of 5A allele in the European population 40-50%.The present study aims to asses the association of 5A and 6A polymorphisms in the MMP-3gene in patients suffering from oral squamous cell carcinoma and compare with that of healthy controls. B. MATERIALS AND METHODS Patients with OSCC and the age and sex matched control from Source of data: department of oral medicine and radiology, SDMCDS, Dharwad and OPD, KCTRI, Navnagar would be taken up for the study. 30 patients with clinically and histopathologically diagnosed Method of collection of data(Including procedure ): OSCC and 20 healthy controls who are willing to participate in the study will be enrolled. Detailed clinical and personal sampling 7 information of each patient will be noted in standardized proforma. 7Information regarding the patients name, age, sex, occupation, personal habits and present complain will be gathered. A detailed 7 information regarding addictions to arecanut, tobacco, gutkha (mixture of arecanut, tobacco and lime) and alcohol consumptions will be noted. Inclusion criteria – 1. Patients with clinically and histopathologically diagnosed OSCC. 2. Age and sex matched healthy individuals without the habits like tobacco, gutkha, arecanut chewing. 3. patients who are willing to participate in the study. Exclusion criteria – 1. patients who have completed or under the course of treatment for OSCC 2. patients suffering from any other malignancies other than OSCC or having suffered from malignancies previously. 3. Individuals with hematological diseases, skin diseases and autoimmune disorders. The blood samples (5ml) will be taken after obtaining patients informed consent. Blood will be drawn into vacutainer tubes containing EDTA and the collected sample will be transported to the laboratory in -200C cryobox and stored at -200C deep freezer. Isolation of Genomic DNA from collected samples: Genomic DNA would be isolated from the samples using standard DNA isolation kit, after standardizing the protocol to the laboratory conditions. DNA quantification: DNA will be quantified using nano drop technique. Primer design: Primers needed to detect MMP-3 would be designed. (FP 5’-GGTTCTCCATTCCTTTGATGGGGGGAAAGA-3’ RP 5’-CTTCCTGGAATTCACATCACTGCCACCACT-3’) PCR amplification based MMP-3 detection and genotyping: MMP-3 gene promoter was genotyped using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). PCR amplification of a 129 bp fragment was performed in the promoter region of MMP-3 for the detection of -1171 5A/6A polymorphism using genomic DNA, forward and reverse primers, IX of Taq buffer, Taq polymerase and PCR conditions would be standardized. PCR based Restriction Analysis: Tth 111 l digestion of MMP-3 The aliquot of PCR-RFLP product was amplified on agarose gel electrophoresis stained with Ethidium Bromide and 5A alleles represented by DNA bands at 97 and 32bp and 6A alleles at 129bp. Gel electrophoresis:10 µl of the product would be loaded into agarose gel containing ethidium bromide for MMP-3 gene amplification. Statistical ananlysis and interpretation: Statistical analysis will be done using chi-square test. Does the study need any investigation or Yes. (5 ml peripheral venous blood) interactions to be conducted on patients or animals? Has the ethical clearance Yes. been obtained from your institution (in case of 7.3)? C. List of references: 1.Chaudhary A ,Singh M, Bharti A, Shukla S, Singh A, Mehrotra R. Genetic polymorphisms of matrix metalloproteinases and their inhibitors in potentially malignant and malignant lesions of the head and neck. Journal of biomedical science2010;17:10 2.Kulkarni B, Hiremath S, Hallikeri U, Patil B, Gai P. Decades of breast cancer-trends in patients profiles attending tertiary cancer care centre in south india.Asian journal of epidemiology 2012.5(4):103-113 3.Chaudhary A ,Singh M, Bharti A, Shukla S, Singh A, Mehrotra R. synergistic effect of stromelysin-1(matrix metalloproteinase-3)promoter(-1171 5A->6A)polymorphism in oral submucous fibrosis and head and neck lesions.BMC Cancer 2010;10:369 Vairaktaris E,Yapijakis C, Vasiliou D, Nkenke E, Skerefoglou Z, Vorris E, vylliotis A, Ragos V, Neukam F,Patsouris E.Association of –1171 Promoter Polymorphism of Matrix Metalloproteinase-3 with Increased Risk for Oral Cancer. Anticancer research.2007.27:4095-4100. 4. 5.Tadbir azadeh A, Purshahidi S, Ebrahimi H, Khademi B, Malekzadeh M, Mardani M, Taghva M, Sardari Y. Serum Level of MMP-3 in Patients with Oral Skquamous Cell Carcinoma - Lack of Association with Clinico-pathological Features. Asian pacific journal cancer .2012.13(9):4545-4548. 6.Zhang C, Li C, Zhu M, Zhang Q, Xie Z, et al. Meta-Analysis of MMP2, MMP3, and MMP9 Promoter Polymorphisms and Head and Neck Cancer risk.2013. PLoS ONE 8(4).