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The influence of Lysyl Oxidase G473A polymorphism on collagen expression and clinical response to Zinc-VitaminA therapy in Oral Submucous Fibrosis patients
Running title: Lysyl oxidase gene polymorphism in OSF 

Jay Gopal Ray MDS,PhD1#, Sanjit Mukherjee PhD2#�, Atul Katarkar MS(Pharm)2 and Keya Chaudhuri PhD2*
1Department of Oral Pathology, Dr. R. Ahmed Dental College & Hospital, Kolkata, India.
2CSIR-Indian Institute of Chemical Biology, Molecular & Human Genetics division, Kolkata, India;   

*Corresponding author:
Dr. Keya ChaudhuriEmeritus Scientist
Molecular & Human Genetics DivisionCSIR-Indian Institute of Chemical Biology,
4 Raja S C Mullick RoadKolkata-700032IndiaTel +91-33-2499-5762
Fax+91-33-2473-5197email-kchaudhuri@iicb.res.inkeya.chaudhuri@gmail.com
# These two authors have equal contribution.
� Present address: Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Room 3128, Building 37, Bethesda, MD 20892, United States



Abstract
Background.
The occurrence of oral submucous fibrosis (OSF), mainly caused by chewing processed arecanut products (betelquids with/without tobacco) has been increasing in number, though several measures have been taken to ban its etiological factor. A significant number of OSF patients turn to malignancy if left untreated. Zinc acetate alongwith VitaminA has shown promise in successful treatment in most of the patients. Lysyl oxidase (LOX) which catalyzes the cross-linking of elastin and collagen, is one of the major factor in increasing the severity of the disease. 
Material and Methods.
In the current study we performed a PCR--RFLP based approach in identifying the putative association of a G473A (Arg158Gln) polymorphism with OSF and its effect on LOX and collagen (type I) expression in pre and post Zinc acetate-Vitamin A treated patients.  
Results.
The prevalence of GA was significantly higher [OR=2.903; (95% CI=1.162-6.216)] in OSF cases, especially among < 30yrs age-group [OR=4.240; (95% CI=1.144-9.6)]. When segregated according to genotype mostly the GG patients responded well to the supplementation. The mean mature LOX: proLOX ratio in GA & AA patients remained significantly higher (p= 0.006 & 0.05) compared to GG patients. This was reflected in either total collagen level or specifically type I where no significant reduction was observed in pre and post treated patients. 
Conclusions.
These findings may propose a prospective relevance of LOX polymorphism in increasing risk of OSF and might indicate one mechanism for collagen accumulation in OSF in a subset of areca chewers. 

Key words: Oral Submucous Fibrosis, Lysyl Oxidase, Genetic Polymorphism, Zinc-Vitamin A supplementation, Collagen Type-1, Precancerous condition, Areca Nut    








Introduction
Arecanut or smokeless tobacco chewing is an integral part of oral habits prevalent in Indian subcontinent. In most habitual chewers, a plethora of Oropharyngeal pathological conditions are noted among which a considerable percentage are designated as potential malignant lesions. Oral submucous fibrosis (OSF) is among one such potentially malignant lesions with a turnover rate of about 7% ADDIN EN.CITE <EndNote><Cite><Author>Reichart</Author><Year>2008</Year><RecNum>28</RecNum><record><rec-number>28</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Reichart, P. A.</author><author>Nguyen, X. H.</author></authors></contributors><auth-address>Charite, Virchow Klinikum, Department of Oral Surgery and Dental Radiology, Berlin, Germany. peter-a.reichart@charite.de</auth-address><titles><title>Betel quid chewing, oral cancer and other oral mucosal diseases in Vietnam: a review</title><secondary-title>J Oral Pathol Med</secondary-title></titles><periodical><full-title>J Oral Pathol Med</full-title></periodical><pages>511-4</pages><volume>37</volume><number>9</number><keywords><keyword>Adolescent</keyword><keyword>Adult</keyword><keyword>Aged</keyword><keyword>Areca/*adverse effects</keyword><keyword>Female</keyword><keyword>Humans</keyword><keyword>Male</keyword><keyword>Middle Aged</keyword><keyword>Mouth Diseases/*chemically induced/epidemiology</keyword><keyword>Mouth Mucosa/drug effects/*pathology</keyword><keyword>Mouth Neoplasms/*chemically induced/epidemiology</keyword><keyword>Precancerous Conditions/*chemically induced/epidemiology</keyword><keyword>Prevalence</keyword><keyword>Vietnam/epidemiology</keyword><keyword>Young Adult</keyword></keywords><dates><year>2008</year><pub-dates><date>Oct</date></pub-dates></dates><isbn>1600-0714 (Electronic)&#xD;0904-2512 (Linking)</isbn><accession-num>18624933</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=18624933 </url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>Pindborg</Author><Year>1966</Year><RecNum>20</RecNum><record><rec-number>20</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Pindborg</author></authors></contributors><titles><title>Oral submucous fibrosis as a precancerous condition</title><secondary-title>J Dental Res</secondary-title></titles><volume>45:54625</volume><dates><year>1966</year></dates><urls></urls></record></Cite></EndNote>[1, 2] This pathological condition is characterized by a fibrotic change in the submucosal layer of the palate, faces, cheek, lips, pharynx, and esophagus with progressive tissue rigidity leading to �trismus� or difficulties in mouth opening  ADDIN EN.CITE <EndNote><Cite><Author>Canniff</Author><Year>1986</Year><RecNum>4</RecNum><record><rec-number>4</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Canniff, J. P.</author><author>Harvey, W.</author><author>Harris, M.</author></authors></contributors><titles><title>Oral submucous fibrosis: its pathogenesis and management</title><secondary-title>Br Dent J</secondary-title><alt-title>British dental journal</alt-title></titles><pages>429-34</pages><volume>160</volume><number>12</number><keywords><keyword>Adolescent</keyword><keyword>Adult</keyword><keyword>Aged</keyword><keyword>Areca</keyword><keyword>Female</keyword><keyword>HLA Antigens/analysis</keyword><keyword>Humans</keyword><keyword>India/ethnology</keyword><keyword>Male</keyword><keyword>Middle Aged</keyword><keyword>Mouth Diseases/*etiology</keyword><keyword>Mouth Mucosa/pathology</keyword><keyword>Oral Submucous Fibrosis/*etiology/immunology/pathology/therapy</keyword><keyword>Pakistan/ethnology</keyword><keyword>Plants, Medicinal</keyword></keywords><dates><year>1986</year><pub-dates><date>Jun 21</date></pub-dates></dates><isbn>0007-0610 (Print)&#xD;0007-0610 (Linking)</isbn><accession-num>3459494</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=3459494 </url></related-urls></urls><language>eng</language></record></Cite></EndNote>[3]. OSF can be described as a collagen metabolic disorder characterized by increased collagen synthesis or reduced collagen degradation in oral subepithelial tissues often attributed to arecanut chewing  ADDIN EN.CITE <EndNote><Cite><Author>Rajendran</Author><Year>1994</Year><RecNum>47</RecNum><record><rec-number>47</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Rajendran, R.</author></authors></contributors><auth-address>Department of Oral Pathology and Microbiology, Medical College, Trivandrum, India.</auth-address><titles><title>Oral submucous fibrosis: etiology, pathogenesis, and future research</title><secondary-title>Bull World Health Organ</secondary-title><alt-title>Bulletin of the World Health Organization</alt-title></titles><pages>985-96</pages><volume>72</volume><number>6</number><keywords><keyword>Female</keyword><keyword>Forecasting</keyword><keyword>Humans</keyword><keyword>Male</keyword><keyword>Mouth Neoplasms/*etiology/pathology</keyword><keyword>Oral Submucous Fibrosis/*etiology/pathology/therapy</keyword><keyword>*Precancerous Conditions</keyword><keyword>Research</keyword></keywords><dates><year>1994</year></dates><isbn>0042-9686 (Print)&#xD;0042-9686 (Linking)</isbn><accession-num>7867145</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=7867145 </url></related-urls></urls><language>eng</language></record></Cite></EndNote>[4]. Lysyl Oxidase (LOX) is one of the key collagen biosynthetic enzymes having a copper binding domain and since arecanut contain high amount of soluble copper an upsurge in LOX production is observed which leads to increased collagen production  ADDIN EN.CITE <EndNote><Cite><Author>Canniff</Author><Year>1981</Year><RecNum>29</RecNum><record><rec-number>29</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Canniff, J. P.</author><author>Harvey, W.</author></authors></contributors><titles><title>The aetiology of oral submucous fibrosis: the stimulation of collagen synthesis by extracts of areca nut</title><secondary-title>Int J Oral Surg</secondary-title><alt-title>International journal of oral surgery</alt-title></titles><pages>163-7</pages><volume>10</volume><number>Suppl 1</number><keywords><keyword>*Areca/analysis</keyword><keyword>Cells, Cultured</keyword><keyword>Chemical Phenomena</keyword><keyword>Chemistry</keyword><keyword>Collagen/*biosynthesis</keyword><keyword>Fibroblasts/metabolism</keyword><keyword>Humans</keyword><keyword>Mouth Diseases/*etiology</keyword><keyword>Oral Submucous Fibrosis/*etiology/metabolism</keyword><keyword>Plant Extracts/analysis/*pharmacology</keyword><keyword>*Plants, Medicinal</keyword></keywords><dates><year>1981</year></dates><isbn>0300-9785 (Print)&#xD;0300-9785 (Linking)</isbn><accession-num>6807873</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=6807873 </url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>Harvey</Author><Year>1986</Year><RecNum>30</RecNum><record><rec-number>30</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Harvey, W.</author><author>Scutt, A.</author><author>Meghji, S.</author><author>Canniff, J. P.</author></authors></contributors><titles><title>Stimulation of human buccal mucosa fibroblasts in vitro by betel-nut alkaloids</title><secondary-title>Arch Oral Biol</secondary-title><alt-title>Archives of oral biology</alt-title></titles><pages>45-9</pages><volume>31</volume><number>1</number><keywords><keyword>Alkaloids/*pharmacology</keyword><keyword>*Areca</keyword><keyword>Arecoline/analogs &amp; derivatives/metabolism</keyword><keyword>Cheek</keyword><keyword>Collagen/biosynthesis</keyword><keyword>Fibroblasts/drug effects/metabolism</keyword><keyword>Humans</keyword><keyword>Hydrolysis</keyword><keyword>Mouth Mucosa/cytology/*drug effects/metabolism</keyword><keyword>*Plants, Medicinal</keyword><keyword>Stimulation, Chemical</keyword></keywords><dates><year>1986</year></dates><isbn>0003-9969 (Print)&#xD;0003-9969 (Linking)</isbn><accession-num>3458437</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=3458437 </url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>Murti</Author><Year>1995</Year><RecNum>9</RecNum><record><rec-number>9</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Murti, P. R.</author><author>Bhonsle, R. B.</author><author>Gupta, P. C.</author><author>Daftary, D. K.</author><author>Pindborg, J. J.</author><author>Mehta, F. S.</author></authors></contributors><auth-address>Basic Dental Research Unit, Tata Institute of Fundamental Research, Bombay, India.</auth-address><titles><title>Etiology of oral submucous fibrosis with special reference to the role of areca nut chewing</title><secondary-title>J Oral Pathol Med</secondary-title></titles><periodical><full-title>J Oral Pathol Med</full-title></periodical><pages>145-52</pages><volume>24</volume><number>4</number><keywords><keyword>Animals</keyword><keyword>*Areca</keyword><keyword>Arecoline/pharmacology</keyword><keyword>Collagen/biosynthesis</keyword><keyword>Cross Reactions</keyword><keyword>Disease Susceptibility</keyword><keyword>Fibroblasts/drug effects/metabolism</keyword><keyword>Humans</keyword><keyword>India/epidemiology</keyword><keyword>Oral Submucous Fibrosis/epidemiology/*etiology</keyword><keyword>*Plants, Medicinal</keyword><keyword>Substance-Related Disorders/*complications/epidemiology</keyword></keywords><dates><year>1995</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>0904-2512 (Print)&#xD;0904-2512 (Linking)</isbn><accession-num>7783003</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=7783003 </url></related-urls></urls><language>eng</language></record></Cite></EndNote>[5-7]. LOX is produced initially as a pre-proenzyme which is converted to proenzyme that finally cleaves between Gly-168 and Asp-169 by peptidase to yield an active lysyl oxidase. A single nucleotide polymorphism of G473A changes Arg at residue 158 to Gln (LOX Arg158Gln) and the site is near peptidase cutting sites of LOX  ADDIN EN.CITE <EndNote><Cite><Author>Shieh</Author><Year>2007</Year><RecNum>42</RecNum><record><rec-number>42</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Shieh, T. M.</author><author>Lin, S. C.</author><author>Liu, C. J.</author><author>Chang, S. S.</author><author>Ku, T. H.</author><author>Chang, K. W.</author></authors></contributors><auth-address>Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan.</auth-address><titles><title>Association of expression aberrances and genetic polymorphisms of lysyl oxidase with areca-associated oral tumorigenesis</title><secondary-title>Clin Cancer Res</secondary-title></titles><periodical><full-title>Clin Cancer Res</full-title></periodical><pages>4378-85</pages><volume>13</volume><number>15 Pt 1</number><keywords><keyword>Adult</keyword><keyword>Aged</keyword><keyword>Aged, 80 and over</keyword><keyword>Animals</keyword><keyword>Areca/*chemistry</keyword><keyword>Carcinoma, Squamous Cell/chemically</keyword><keyword>induced/*enzymology/genetics/prevention &amp; control</keyword><keyword>Cell Movement</keyword><keyword>*Cell Transformation, Neoplastic</keyword><keyword>Gene Expression Regulation, Neoplastic</keyword><keyword>Humans</keyword><keyword>Keratinocytes/cytology/drug effects/metabolism</keyword><keyword>Male</keyword><keyword>Mice</keyword><keyword>Mice, Nude</keyword><keyword>Middle Aged</keyword><keyword>Mouth Mucosa/*drug effects/metabolism</keyword><keyword>Mouth Neoplasms/chemically induced/*enzymology/genetics/prevention &amp;</keyword><keyword>control</keyword><keyword>Neoplasm Invasiveness/pathology</keyword><keyword>Plant Extracts/*toxicity</keyword><keyword>*Polymorphism, Genetic</keyword><keyword>Protein-Lysine 6-Oxidase/antagonists &amp; inhibitors/*genetics/metabolism</keyword><keyword>RNA, Messenger/metabolism</keyword><keyword>RNA, Small Interfering/pharmacology</keyword><keyword>Tissue Array Analysis</keyword><keyword>Transplantation, Heterologous</keyword><keyword>Tumor Cells, Cultured</keyword></keywords><dates><year>2007</year><pub-dates><date>Aug 1</date></pub-dates></dates><isbn>1078-0432 (Print)&#xD;1078-0432 (Linking)</isbn><accession-num>17671119</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=17671119 </url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>Shieh</Author><Year>2009</Year><RecNum>18</RecNum><record><rec-number>18</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Shieh, T. M.</author><author>Tu, H. F.</author><author>Ku, T. H.</author><author>Chang, S. S.</author><author>Chang, K. W.</author><author>Liu, C. J.</author></authors></contributors><auth-address>Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan.</auth-address><titles><title>Association between lysyl oxidase polymorphisms and oral submucous fibrosis in older male areca chewers</title><secondary-title>J Oral Pathol Med</secondary-title></titles><periodical><full-title>J Oral Pathol Med</full-title></periodical><pages>109-13</pages><volume>38</volume><number>1</number><keywords><keyword>Adenine</keyword><keyword>Age Factors</keyword><keyword>Alleles</keyword><keyword>Amino Acid Sequence</keyword><keyword>*Areca</keyword><keyword>Arginine/genetics</keyword><keyword>Genetic Predisposition to Disease/genetics</keyword><keyword>Genotype</keyword><keyword>Glutamine/genetics</keyword><keyword>Guanine</keyword><keyword>Heterozygote</keyword><keyword>Humans</keyword><keyword>Male</keyword><keyword>Middle Aged</keyword><keyword>Oral Submucous Fibrosis/*enzymology</keyword><keyword>Polymorphism, Single Nucleotide/*genetics</keyword><keyword>Precancerous Conditions/genetics</keyword><keyword>Protein-Lysine 6-Oxidase/*genetics</keyword><keyword>Risk Factors</keyword></keywords><dates><year>2009</year><pub-dates><date>Jan</date></pub-dates></dates><isbn>1600-0714 (Electronic)&#xD;0904-2512 (Linking)</isbn><accession-num>18764858</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=18764858 </url></related-urls></urls><language>eng</language></record></Cite></EndNote>[8, 9]. The present study examines the association of G473A LOX polymorphism with OSF, and the expression of LOX among different genotypes. Previously, an elemental profile of OSF tissue revealed significant depletion of Zinc in mucosal tissues of OSF patients, following this we have developed a unique and successful treatment strategy of OSF patients with supplementation of Zinc acetate-Vitamin A  ADDIN EN.CITE <EndNote><Cite><Author>Dhariwal</Author><Year>2010</Year><RecNum>24</RecNum><record><rec-number>24</rec-number><ref-type name="Generic">13</ref-type><contributors><authors><author>Dhariwal, R</author><author>Mukherjee, S</author><author>Pattanayak, S</author><author>Chakraborty, A</author><author>Ray, J G</author><author>Chaudhuri, K</author></authors></contributors><titles><title>Zinc and vitamin A can minimise the severity of oral submucous fibrosis</title></titles><volume>2010</volume><dates><year>2010</year><pub-dates><date>January 1, 2010</date></pub-dates></dates><pub-location>London</pub-location><publisher>BMJ Case Rep. d.o.i. 10.1136/bcr.10.2009.2348</publisher><urls><related-urls><url>http://casereports.bmj.com/content/2010/bcr.10.2009.2348.abstract </url></related-urls></urls></record></Cite><Cite><Author>Paul</Author><Year>2002</Year><RecNum>52</RecNum><record><rec-number>52</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Paul, RanjanRashmi</author><author>Chatterjee, Jyotirmoy</author><author>Das, ArabindaK</author><author>Cervera, M. Luisa</author><author>Guardia, Miguel</author><author>Chaudhuri, Keya</author></authors></contributors><titles><title>Altered elemental profile as indicator of homeostatic imbalance in pathogenesis of oral submucous fibrosis</title><secondary-title>Biological Trace Element Research</secondary-title></titles><pages>45-56</pages><volume>87</volume><number>1-3</number><keywords><keyword>Oral submucous fibrosis</keyword><keyword>precancerous condition</keyword><keyword>elemental profile</keyword><keyword>inductively coupled plasma-mass spectrometry</keyword><keyword>homeostasis</keyword></keywords><dates><year>2002</year></dates><publisher>Humana Press</publisher><urls><related-urls><url>http://dx.doi.org/10.1385/BTER%3A87%3A1-3%3A045 </url></related-urls></urls></record></Cite></EndNote>[10, 11]. However, though most of the patients were benefited with this supplementation, some remained unresponsive. Hence the aim of present study to investigate the role of LOX G473A polymorphism and increases risk of developing OSF in habitual processed arecanut and betel quid (with or without tobacco) chewers. Also to investigate the influences and effectiveness of Zinc acetate-Vitamin A therapy in OSF patients with LOX G473A polymorphism.
Material and Methods:
Ethics statement
Human blood and oral biopsy tissue was collected by authorized medical practitioners only as a part of routine pathological examinations, a part of which was used in this study. The study was duly approved by the institutional ethical committee for performing human experiments as per guidelines.
Study population
Two hundred and fifty consecutive patients over a period of 24 months (male=157, females=93), diagnosed and histopahtologically confirmed with oral submucous fibrosis at Dr. R Ahmed Dental College & Hospital, Kolkata, India were selected for this study. At the same time 250 volunteers who underwent regular physical examination (male=172, female=78) (Table 1) and having at least more than a year of habitual chewing of betel quid with or without tobacco, were recruited as controls. Controls having familial history of cancer syndromes or any other systemic disorders were excluded from this study. All the control subjects were matched with patient population in terms of age and sex. About 2-5ml of blood sample was collected from antecubital vein from all study participants after obtaining an informed consent from each participant. Record of clinical photographs and inter-incisal distance (IID) with vernier calipers was maintained. 
Treatment
The OSF patients were advised supplementation of zinc actetate tablets, 50 mg three times daily, and vitamin A 25000 IU, once daily. A regular follow-up at an interval of 1 month upto one year was noted with an initial improvement in symptoms in most of the patients were noted after 4 months of therapy. 
Polymorphism Genotyping
Genomic DNA was isolated from the peripheral blood by proteinase-K treatment and salt extraction procedure method  ADDIN EN.CITE <EndNote><Cite><Author>MANIATIS T</Author><Year>1982</Year><RecNum>48</RecNum><record><rec-number>48</rec-number><ref-type name="Book">6</ref-type><contributors><authors><author>MANIATIS T, FRITSCH, E. F., AND SAMBROOK, J. </author></authors></contributors><titles><title>Molecular Cloning. A Laboratory Manual.</title></titles><section>458-460.</section><dates><year>1982</year></dates><publisher>Cold Spring Harbor Laboratory, : Cold Spring Harbor, NY.</publisher><urls></urls></record></Cite></EndNote>[12]. LOX gene encompassing the polymorphic region (G473A) was amplified using primers forward: 5`-CACTGGTTCCAAGCTGGCTA-3` reverse:5`-GGAAGTAGCCAGTGCCGTAT-3`. The reaction-mix contained 100pg DNA, 2�l 2.5mM dNTPs, 1 pg of each primer, 3U Taq Polymerase, 3�l 10X Taq buffer, 0.6�l 50mM MgCl2 and 21.1�l PCR-water in 30�l. The reaction conditions were: initial denaturation at 95�C(2min), followed by 35cycles of 94�C(30sec)-60�C(30sec)-72�C(1min) and final extension at 72�C(1min). For genotyping, the PCR product was digested with restriction enzyme Pst1(CTGCAG) and resolved on 6% polyacrylamide gel. Wild (G/G), homozygous mutant (A/A) and heterozygous mutant (G/A) gave one (259 bp), two (149 & 110 bp) and three (259, 149, 110 bp) bands respectively. Direct sequencing was done with 10 random samples for verification.
RNA isolation from oral mucosal tissue 
Total RNA was extracted using Trizol reagent (Gibco BRL, Gaithersburg, MD) according to the manufacturer's protocol. Approximately 50 mg of tissue was collected in 1 ml Trizol reagent and homogenized using a handheld homogenizer. After the addition of 0.2 ml chloroform, the mixture was vigorously shaken for 3 min at 22�C and centrifuged at 13000 rpm for 15 min at 4�C. An equal volume of isopropanol (chilled) was added and was kept in cold condition for 10 min, RNA was precipitated by centrifugation at 13000 rpm for 10 min at 4�C. The pellet was washed twice with 70% ethanol, briefly dried under air, and dissolved in 100 �l of diethylprocarbonate- treated water.
Semiquantitative and realtime RT PCR of lysyl oxidase
Isolated RNA from OSF and control tissues was immediately subjected to  realtime RT PCR (using Takara PrimescriptTM one step realtime RT PCR kit) (Takara, Japan) analysis for detection of changes in expression of  LOX mRNA and the same was visualized using semiquantitative RT PCR (using Qiagen one step RT PCR kit) (Qiagen, Hilden, Germany), both according to the manufacturers protocol. Briefly, the PCR was carried out using following primers LOX forward 5�-GATCCTGCTGATCCGCGACAA-3� and reverse 5�-GGGAGACCGTACTGGAAGTAG-3� (369 bp) while for GAPDH the forward and reverse primers were 5�-ATGGGGAAGGTGAAGGTCGG-3� and 5�-GGATGCTAAGCAGTTGGT-3� respectively, yielding a 470 bp product. The reaction mix contained 200 ng of RNA; �l of PrimeScript One Step Enzyme Mix; �l 2X One Step Buffer; 20 �M of each of the primers and RNase Free dH2O. The real-time RT-PCR cycling program involved an initial denaturation step at 95�C for 2 min, followed by 40 cycles of 15 s at 95�C and 30 s at 60�C. Thermal cycling was performed in a BioRad iQ5 Real-Time PCR Detection System (Hercules, CA, USA). 
Lysyl Oxidase and collagen (Type I) expression by Western Blot analysis
A part of the biopsy tissue sample, collected  in 1ml lysis buffer (1.5mM Tris-HCl/pH 6.8, 10% SDS, 10% glycerol) containing protease-inhibitor cocktail (Sigma Aldrich, USA), was homogenized with glass hand-held homogenizer, centrifuged (13,000rpm) and total protein was estimated by Bradford reagent (BioRad Inc, USA).  50�g protein samples were boiled (100�C, 10 min) with �-mercaptoethanol (Sigma-Aldrich, USA) and cooled on ice; and separated on 12% SDS-PAGE with prestained protein molecular weight marker (GENEI, Bangalore, India). Western blotting was done using rabbit polyclonal anti-LOX (1:1000, Novus Biologicals, USA, NB100-2530). The immunogen is A cocktail of two synthetic peptides; one made to a region of the human LOX protein within residues 300-350 (NB100-2528) and one within residues 200-300 (NB100-2527). [Swiss-Prot P28300]. In Western blot a band is seen at ~58 kDa. This is a highly glycosylated protein; therefore bands are seen at ~32 kDa, ~51 kDa and ~58 kDa representing the mature (secreted) (mLOX) form, the pro (pLOX) form and the glycosylated forms respectively. Mouse �-actin antibodies (1:2000, Sigma-Aldrich) and mouse monoclonal COL1A Antibody (COL-1) (Santacruz Biotechnology, Dallas, USA sc-59772)  in NaCl-TBST buffer-5%BSA; followed by incubation with alkaline phosphatase-conjugated goat anti-rabbit IgG(GENEI) or rabbit anti-mouse IgG(GENEI) at 1:2000 dilution in NaCl-TBST buffer-5% BSA. The alkaline phosphatase-positive bands were visualized using a developing solution containing 5-bromo,4-chloro,3-indolylphosphate/nitrobluetetrazolium(BCIP/NBT)(GENEI), 1.5 mm Tris-HCl(pH 8.8) and water in the dark at RT for 10min. The bands intensities were expressed in densitometric units (DU) using ImageJ (http://rsbweb.nih.gov/ij/download.html).  
Total collagen assay
Total collagen was extracted and assayed using the Sircol Collagen Assay (Biocolor, Northern Ireland) from homogenate containing 50�g of protein according to the manufactures protocol. Briefly, Acid Neutralising Reagent was added to the samples following protein isolation to each tube Isolation & Concentration Reagent was added and the microcentrifuge tube was incubated overnight at 4�C. The tubes were then removed and centrifuged tubes at 12,000 rpm for 10 min the supernatant was discarded carefully. Sircol Dye Reagent (1.0ml) was added and incubated for 30 min, collagen-dye complex was centrifuged, supernantant was carefully discarded. The pellets were dissolved using the Alkali Reagents and was left for 5 min and then reading was observed using a microplate reader to 555nm, or the closest matching blue-green color filter. Collagen concentrations of the test sample can be obtained from the standard curve. Immunohistochemistry 
Oral biopsy specimens collected were paraffin embedded and 4-5 �m thickness sections were collected on poly-L-lysine coated slides, after paraffin removal by xylene and rehydration (100%, 90%, and 70% of ethanol for 5 min, treated with deionised water for 10 min), the slides were treated with citrate buffer for unmasking the antigen. The further immunostaining were performed using Novolink polymer detection system (Leica Microsystems, Switzerland). The endogenous peroxidase and protein were blocked using supplied blockers. The Expression Collagen type I protein was detected with the same primary antibody of Collagen type I (1:200, Santacruz Biotech.) used for Western blotting. After post primary blocking the sections were incubated with novolink polymer and were then developed with DAB using supplied DAB substrate buffer.  The sections were counterstained with haematoxylin and were observed under LEICA DM 3000 microscope (Leica Microsystems, Switzerland).
Statistical Analysis
The Odd�s Ratio (OR) and 95% CI was calculated using JAVASTAT 2-way contingency table analysis software (http://statpages.org/ctab2x2.html). The difference in protein expression was analyzed using Student�s t test. Student�s t-test was performed to observe the differences between protein expressions and p value was calculated. A p value of <0.05 was considered significant.
RESULTS 
Association LOX G473C polymorphism with OSF
The demographic variables are presented in Table-1. Genotyping was performed using genomic DNA obtained from 250 OSF patients (cases) and age sex and habit matched controls (n=250).  62.8% of the population was male and most of them reported with a much advanced stage of the disease (83%). The frequency of the polymorphic 473A allele was 0.20 in control eastern Indian population. A significantly higher frequency of homozygous minor A allele (25.2%) was found in OSF cases compared to the controls (16.4%) [OR = 1.798, 95% CI = 1.088-2.975, p= 0.021] (Table-2). Age stratified analysis indicated a statistically significant (p=0.05) increase in the frequency of heterozygous genotype in patients above 31 years of age group (OR=2.031, 95% CI = 1.001-4.138). Moreover, a statistically significant higher frequency of heterozygous genotype was seen in female cases (OR =2.118, 95% CI=1.042-4.321, p= 0.037) (Table-2). 
Lysyl Oxidase expression in OSF patients supplemented with Zn acetate � Vitamin A
Mucosal tissues obtained from all OSF patients showed mRNA expression of LOX at a much higher level compared to healthy non-chewers mucosa (Figure 1A). Western blot analysis confirmed that mature LOX (mLOX) have consistently higher expression in OSF than normal healthy mucosa (Figure 2). The mucosal tissue obtained from post Zn acetate -Vitamin A (4 months follow-up) treated patients presented a varied activity of LOX both at mRNA level and as expressed as the ratio between the mLOX and pLOX (pro-LOX). The semi-quantitative RT-PCR analysis shows the significantly higher expression of LOX mRNA in OSF patient (p<0.0001) but there was no significant change was observed in post supplemented patients compared to normal (figure 1B). A quantitative realtime RT PCR analysis confirmed a general decrease of LOX mRNA expression in post supplemented patients (10.552�1.7) compared to the pre supplemented patients (12.19�1.13) (Figure 2C) which was near to the normal non-chewers mucosal expression (9.40 � 1.5). A general decrease in mLOX level was noted in most of the patients; however this was not uniform in all of them.  When the total populations were segregated according to the stages/grades and further by genotypes this non-uniformity of LOX activity was explained. Interestingly the patients reporting with advanced stages of the disease (n=166) either in grade II or III showed significant increase in mLOX:pLOX ratio (p=0.023 & 0.05 respectively) in their 4 month follow-up course, though the normal non-chewers mucosal mLOX:pLOX ratio was much lower (0.54�0.15; n=25). The mLOX:pLOX ratio was observed to be significantly higher in both the GA (p =0.006) and AA (p=0.05) post supplemented patients. The wild type (GG) patients presented a significant decrease in the mLOX:pLOX ratio in a follow-up of 4 months was observed which indicated these patients responding well to Zn acetate � Vitamin A therapy (Figure 2B & 2C).
Influence of LOX G473A on collagen expression in patients supplemented with Zn acetate � Vitamin A
With our initial observation of presence of polymorphism and its influence on post supplementation LOX expression we further wanted to observe the total collagen content and especially collagen type I expression pattern in these patients. A total tissue collagen assay performed revealed a general reduction in its level in post supplemented patients (25.26�4.15 �g/mg tissue) compared to its pre supplemented state (42.02�6.56 �g/mg tissue) (Figure 3A). However, when segregated according to genotypes the wild type (GG) patients revealed a significant reduction of total tissue collagen level (10.625�0.611 �g/mg tissue) compared to its pre supplemented state (20.38�5.05 �g/mg tissue). The GA and AA patients though presented general reduced collagen content (18.14�1.46 & 35.75�10.32 �g/mg tissue) compared to their pre supplemented state (19.71�3.85 & 47.03�7.89 �g/mg tissue) respectively, but it was not significant. The collagen content was observed to be significantly reduced after supplementation in wild type (GG) patients to 10.625�0.611 �g/ mg tissue in post supplemented group from 20.38 � 5.05 �g/ mg tissue in pre supplemented group (p < 0.01) whereas either a minimal reduction or no change was observed in GA patients (18.13� 1.47 �g/ mg tissue  compared to 19.71 � 3.86 �g/ mg tissue)  or AA (35.75� 10.3 �g/ mg tissue compared to 47.03 � 7.9 �g/ mg tissue) (Figure 3A). As accumulation and overexpression of collagen type-I is mainly associated with the pathogenesis of OSF, further influence of Zn acetate�Vitamin A supplementation on collagen Type-I expression was evaluated by western blot analysis (Figure 3B). There was overall significant decrease in the collagen type-I expression was observed (p<0.01). Likely, there was significant downregulation of collagen type-1 was observed in post supplemented group with GG genotype (p<0.0001). But there was no significant change was observed in post supplemented group with GA and AA genotype (Figure 3C). Moreover, this observation was also reflected immunohistochemically where a general reduction of collagen type I was observed around fibroblast cells in the submucosal area (marked with arrow) in patients with GG genotype (Figure 3D & E) compared to GA and AA patients.
The response of Zn acetate�Vitamin A supplementation with LOX G473A polymorphism
We have observed the critical and LOX genotype dependent response to Zn acetate�Vitamin A supplementation in the OSF patients. The continue accumulation of collagen Type-I in the OSF leads to severe fibrosis and reduction in mouth opening. The reduction in mouth opening is due to tidily cross linking of overexpressed collagen Type-I by LOX. Hence the further response of Zn acetate�Vitamin A supplementation was correlated with change in mouth opening in terms of inter-incisional distances (IID in cm). There was significant increase IID in post supplemented group (2.96�0.68) with GG genotype (p<0.0001) compared to pretreated OSF patient (2.427�0.78) but not in post supplemented group with GA (2.21�0.60; 2.40�0.56) and AA (1.75�0.46; 1.62�0.29) genotype (Figure 4A). Also there was significant change in IID in post supplemented group with GG genotype (p<0.01) compared to GA (2.21�0.60; 2.40�0.56) and AA genotype (Figure 4B). There was significant difference (p<0.0001) in change in IID compared to GA and AA which suggested that there was some patients with GA genotype had response to Zn acetate�Vitamin A supplementation. There was no significant effect in change of IID was observed when stratified with grade (Figure 4C). This suggested that the response of Zn acetate�Vitamin A supplementation may be depends on LOX genotypes. To rule out this the % change in mLOX:pLOX ratio were analysed followed post Zn acetate�Vitamin A supplementation (Figure 4D). The overall 39.75% OSF patients was responded well to Zn acetate�Vitamin A supplementation. Among them, 71.42% (40/56) OSF patient with GG genotype (overall 24.09%), 12.65% (21/71) with GA genotype (overall 12.65%) and 3.01% (5/39) with AA genotype (overall 12.65%) shows the reduction in the % change of mLOX:pLOX followed by Zn acetate�Vitamin A supplementation. This clearly suggested that LOX genotype plays significant role in Zn acetate�Vitamin A therapy. 
Discussion 
Oral submucous fibrosis was classified as an idiopathic disorder when first described but later on a multifactorial etiology was proposed with arecanut as the most compelling factor. The commonly accepted hypothesis in the pathogenesis of OSF is increased collagen synthesis and/or reduced collagen degradation in the oral sub-epithelial tissues because of chemical, physical or inflammatory irritation with involvement of various biological pathways  ADDIN EN.CITE <EndNote><Cite><Author>Canniff</Author><Year>1981</Year><RecNum>29</RecNum><record><rec-number>29</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Canniff, J. P.</author><author>Harvey, W.</author></authors></contributors><titles><title>The aetiology of oral submucous fibrosis: the stimulation of collagen synthesis by extracts of areca nut</title><secondary-title>Int J Oral Surg</secondary-title><alt-title>International journal of oral surgery</alt-title></titles><pages>163-7</pages><volume>10</volume><number>Suppl 1</number><keywords><keyword>*Areca/analysis</keyword><keyword>Cells, Cultured</keyword><keyword>Chemical Phenomena</keyword><keyword>Chemistry</keyword><keyword>Collagen/*biosynthesis</keyword><keyword>Fibroblasts/metabolism</keyword><keyword>Humans</keyword><keyword>Mouth Diseases/*etiology</keyword><keyword>Oral Submucous Fibrosis/*etiology/metabolism</keyword><keyword>Plant Extracts/analysis/*pharmacology</keyword><keyword>*Plants, Medicinal</keyword></keywords><dates><year>1981</year></dates><isbn>0300-9785 (Print)&#xD;0300-9785 (Linking)</isbn><accession-num>6807873</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=6807873 </url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>Harvey</Author><Year>1986</Year><RecNum>30</RecNum><record><rec-number>30</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Harvey, W.</author><author>Scutt, A.</author><author>Meghji, S.</author><author>Canniff, J. P.</author></authors></contributors><titles><title>Stimulation of human buccal mucosa fibroblasts in vitro by betel-nut alkaloids</title><secondary-title>Arch Oral Biol</secondary-title><alt-title>Archives of oral biology</alt-title></titles><pages>45-9</pages><volume>31</volume><number>1</number><keywords><keyword>Alkaloids/*pharmacology</keyword><keyword>*Areca</keyword><keyword>Arecoline/analogs &amp; derivatives/metabolism</keyword><keyword>Cheek</keyword><keyword>Collagen/biosynthesis</keyword><keyword>Fibroblasts/drug effects/metabolism</keyword><keyword>Humans</keyword><keyword>Hydrolysis</keyword><keyword>Mouth Mucosa/cytology/*drug effects/metabolism</keyword><keyword>*Plants, Medicinal</keyword><keyword>Stimulation, Chemical</keyword></keywords><dates><year>1986</year></dates><isbn>0003-9969 (Print)&#xD;0003-9969 (Linking)</isbn><accession-num>3458437</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=3458437 </url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>Rajalalitha</Author><Year>2005</Year><RecNum>8</RecNum><record><rec-number>8</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Rajalalitha, P.</author><author>Vali, S.</author></authors></contributors><auth-address>Institute of Bioinformatics and Applied Biotechnology, Tech Park Mall, Bangalore, India. lailitha_am11@yahoo.co.in</auth-address><titles><title>Molecular pathogenesis of oral submucous fibrosis--a collagen metabolic disorder</title><secondary-title>J Oral Pathol Med</secondary-title></titles><periodical><full-title>J Oral Pathol Med</full-title></periodical><pages>321-8</pages><volume>34</volume><number>6</number><keywords><keyword>Areca/adverse effects</keyword><keyword>Collagen/metabolism</keyword><keyword>Collagen Diseases/*etiology/metabolism</keyword><keyword>Humans</keyword><keyword>Oral Submucous Fibrosis/*etiology/metabolism</keyword><keyword>Signal Transduction/physiology</keyword><keyword>Transforming Growth Factor beta/physiology</keyword></keywords><dates><year>2005</year><pub-dates><date>Jul</date></pub-dates></dates><isbn>0904-2512 (Print)&#xD;0904-2512 (Linking)</isbn><accession-num>15946178</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=15946178 </url></related-urls></urls><language>eng</language></record></Cite></EndNote>[5, 6, 13]. However, all the areca nut chewers do not develop the disease and it is not necessarily a dose response phenomenon. Even cessation of the habit does not influence the characteristics of the disease once it is established indicating towards genetic predisposition as a plausible cause for such variability  ADDIN EN.CITE <EndNote><Cite><Author>Li</Author><Year>2008</Year><RecNum>7</RecNum><record><rec-number>7</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Li, N.</author><author>Jian, X.</author><author>Hu, Y.</author><author>Xu, C.</author><author>Yao, Z.</author><author>Zhong, X.</author></authors></contributors><auth-address>Department of Oral and Maxillofacial Surgery, Xiangya Hospital, Central South University, Changsha, China.</auth-address><titles><title>Discovery of novel biomarkers in oral submucous fibrosis by microarray analysis</title><secondary-title>Cancer Epidemiol Biomarkers Prev</secondary-title></titles><pages>2249-59</pages><volume>17</volume><number>9</number><keywords><keyword>Adolescent</keyword><keyword>Adult</keyword><keyword>Areca/adverse effects</keyword><keyword>Blotting, Western</keyword><keyword>Chi-Square Distribution</keyword><keyword>Female</keyword><keyword>Gene Expression Regulation, Neoplastic</keyword><keyword>Humans</keyword><keyword>Male</keyword><keyword>*Microarray Analysis</keyword><keyword>Oral Submucous Fibrosis/etiology/*genetics</keyword><keyword>Precancerous Conditions/*genetics</keyword><keyword>RNA, Messenger/genetics</keyword><keyword>Reverse Transcriptase Polymerase Chain Reaction</keyword><keyword>Tumor Markers, Biological/*analysis</keyword></keywords><dates><year>2008</year><pub-dates><date>Sep</date></pub-dates></dates><isbn>1055-9965 (Print)&#xD;1055-9965 (Linking)</isbn><accession-num>18768491</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=18768491 </url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>Mukherjee</Author><RecNum>49</RecNum><record><rec-number>49</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Mukherjee, Sanjit</author><author>Bhowmik, Aneek Das</author><author>Roychoudhury, Paromita</author><author>Mukhopadhyay, Kanchan</author><author>Ray, Jay Gopal</author><author>Chaudhuri, Keya</author></authors></contributors><titles><title>Association of XRCC1, XRCC3, and NAT2 polymorphisms with the risk of oral submucous fibrosis among eastern Indian population</title><secondary-title>Journal of Oral Pathology &amp; Medicine</secondary-title></titles><pages>292-302</pages><keywords><keyword>areca</keyword><keyword>NAT</keyword><keyword>oral submucous fibrosis</keyword><keyword>single nucleotide polymorphism</keyword><keyword>XRCC</keyword></keywords><dates><year>2012</year></dates><publisher>Blackwell Publishing Ltd</publisher><urls><related-urls><url>http://dx.doi.org/10.1111/j.1600-0714.2011.01097.x </url></related-urls></urls></record></Cite></EndNote>[14, 15].
The present study investigated the association of LOX Arg158Gln polymorphism with the risk of OSF in the Eastern Indian population. Previously, a study on Taiwanese population reported LOX Arg158Gln allelotype in approximately 20% of areca-chewers while a higher prevalence of the same alleotype was noted in OSF cases >50 years age  ADDIN EN.CITE <EndNote><Cite><Author>Shieh</Author><Year>2009</Year><RecNum>18</RecNum><record><rec-number>18</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Shieh, T. M.</author><author>Tu, H. F.</author><author>Ku, T. H.</author><author>Chang, S. S.</author><author>Chang, K. W.</author><author>Liu, C. J.</author></authors></contributors><auth-address>Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan.</auth-address><titles><title>Association between lysyl oxidase polymorphisms and oral submucous fibrosis in older male areca chewers</title><secondary-title>J Oral Pathol Med</secondary-title></titles><periodical><full-title>J Oral Pathol Med</full-title></periodical><pages>109-13</pages><volume>38</volume><number>1</number><keywords><keyword>Adenine</keyword><keyword>Age Factors</keyword><keyword>Alleles</keyword><keyword>Amino Acid Sequence</keyword><keyword>*Areca</keyword><keyword>Arginine/genetics</keyword><keyword>Genetic Predisposition to Disease/genetics</keyword><keyword>Genotype</keyword><keyword>Glutamine/genetics</keyword><keyword>Guanine</keyword><keyword>Heterozygote</keyword><keyword>Humans</keyword><keyword>Male</keyword><keyword>Middle Aged</keyword><keyword>Oral Submucous Fibrosis/*enzymology</keyword><keyword>Polymorphism, Single Nucleotide/*genetics</keyword><keyword>Precancerous Conditions/genetics</keyword><keyword>Protein-Lysine 6-Oxidase/*genetics</keyword><keyword>Risk Factors</keyword></keywords><dates><year>2009</year><pub-dates><date>Jan</date></pub-dates></dates><isbn>1600-0714 (Electronic)&#xD;0904-2512 (Linking)</isbn><accession-num>18764858</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=18764858 </url></related-urls></urls><language>eng</language></record></Cite></EndNote>[9]. The frequency of minor A allele carrier in our population was about 35%, which was about 43% in case of OSF patients. A significant association of the heterozygous (GA) LOX genotype in patients of age 31 years or more indicates the susceptibility of heterozygous individuals and goes in line with the previous report by Shieh et al among 83 OSF patients  ADDIN EN.CITE <EndNote><Cite><Author>Shieh</Author><Year>2009</Year><RecNum>18</RecNum><record><rec-number>18</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Shieh, T. M.</author><author>Tu, H. F.</author><author>Ku, T. H.</author><author>Chang, S. S.</author><author>Chang, K. W.</author><author>Liu, C. J.</author></authors></contributors><auth-address>Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan.</auth-address><titles><title>Association between lysyl oxidase polymorphisms and oral submucous fibrosis in older male areca chewers</title><secondary-title>J Oral Pathol Med</secondary-title></titles><periodical><full-title>J Oral Pathol Med</full-title></periodical><pages>109-13</pages><volume>38</volume><number>1</number><keywords><keyword>Adenine</keyword><keyword>Age Factors</keyword><keyword>Alleles</keyword><keyword>Amino Acid Sequence</keyword><keyword>*Areca</keyword><keyword>Arginine/genetics</keyword><keyword>Genetic Predisposition to Disease/genetics</keyword><keyword>Genotype</keyword><keyword>Glutamine/genetics</keyword><keyword>Guanine</keyword><keyword>Heterozygote</keyword><keyword>Humans</keyword><keyword>Male</keyword><keyword>Middle Aged</keyword><keyword>Oral Submucous Fibrosis/*enzymology</keyword><keyword>Polymorphism, Single Nucleotide/*genetics</keyword><keyword>Precancerous Conditions/genetics</keyword><keyword>Protein-Lysine 6-Oxidase/*genetics</keyword><keyword>Risk Factors</keyword></keywords><dates><year>2009</year><pub-dates><date>Jan</date></pub-dates></dates><isbn>1600-0714 (Electronic)&#xD;0904-2512 (Linking)</isbn><accession-num>18764858</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=18764858 </url></related-urls></urls><language>eng</language></record></Cite></EndNote>[9]. Though most of the patients reported was males, a borderline association of GA genotype was noted (p= 0.06), on the other hand a highly significant association of GA genotype was noted among the female patients. (p=0.037). 
An elemental profile of OSF tissues from our earlier studies revealed a severe depletion of elemental Zinc was noted  ADDIN EN.CITE <EndNote><Cite><Author>Paul</Author><Year>2002</Year><RecNum>52</RecNum><record><rec-number>52</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Paul, RanjanRashmi</author><author>Chatterjee, Jyotirmoy</author><author>Das, ArabindaK</author><author>Cervera, M. Luisa</author><author>Guardia, Miguel</author><author>Chaudhuri, Keya</author></authors></contributors><titles><title>Altered elemental profile as indicator of homeostatic imbalance in pathogenesis of oral submucous fibrosis</title><secondary-title>Biological Trace Element Research</secondary-title></titles><pages>45-56</pages><volume>87</volume><number>1-3</number><keywords><keyword>Oral submucous fibrosis</keyword><keyword>precancerous condition</keyword><keyword>elemental profile</keyword><keyword>inductively coupled plasma-mass spectrometry</keyword><keyword>homeostasis</keyword></keywords><dates><year>2002</year></dates><publisher>Humana Press</publisher><urls><related-urls><url>http://dx.doi.org/10.1385/BTER%3A87%3A1-3%3A045 </url></related-urls></urls></record></Cite></EndNote>[11]. This observation directed us for an oral supplementation of Zinc in form of Zinc acetate in combination with Vitamin A. Interactions between Zinc and Vitamin A is well established ADDIN EN.CITE <EndNote><Cite><Author>Christian</Author><Year>1998</Year><RecNum>53</RecNum><record><rec-number>53</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Christian, P.</author><author>West, K. P.</author></authors></contributors><titles><title>Interactions between zinc and vitamin A: an update</title><secondary-title>Am J Clin Nutr</secondary-title></titles><pages>435S-441S</pages><volume>68</volume><number>2</number><dates><year>1998</year><pub-dates><date>August 1, 1998</date></pub-dates></dates><urls><related-urls><url>http://ajcn.nutrition.org/content/68/2/435S.abstract </url></related-urls></urls></record></Cite></EndNote>[16], whereas role of Vitamin A in maintenance of epithelial integrity has been demonstrated earlier in oral precancerous conditions such as leukoplakia ADDIN EN.CITE <EndNote><Cite><Author>Stich</Author><Year>1988</Year><RecNum>54</RecNum><record><rec-number>54</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Stich, Hans F.</author><author>Rosin, Miriam P.</author><author>Hornby, A. Paul</author><author>Mathew, Babu</author><author>Sankaranarayanan, R.</author><author>Nair, M. Krishnan</author></authors></contributors><titles><title>Remission of oral leukoplakias and micronuclei in tobacco/betel quid chewers treated with beta-carotene and with beta-carotene plus vitamin A</title><secondary-title>International Journal of Cancer</secondary-title></titles><pages>195-199</pages><volume>42</volume><number>2</number><dates><year>1988</year></dates><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><urls><related-urls><url>http://dx.doi.org/10.1002/ijc.2910420209 </url></related-urls></urls></record></Cite></EndNote>[17], lichen planus  ADDIN EN.CITE <EndNote><Cite><Author>Gunther</Author><Year>1973</Year><RecNum>55</RecNum><record><rec-number>55</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Gunther, S. H.</author></authors></contributors><titles><title>VItamin a acid in treatment of oral lichen planus</title><secondary-title>Archives of Dermatology</secondary-title></titles><pages>277-277</pages><volume>107</volume><number>2</number><dates><year>1973</year></dates><urls><related-urls><url>http://dx.doi.org/10.1001/archderm.1973.01620170085024 </url></related-urls></urls></record></Cite></EndNote>[18] and in betel quid chewers mucosa  ADDIN EN.CITE <EndNote><Cite><Author>Stich</Author><Year>1984</Year><RecNum>56</RecNum><record><rec-number>56</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Stich, Hans F.</author><author>Stich, Waltraud</author><author>Rosin, Miriam P.</author><author>Vallejera, Mythyl O.</author></authors></contributors><titles><title>Use of the micronucleus test to monitor the effect of vitamin A, beta-carotene and canthaxanthin on the buccal mucosa of betel nut/tobacco chewers</title><secondary-title>International Journal of Cancer</secondary-title></titles><pages>745-750</pages><volume>34</volume><number>6</number><dates><year>1984</year></dates><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><urls><related-urls><url>http://dx.doi.org/10.1002/ijc.2910340602 </url></related-urls></urls></record></Cite></EndNote>[19]. A follow-up of 4 months to 1 year of supplementation revealed majority of the patients to be benefitted as they reported with improved oral conditions and reduced symptoms  ADDIN EN.CITE <EndNote><Cite><Author>Dhariwal</Author><Year>2010</Year><RecNum>24</RecNum><record><rec-number>24</rec-number><ref-type name="Generic">13</ref-type><contributors><authors><author>Dhariwal, R</author><author>Mukherjee, S</author><author>Pattanayak, S</author><author>Chakraborty, A</author><author>Ray, J G</author><author>Chaudhuri, K</author></authors></contributors><titles><title>Zinc and vitamin A can minimise the severity of oral submucous fibrosis</title></titles><volume>2010</volume><dates><year>2010</year><pub-dates><date>January 1, 2010</date></pub-dates></dates><pub-location>London</pub-location><publisher>BMJ Case Rep. d.o.i. 10.1136/bcr.10.2009.2348</publisher><urls><related-urls><url>http://casereports.bmj.com/content/2010/bcr.10.2009.2348.abstract </url></related-urls></urls></record></Cite></EndNote>[10]. However, some patient did not respond well, in fact the severity of the disease increased over time. When categorized according to genotype, most of these patients were carriers of the minor A allele of LOX G473A polymorphism. An overall increase in active lysyl oxidase and in turn collagen (Type I) was noted among them. Thus our findings would further support the hypothesis that accumulation of collagen seen in advanced cases of OSF is not only due to fibrogenesis, but may also be due to decrease in the breakdown of collagen earlier cross-linked by lysyl oxidase and thus overall turnover  ADDIN EN.CITE <EndNote><Cite><Author>Trivedy</Author><Year>1999</Year><RecNum>14</RecNum><record><rec-number>14</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Trivedy, C.</author><author>Warnakulasuriya, K. A.</author><author>Hazarey, V. K.</author><author>Tavassoli, M.</author><author>Sommer, P.</author><author>Johnson, N. W.</author></authors></contributors><auth-address>Department of Oral Medicine and Pathology, The Guy&apos;s School of Medicine and Dentistry of King&apos;s College London, England.</auth-address><titles><title>The upregulation of lysyl oxidase in oral submucous fibrosis and squamous cell carcinoma</title><secondary-title>J Oral Pathol Med</secondary-title></titles><periodical><full-title>J Oral Pathol Med</full-title></periodical><pages>246-51</pages><volume>28</volume><number>6</number><keywords><keyword>Carcinoma, Squamous Cell/*enzymology</keyword><keyword>Hepatolenticular Degeneration/enzymology</keyword><keyword>Humans</keyword><keyword>Immunohistochemistry</keyword><keyword>Liver/enzymology</keyword><keyword>Mouth Neoplasms/*enzymology</keyword><keyword>Oral Submucous Fibrosis/*enzymology</keyword><keyword>Polyps/enzymology</keyword><keyword>Protein-Lysine 6-Oxidase/*metabolism</keyword><keyword>Up-Regulation</keyword></keywords><dates><year>1999</year><pub-dates><date>Jul</date></pub-dates></dates><isbn>0904-2512 (Print)&#xD;0904-2512 (Linking)</isbn><accession-num>10426196</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=10426196 </url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>Trivedy</Author><Year>2001</Year><RecNum>43</RecNum><record><rec-number>43</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Trivedy, C.</author><author>Meghji, S.</author><author>Warnakulasuriya, K. A.</author><author>Johnson, N. W.</author><author>Harris, M.</author></authors></contributors><auth-address>Department of Oral Medicine and Pathology/WHO Collaborating Center for Oral Cancer and Precancer, The Guy&apos;s, King&apos;s, St Thomas&apos; School of Dentistry, King&apos;s College London, England.</auth-address><titles><title>Copper stimulates human oral fibroblasts in vitro: a role in the pathogenesis of oral submucous fibrosis</title><secondary-title>J Oral Pathol Med</secondary-title></titles><periodical><full-title>J Oral Pathol Med</full-title></periodical><pages>465-70</pages><volume>30</volume><number>8</number><keywords><keyword>Areca/adverse effects/chemistry</keyword><keyword>Cell Culture Techniques</keyword><keyword>Cell Division/drug effects</keyword><keyword>Collagen/biosynthesis/drug effects</keyword><keyword>Copper/administration &amp; dosage/*adverse effects/analysis</keyword><keyword>Dose-Response Relationship, Drug</keyword><keyword>Fibroblasts/*drug effects</keyword><keyword>Humans</keyword><keyword>Mouth Mucosa/cytology/*drug effects</keyword><keyword>Oral Submucous Fibrosis/*etiology</keyword><keyword>Plants, Medicinal</keyword><keyword>Proline/diagnostic use</keyword><keyword>Protein Biosynthesis</keyword><keyword>Proteins/drug effects</keyword><keyword>Radiopharmaceuticals/diagnostic use</keyword><keyword>Solubility</keyword><keyword>Statistics as Topic</keyword><keyword>Thymidine/diagnostic use</keyword><keyword>Tritium/diagnostic use</keyword><keyword>Up-Regulation</keyword></keywords><dates><year>2001</year><pub-dates><date>Sep</date></pub-dates></dates><isbn>0904-2512 (Print)&#xD;0904-2512 (Linking)</isbn><accession-num>11545237</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=11545237 </url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>Rajalalitha</Author><Year>2005</Year><RecNum>8</RecNum><record><rec-number>8</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Rajalalitha, P.</author><author>Vali, S.</author></authors></contributors><auth-address>Institute of Bioinformatics and Applied Biotechnology, Tech Park Mall, Bangalore, India. lailitha_am11@yahoo.co.in</auth-address><titles><title>Molecular pathogenesis of oral submucous fibrosis--a collagen metabolic disorder</title><secondary-title>J Oral Pathol Med</secondary-title></titles><periodical><full-title>J Oral Pathol Med</full-title></periodical><pages>321-8</pages><volume>34</volume><number>6</number><keywords><keyword>Areca/adverse effects</keyword><keyword>Collagen/metabolism</keyword><keyword>Collagen Diseases/*etiology/metabolism</keyword><keyword>Humans</keyword><keyword>Oral Submucous Fibrosis/*etiology/metabolism</keyword><keyword>Signal Transduction/physiology</keyword><keyword>Transforming Growth Factor beta/physiology</keyword></keywords><dates><year>2005</year><pub-dates><date>Jul</date></pub-dates></dates><isbn>0904-2512 (Print)&#xD;0904-2512 (Linking)</isbn><accession-num>15946178</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=15946178 </url></related-urls></urls><language>eng</language></record></Cite></EndNote>[13, 20, 21]. This cross-linking of collagen fibrils may be affected by various polymorphic lysyl oxidases  ADDIN EN.CITE <EndNote><Cite><Author>Consuegra</Author><Year>2006</Year><RecNum>57</RecNum><record><rec-number>57</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Consuegra, Sofia</author><author>Johnston, Ian A.</author></authors></contributors><titles><title>Polymorphism of the lysyl oxidase gene in relation to muscle collagen cross-link concentration in Atlantic salmon</title><secondary-title>Aquaculture Research</secondary-title></titles><pages>1699-1702</pages><volume>37</volume><number>16</number><keywords><keyword>Atlantic salmon</keyword><keyword>Salmo salar</keyword><keyword>lysyl oxidase</keyword><keyword>collagen cross-links</keyword><keyword>marker-assisted selection</keyword><keyword>adaptation</keyword></keywords><dates><year>2006</year></dates><publisher>Blackwell Publishing Ltd</publisher><urls><related-urls><url>http://dx.doi.org/10.1111/j.1365-2109.2006.01609.x </url></related-urls></urls></record></Cite></EndNote>[22], however its exact role is still elusive. Existing literature suggests that OSF fibroblasts exhibit increased TIMP (1 & 2) and relatively unchanged MMP (1 & 2)  ADDIN EN.CITE <EndNote><Cite><Author>Manka</Author><Year>2012</Year><RecNum>58</RecNum><record><rec-number>58</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Manka, Szymon W.</author><author>Carafoli, Federico</author><author>Visse, Robert</author><author>Bihan, Dominique</author><author>Raynal, Nicolas</author><author>Farndale, Richard W.</author><author>Murphy, Gillian</author><author>Enghild, Jan J.</author><author>Hohenester, Erhard</author><author>Nagase, Hideaki</author></authors></contributors><titles><title>Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1</title><secondary-title> Proc. Natl. Acad. Sci. </secondary-title></titles><pages>12461-12466</pages><volume>109</volume><number>31</number><dates><year>2012</year><pub-dates><date>July 31, 2012</date></pub-dates></dates><urls><related-urls><url>http://www.pnas.org/content/109/31/12461.abstract </url></related-urls></urls><electronic-resource-num>10.1073/pnas.1204991109</electronic-resource-num></record></Cite></EndNote>[23]. Recent advances in pharmacogenomics suggest polymorphisms of the �candidate gene� either in group or individually might affect the disposition or response to a given drug  ADDIN EN.CITE <EndNote><Cite><Author>Evans</Author><Year>2004</Year><RecNum>59</RecNum><record><rec-number>59</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Evans, William E.</author><author>Relling, Mary V.</author></authors></contributors><titles><title>Moving towards individualized medicine with pharmacogenomics</title><secondary-title>Nature</secondary-title></titles><pages>464-468</pages><volume>429</volume><number>6990</number><dates><year>2004</year></dates><publisher>Nature Publishing Group</publisher><isbn>0028-0836</isbn><urls></urls></record></Cite></EndNote>[24].  Several studies related to Cytochrome P450, Glutatione S transferases and many other key enzyme polymorphisms affecting the medications in diseases like cardiovascular, glioma, toxicity, Parkinson�s and others has been reported  ADDIN EN.CITE <EndNote><Cite><Author>Evans</Author><Year>2004</Year><RecNum>59</RecNum><record><rec-number>59</rec-number><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Evans, William E.</author><author>Relling, Mary V.</author></authors></contributors><titles><title>Moving towards individualized medicine with pharmacogenomics</title><secondary-title>Nature</secondary-title></titles><pages>464-468</pages><volume>429</volume><number>6990</number><dates><year>2004</year></dates><publisher>Nature Publishing Group</publisher><isbn>0028-0836</isbn><urls></urls></record></Cite></EndNote>[24]. Supplementation of zinc may enhance the activity of MMPs (a zinc dependent enzyme) in turn reducing accumulation of collagen and minimizing the severity of fibrosis. We observed the 40% overall responsiveness to Zn acetate�Vitamin A supplementation. As so far there is no drug in the present scenario available for the treatment of OSF. Zn acetate�Vitamin A supplementation may serve as first line treatment strategy along with the LOX G473A status. The response of Zn acetate�Vitamin A supplementation to the wild type LOX up-regulation may further suggests antagonizing effect of Cu which is the co-factor for the LOX activity by maintaining the delicate Zn-Cu balance at the site of fibrosis. Though there was unresponsiveness to the Zn acetate�Vitamin A supplementation in rest wild type patient or those shown by polymorphic LOX patients alternatively suggests the LOX variant alleles may be functionally relevant or in linkage disequilibrium with other functional variants of LOX or with alleles at other nearby loci. However, this hypothesis remains to be tested in OSF. Apart from that the response of Zn acetate�Vitamin A supplementation in OSF need to be studied in the more detailed so combination therapy could be design with knowing molecular details which is  need of hour and essential for the treatment of OSF. 
In conclusion, we have observed chewers carrying Lysyl Oxidase (G473A) genotype are more susceptible to developing OSF. We have been using a combination therapy of Zinc acetate and Vitamin A for treatment of OSF and symptoms significantly improved in most of the patients after a follow-up of 4 months. When the population was segregated according to the LOX genotypes, the wild types presented with greater improvement compared to heterozygous (GA) or homozygous carrier (AA) of the genotype. This result is first to link the treatment response of  OSF patients with its genotypic status and emphasizes that candidate gene polymorphisms can be explored to further establish genetic variability as a determinant of specific drug effects in treatment of OSF patients who do not respond to conventional therapies. Present therapeutic approach shows promise to minimize symptoms of OSF, however presence of risk genotypes may be a determining factor and indicate towards further investigation and identification of these genotypes. Such investigations may aid in designing personalized therapy for disease treatment in near future. 

Acknowledgement: 
This study is supported by the Council of Scientific and Industrial Research (CSIR, Govt of India) for financial support (21(0932)/12/EMR-II). AK is grateful to Indian Council of Medical Research (ICMR) for providing Senior Research fellowship.

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Table Legends 
Table1: Demographic status of cases (OSF) and controls
Table 2: Genotypic distribution of LOX G473A polymorphism among the population 

Figure Legends
Figure 1: Lysyl oxidase (LOX) mRNA expression and quantification.
Representative semiquantitative RT-PCR expression of LOX mRNA in OSF patients (PT), Zinc acetate-Vitamin A supplementation (POT) and controls (N). The expression of GAPDH is as internal control. 
The quantitative representation of mRNA expression of LOX by semiquantitative RT-PCR analysis. 
The quantitative representation qRT-PCR LOX effect of Zinc acetate-Vitamin A supplemetation upon LOX mRNA expression of indicating a clear decrease in LOX expression upon treatment.
Figure 2: Lysyl oxidase (LOX) protein analysis by western blot. 
Representative Western blots showing the pro-& mature-Lysyl Oxidase (pLOX and mLOX) enzyme expression in oral mucosa of OSF (PT), Zinc acetate-Vitamin A supplementation (POT) patients and controls (N). A clear reduction of mLOX can be observed in post treated patients.
Stratified expression of mLOX, pLOX and mLOX:pLOX expression according to different genotypes of LOX G 473 A polymorphism and different grades of the disease.
Stratified expression of mLOX, pLOX and mLOX:pLOX expression according to different genotypes of LOX G 473 A polymorphism and different grades of the disease.






Figure 3: Influence of LOX G473A on collagen content and expression in OSF and supplemented with Zn acetate � Vitamin A. 
Collagen content in pre and post Zinc acetate-Vitamin A supplementation in combined and segregated (according to LOX G 473 A polymorphism). 
Representative Western blots effect of Zinc acetate-Vitamin A supplemetation upon Collagen Type I expression in oral mucosa of OSF patients.
Quantitative representation of corresponding change in expression of collagen type-I in pre and post Zinc acetate-Vitamin A supplementation with LOX G473A polymorphisms. 
Representative immunohistochemical expression of Collagen Type I in biopsied pre and post Zinc acetate-Vitamin A supplemented oral mucosal tissue. Reduced expression of collagen along the fibroblastic cells (shown by arrow) can be observed in wild type (GG) patients. 
Quantitative estimation of corresponding change in immunohistochemical expression of collagen type-#1D����������				����ᴥ���tdUC�td0%hF?�hn�5�CJH*OJQJ\�aJ"h�yhn�5�CJOJQJ\�aJhn�5�CJOJQJ\�aJhn�5�CJH*OJQJ\�aJh�Rr5�CJOJQJ\�aJ"hF?�hn�5�CJOJQJ\�aJhF?�hn�CJOJQJ\�aJhF?�hn�CJOJQJaJhF?�hn�5�CJOJQJaJh=w`5�CJOJQJaJhF?�h=w`5�CJOJQJaJhF?�hn�5�CJOJQJaJh�h�CJOJQJaJ���J	�	


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Figure 4: Inter-incisional distance (IID) and % chang in LOX expression.
Whisker box plot representation of the IID in pre and post Zinc acetate-Vitamin A supplement patients according their respective LOX A473G.
Whisker box plot representation of the change in IID in pre and post Zinc acetate-Vitamin A supplement patients according to their respective LOX A473G  genotype.
Whisker box plot representation of the change in IID in pre and post Zinc acetate-Vitamin A supplement patients according to grade.
Vertical scatter plot of percentage change in mLOX:pLOX ratio among patients carrying different genotypes. 
















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