|Ahead of print publication
Characterization of genetic polymorphisms in oral cancer-related genes pertaining to oxidative stress, carcinogen detoxifying, and DNA repair: A case–control study
Neville Hoshedar Tata1, Ashok Kshirsagar2, Nitin Nangare2
1 Department of General Surgery, KIMSDU, Karad, Maharashtra, India
2 Department of Surgery, Krishna Institute of Medical Sciences, Karad, Maharashtra, India
|Date of Submission||27-Jul-2020|
|Date of Decision||11-Sep-2020|
|Date of Acceptance||25-Dec-2020|
|Date of Web Publication||24-Jul-2021|
Department of Surgery, Krishna Institute of Medical Sciences, Karad - 415 110, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: The genetic polymorphism in the DNA repair and maintenance genes leads to mutations and deregulated growth hormones which have implications in cancer. Apart from identified carcinogens such as tobacco, specific genetic polymorphisms correspond to an individual's risk of oral cancer. The current study aims at identification of differences in genetic polymorphisms in subjects with and without oral cancer in Karad, India.
Aim/Objectives: The aim of the study was to characterize genetic polymorphisms in oral cancer-related genes pertaining to oxidative stress, carcinogen detoxifying, and DNA repair.
Methodology: A hospital-based case–control was conducted with 150 subjects sorted into cases (n = 75) and controls (n = 75). The polymerase chain reaction-based restriction fragment length polymorphism assay was used to genotype the polymorphisms of selected DNA repair, detoxifying, and oxidative stress-related genes.
Results: In the cases group, among the DNA repair set, Gene-1 (XRCC1), Gene-3 (XRCC3), Xeroderma Pigmentosum Group-D gene (XPD), and human 8-oxoguanine DNA glycosylase (hOGG1) showed significant genetic polymorphism. Similarly, the genetic polymorphism in the carcinogen detoxifying genes-n-acetyl transferase, GSTP1, and oxidative stress-related gene catalase were noted.
Statistical Analysis: The Cramer's V/odds ratio was applied to estimate the association of genetic risk factors with oral cancer.
Conclusion: The polymorphisms of XRCC1, XRCC3, XPD, and hOGG1 genes were associated with a higher susceptibility to oral cancer as compared to controls. This information may be a useful novel marker in oral oncology for primary prevention and intervention.
Keywords: Genetic polymorphisms, oral cancer, polymerase chain reaction
|How to cite this URL:|
Tata NH, Kshirsagar A, Nangare N. Characterization of genetic polymorphisms in oral cancer-related genes pertaining to oxidative stress, carcinogen detoxifying, and DNA repair: A case–control study. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=322267
| > Introduction|| |
The oral cancer represents the most common cancer in men in India with an incidence of 56,000 every year. It accounts for 30%–40% of all head-and-neck cancers with a global prevalence of 8%., The causal etiology of oral cancer is linked to tobacco-based addictions, poor oral hygiene, poor nutrition, poor immunity, and chronic infections., Oral precancers and cancer lack symptoms in early stages and are often neglected often leading to diagnosis in advanced stages. Early detection is essential for good prognosis. The management of oral cancer may include surgical excision/radiation therapy, chemotherapy, advanced immunotherapy, or a combination of these modalities. These morbidity associated with these modalities often result in reduced quality of life.
The genetic makeup determines that the susceptibility one has toward the development of a particular cancer. This genetic vulnerability is linked to chromosomal abnormalities or genetic polymorphisms of DNA repair and maintenance genes. These genetic polymorphisms may correspond to mutation of oncogenes, deletions of cell surface receptors, and dysregulated growth hormones in oral cancer. The polymorphisms of the DNA repair genes are associated with an individual's risk of cancer.
It must be emphasized that oral cancer is one of the potentially preventable cancers and yet has a high incidence and in late stages is associated with poor prognosis. Early detection by gene polymorphism corresponding to molecular alterations may aid in prompt diagnosis and thereby improve prognosis. Molecular signaling defects leading to dysregulation, mutation, and overexpression may lead to changes in tumor microenvironment like raised markers of oxidative stress and failed DNA repair. Identification of these may aid in early identification of cancer, having prognostic implications., Thus, the study aimed at understanding the genetic polymorphisms pertaining to malignancies of the oral cavity-related genes (oxidative stress, carcinogen detoxifying, and DNA repair mechanism genes) in a Indian state with a high risk of oral cancer.
| > Methodology|| |
The hospital-based case–control study was conducted following approval by the institutional ethics committee on 150 individuals equally divided into case (n = 75) and healthy age- and sex-matched controls (n = 75) selected by purposive sampling. Cases already on therapy for malignancy were excluded. Following recording of demographic data, 5 ml of whole blood and tissue specimens (biopsy samples) were collected.
Genomic DNA isolation from whole blood
Genomic DNA isolation was performed from the tissue samples using HiPurA Mammalian genomic DNA extraction and purification Kit (Hi-Media Laboratories, Bengaluru, India) following the manufacturer's instructions. DNA isolation was performed from peripheral blood samples using Purelink genomic DNA mining and purification Kit (Invitrogen, Life technologies, USA) following the manufacturer's instructions. After quantitative and qualitative analysis of genomic DNA, the final samples were kept in Tris-EDTA buffer (pH 8) at −20°C until use.
Genotyping of DNA repair genes (XRCC1, XRCC2, XPD, APE1, and hOGG1), carcinogen detoxifying genes (GSTM1, GSTP1, GSTT1, and NAT), and oxidative stress-related genes (SOD1, SOD2, and catalase) were performed by the polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) method with appropriate primer sets. The primers were designed to amplify the regions of DNA that contain polymorphic sites of interest. PCR amplification was carried out separately under different conditions in 20 μL reaction mixtures containing 1X PCR buffer 0.2 mM each dNTP, 10 picomoles of each of selected primers, 1U Taq DNA polymerase (GeNei, Merck Bioscience), and 100 nanogram of purified genomic DNA template. The obtained mixtures were imperilled to PCR amplification with a Master cycler Gradient PCR (Eppendorf). The PCR products, obtained for each reaction were analyzed using 2% agarose gel electrophoresis (1X TAE buffer), which were stained with ethidium bromide (10 mg/mL) and later envisioned under UV Transilluminator and saved (photographed) in gel documentation system (Bio Rad Laboratories). After confirmation of DNA amplification, each PCR product was digested with an appropriate restriction enzyme for genotyping.
Statistical analysis was carried out using R statistical software (version 3.6.3; USA). Categorical variables are represented by frequency tables. Continuous variables are represented in mean ± standard deviation form. P ≤ 0.05 indicates statistical significance. Strength of association was obtained by Cramer's V/odds ratio (OR).
| > Results|| |
Age, gender, and alcohol status had no significant association with the groups. It was observed that there was significant association (P = 0.001) of habit history related to tobacco usage with the groups. Around 97.33% (n = 73) of the cases used tobacco as opposed to 22.3% (n = 22) of the controls. The strength of association between tobacco habit and cancer was found to be very high (Cramer's V). The odds of not having oral cancer was 87.93 times more for the subjects who did not use tobacco compared to the subjects who used tobacco. [Table 1] shows the distribution and comparison of selected demographic characteristics of oral cancer in cases and controls.
|Table 1: The distribution and comparison of selected demographic characteristics of oral cancer in cases and controls|
Click here to view
Considering the site specificity of untreated cancers, the 60% (n = 45) had presented on the buccal mucosa. The other intraoral sites for cancer occurrence are shown in [Figure 1]. The associations between the genotypes and risk of oral cancer with or without tobacco were studied using OR. Logistic regression analyses (univariate and multivariate) were employed to determine the cancer risk associated with gene types. [Table 2] shows the frequency of genotypes pertaining to DNA repair genes in cases and controls.
|Figure 1: Distribution of untreated oral cancer patients based on site of lesion|
Click here to view
|Table 2: The genotype frequencies of DNA repair gene variants in untreated oral cancer patients and healthy controls|
Click here to view
In the cases group, 33.34% (n = 25) showed significant genetic polymorphism in the DNA repair gene-1 (XRCC1), 75 53.34% (n = 40) showed significant genetic polymorphism in the gene-3 (XRCC3). Furthermore, 80% (n = 60) showed significant genetic polymorphism in the DNA repair gene Xeroderma Pigmentosum Group-D gene (XPD) and 77.34% (n = 58) showed significant genetic polymorphism in the repair gene human 8-oxoguanine DNA glycosylase (hOGG1).
The univariate analysis revealed that, in Hogg1 Cd 326, Odds of Having Oral Cancer is 37.65 times more for the subjects with genotypes Cys/Cys compared to genotype Ser/Ser. The XRCC1 Exon 6, XRCC1 Exon 9, XRCC3 Exon 7, and XPD Exon 23 had significant representations for odds of developing oral cancer. The detail of these genes with corresponding genotypes is also presented in [Table 2].
Multivariate analysis showed that, in hOGG1 Cd 326, odds of having oral cancer is 29.81 times more for the subjects with genotypes Cys/Cys compared to genotype Ser/Ser. Furthermore, the odds of having oral cancer is 3.20 times more for the subjects with genotypes Ser/Cys OR Cys/Cys compared to genotype Ser/Ser. Similarly, the genes XRCC1 exon 9, XPD exon 23 and APE1 exon 5 had represented with significant odds of developing oral cancer. The detail of these genes with corresponding genotypes is also presented in [Table 2].
In the cases, 81% (n = 61) showed significant genetic polymorphism in the carcinogen detoxifying Gene n-acetyl transferase (NAT) and around 40% (n = 30) showed significant genetic polymorphism in the carcinogen detoxifying gene GSTP1. Univariate analysis showed that, in NAT, odds of having cancer is 6.09 times more for the subjects with genotypes G/A compared to genotype G/G. Odds of having cancer is 44.29 times more for the subjects with genotypes A/A compared to genotype G/G. Odds of having cancer is 20.78 times more for the subjects with genotypes G/A OR A/A compared to genotype G/G. Similarly, the GSTM1, GSTP1 exon 5, and GSTP1 exon 6 had represented with significant odds of developing oral cancer. The details of these genes with corresponding genotypes are presented in [Table 3].
|Table 3: The genotype frequencies of carcinogen detoxifying gene variants in untreated oral cancer patients and healthy controls|
Click here to view
Multivariate analysis showed that, in NAT, odds of having oral cancer is 4.89 times more for the subjects with genotypes G/A compared to genotype G/G. Odds of having oral cancer is 151.45 times more for the subjects with genotypes A/A compared to genotype G/G. Odds of having oral cancer is 35.41 times more for the subjects with genotypes G/A OR A/A compared to genotype G/G. Similarly, in GSTM1, in GSTP1 exon 5, in GSTP1 exon 6, and in GSTT1 presented with significant odds of developing oral cancer. The details of these genes with corresponding genotypes are presented in [Table 3].
Around 76% (n = 57) of cases showed significant genetic polymorphism in the oxidative stress-Related gene catalase. Univariate analysis showed that, in CAT exon 7, odds of having oral cancer is 20.28 times more for the subjects with genotypes A/T compared to genotype A/A. Odds of having oral cancer is 115.6 times more for the subjects with genotypes A/T OR T/T compared to genotype A/A. The SOD1 exon 10, SOD2 codon 58, and GPX exon 10 represented with significant odds of developing oral cancer. The details of these genes with corresponding genotypes are presented in [Table 4].
|Table 4: The genotype frequencies of oxidative stress related gene variants in untreated oral cancer patients and healthy controls|
Click here to view
The multivariate analysis showed that, in CAT exon 7, odds of having oral cancer is 36.1 times more for the subjects with genotypes A/T compared to genotype A/A. Odds of having oral cancer is 165.4 times more for the subjects with genotypes A/T OR T/T compared to genotype A/A. In SOD2 codon 58 and GPX exon 10 depicted significant odds of developing oral cancer. The details of these genes with corresponding genotypes are presented in [Table 4].
| > Discussion|| |
Sociodemographics of the study
The incidence of oral cavity cancer, distribution, and prognosis is influenced by various sociodemographic and habitual risk factors such as age, sex ratio, region, ethnicity, nutritional status, addictions to harmful habits, occupation, and awareness of general health and hygiene. The mean age of cases was found to be 54.88 years, ranging between 50 and 70 years, which is in line with previous reports. In India, oral cancer accounts for 45% and 47% of total cancers.
A male predominance existed for cases as per the study which is consistent with literature. Tobacco habits were higher in cases than controls with significant odds of cancer in cases. This association reported in the current study is a recognized hazard for oral cancer.,, The distribution of oral cancer in males is attributable to higher habit history.,,, The preponderance of the buccal mucosa and tongue reported in this study were cited in previous reports. These are sites for keeping chewable forms of tobacco explaining site specificity of oral precancers.,
DNA repair gene variants (XRCC1, XRCC2, XPD, hOGG1, and SNP)
There is increasing evidence that reduced DNA repair capacity, resulting from genetic polymorphisms of various DNA repair genes, is linked with high risk and susceptibility to various types of human cancers.,, The polymorphism in DNA repair genes has been comprehensively investigated for its associations with cancer risk, and the results were conflicting in different types of cancer or different population., There are multiple genotypes studied under this category, of which the important ones, i.e., XRCC1, XRCC2, XPD, hOGG1, and SNP are discussed in brief.
Significant association was reported between the XRCC3 722C>T polymorphism, with an increased the risk of HNSCC. A genomic study had reported that combination of XRCC1 to XRCC4 single nucleotide polymorphisms is useful biomarkers of oral cancer. Furthermore, an Indian study showed significant difference between cases and controls in case of genetic polymorphs of XRCC1-Arg194Trp and XRCC-Gln399Arg and SNPs; however, the same study had reported no significant difference for XRCC1 Arg280His. The results of the current study showed that the XRCC1, XRCC3, XPD, and hOGG1 gene polymorphisms are associated with the risk of oral cancer. Furthermore, the current study shares the findings with a previously reported study on colorectal adenomas.
The role for XPD as a modifier of the effects of alcohol is biologically plausible. An interaction between the XPD codon 751 SNP and alcohol has previously been reported for head-and-neck cancers. Furthermore, the association between the hOGG1 codon 326 at Ser 326 Cys polymorphism and smoking and drinking-related oral cancer has been reported. The results of the current study, XRCC3 codon 241 SNP modifications are unrelated to alcohol or smoking habits is in agreement with the report by Tranah et al.
A functional effect for the codon 280 SNP, which is in connection disequilibrium with the codon 194 and codon 399 SNPs is reported in literature. Therefore, the results of the current study may be driven by the SNP, which was not examined among these subjects.
Oxidative stress-related gene variants (GSTM1, GSTP1, and GSTT1)
The oxidative stress-related markers are important biomarkers for oral cancers. The important genotypes noted in cases for associations in the current study were GSTM1, GSTP1, and GSTT1. The GSTM1 enzyme had been reported to be present in oral and laryngeal cancer patients exhibiting detectable levels of GST μ protein, a useful biomarker. The current study showed that null genotypes of GSTM1 and GSTT1, individually or in combination, are the risk factors for oral malignancy. These findings are concurrent with studies conducted on GSTM1 and GSTT1 in South India which find that genetic polymorphism in these genes is closely linked to the occurrence of leukoplakia and in turn contributes to the occurrence of oral malignancy as a consequence. GSTP1 assessment in the current study showed significant polymorphism and an increase in the risk of malignancy as compared to healthy individuals. These findings are consistent with studies done on GSTP1 in Denmark which reveal significant polymorphism in the gene.
Carcinogen detoxifying gene variants (SOD1 and SOD2)
Genetic polymorphisms in SOD, CAT, and GPX have been implicated in proneness to cancer and other diseases. This was observed in case of genes coding for SOD2, CATALASE, and GPx with a noteworthy surge in the risk of malignancy as per the current study. Multivariate analysis of the gene group adjusting for tobacco use revealed the polymorphism in the gene coding for SOD2 enzyme was associated with a significant rise in the risk of malignancy. Similar results were observed in a study on SOD 1 and SOD2 polymorphism and its correlation with oral cancer.
The strength of the study is the methodology involving molecular biological indices for oral cancer. Genetic polymorphisms with other possible genes and correlations with grades/stages of cancer and to geographical areas are much awaited.
| > Conclusion|| |
This study is the first to report the combined effects of XRCC gene polymorphisms as a risk factor for oral cancer in Maharashtra population. The XRCC1, XRCC3, XPD, and hOGG1 genes were associated with a higher susceptibility as compared to the wild type. This information may be a useful novel marker in oral oncology for primary prevention and intervention.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Chung CH, Yang YH, Wang TY, Shieh TY, Warnakulasuriya S. Oral precancerous disorders associated with areca quid chewing, smoking, and alcohol drinking in southern Taiwan. J Oral Pathol Med 2005;34:460-6.
García-Pola Vallejo MJ, Martínez Díaz-Canel AI, García Martín JM, González García M. Risk factors for oral soft tissue lesions in an adult Spanish population. Community Dent Oral Epidemiol 2002;30:277-85.
Kalyanpur R, Pushpanjali K, Prasad KV, Chhabra KG. Tobacco cessation in India: A contemporary issue in public health dentistry. Indian J Dent Res 2012;23:123. [Full text]
Gelband H, Jha P, Sankaranarayanan R, Horton S, editors. Cancer: Disease Control Priorities. 3rd
ed., Vol. 3. Washington (DC): The International Bank for Reconstruction and Development/The World Bank; 2015.
Franceschi S, Barra S, La Vecchia C, Bidoli E, Negri E, Talamini R. Risk factors for cancer of the tongue and the mouth. A case-control study from northern Italy. Cancer 1992;70:2227-33.
Peacock ZS, Pogrel MA, Schmidt BL. Exploring the reasons for delay in treatment of oral cancer. J Am Dent Assoc 2008;139:1346-52.
Jurel SK, Gupta DS, Singh RD, Singh M, Srivastava S. Genes and oral cancer. Indian J Hum Genet 2014;20:4-9.
] [Full text]
Cadoni G, Boccia S, Petrelli L, Di Giannantonio P, Arzani D, Giorgio A, et al
. A review of genetic epidemiology of head and neck cancer related to polymorphisms in metabolic genes, cell cycle control and alcohol metabolism. Acta Otorhinolaryngol Ital 2012;32:1-1.
Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J. et al
. Molecular Cell Biology. 4th
edition. New York: W. H. Freeman; 2000. Available from: https://www.ncbi. nlm.nih.gov/books/NBK21475/
[Last accessed on 01 Mar 2020].
Mascolo M, Siano M, Ilardi G, Russo D, Merolla F, De Rosa G, et al
. Epigenetic dysregulation in oral cancer. Int J Mol Sci 2012;13:2331-253.
Sharma S, Satyanarayana L, Asthana S, Shivalingesh KK, Goutham BS, Ramachandra S. Oral cancer statistics in India on the basis of first report of 29 population-based cancer registries. J Oral Maxillofac Pathol 2018;22:18-26.
] [Full text]
Shen H, Xu Y, Qian Y, Yu R, Qin Y, Zhou L, et al
. Polymorphisms of the DNA repair gene XRCC1 and risk of gastric cancer in a Chinese population. Int J Cancer 2000;88:601-6.
David-Beabes GL, Lunn RM, London SJ. No association between the XPD (Lys751G1n) polymorphism or the XRCC3 (Thr241Met) polymorphism and lung cancer risk. Cancer Epidemiol Biomarkers Prev 2001;10:911-2.
Ratnasinghe D, Yao SX, Tangrea JA, Qiao YL, Andersen MR, Barrett MJ, et al
. Polymorphisms of the DNA repair gene XRCC1 and lung cancer risk. Cancer Epidemiol Biomarkers Prev 2001;10:119-23.
Hu Z, Ma H, Chen F, Wei Q, Shen H. XRCC1 polymorphisms and cancer risk: A meta-analysis of 38 case-control studies. Cancer Epidemiol Biomarkers Prev 2005;14:1810-8.
Zheng H, Wang Z, Shi X, Wang Z. XRCC1 polymorphisms and lung cancer risk in Chinese populations: A meta-analysis. Lung Cancer 2009;65:268-73.
Werbrouck J, De Ruyck K, Duprez F, Van Eijkeren M, Rietzschel E, Bekaert S, et al
. Single-nucleotide polymorphisms in DNA double-strand break repair genes: Association with head and neck cancer and interaction with tobacco use and alcohol consumption. Mutat Res 2008;656:74-81.
Yen CY, Liu SY, Chen CH, Tseng HF, Chuang LY, Yang CH, et al
. Combinational polymorphisms of four DNA repair genes XRCC1, XRCC2, XRCC3, and XRCC4 and their association with oral cancer in Taiwan. J Oral Pathol Med 2008;37:271-7.
Kumar A, Pant MC, Singh HS, Khandelwal S. Associated risk of XRCC1 and XPD cross talk and lifestyle factors in progression of head and neck cancer in north Indian population. Mutat Res Fund Mol M 2012;729:24-34.
Longnecker MP, Chen MJ, Probst-Hensch NM, Harper JM, Lee ER, Frankl HD, et al
. Alcohol and smoking in relation to the prevalence of adenomatous colorectal polyps detected at sigmoidoscopy. Epidemiology 1996;7:275-80.
Huang WY, Olshan AF, Schwartz SM, Berndt SI, Chen C, Llaca V, et al
. Selected genetic polymorphisms in MGMT, XRCC1, XPD, and XRCC3 and risk of head and neck cancer: A pooled analysis. Cancer Epidemiol Biomarkers Prev 2005;14:1747-53.
Datkhile KD, Patil MN, Khamkar TS, Vhaval RD, Durgawale PP, Chougule PG, et al
. Genetic polymorphisms in DNA repair genes (hOGG1 & APE1) and their association with oral cancer susceptibility in rural Indian population: A hospital based case-control study. Int J Med Sci Public Health 2017;6:23-8.
Tranah GJ, Giovannucci E, Ma J, Fuchs C, Hankinson SE, Hunter DJ. XRCC2 and XRCC3 polymorphisms are not associated with risk of colorectal adenoma. Cancer Epidemiol Biomarkers Prev 2004;13:1090-1.
Takanami T, Nakamura J, Kubota Y, Horiuchi S. The Arg280His polymorphism in X-ray repair cross-complementing gene 1 impairs DNA repair ability. Mutat Res 2005;582:135-45.
Katiyar T, Yadav V, Maurya SS, Ruwali M, Singh M, Hasan F, et al
. Interaction of glutathione-s-transferase genotypes with environmental risk factors in determining susceptibility to head and neck cancer and treatment response and survival outcome. Environ Mol Mutagen 2020; 61:574-84.
Saravani S, Miri-Moghaddam M, Bazi A, Miri-Moghaddam E. Association of glutathione-S-transferases M1 and T1 deletional Variants with development of oral squamous cell carcinoma: A study in the south-east of Iran. Asian Pac J Cancer Prev 2019;20:1921-6.
Rao AK, Parameswar P, Majumdar S, Uppala D, Kotina S, Vennamaneni NH. Evaluation of genetic polymorphisms in glutathione S-transferase Theta 1, glutathione S-transferase Mu1, and glutathione S-transferase Mu3 in Oral leukoplakia and Oral squamous cell carcinoma with deleterious habits using polymerase chain reaction. Int J Appl Basic Med Res 2017;7:181-5.
Zhang Y, Zhang L, Sun D, Li Z, Wang L, Liu P. Genetic polymorphisms of superoxide dismutases, catalase, and glutathione peroxidase in age-related cataract. Mol Vis 2011;17:2325-32.
[Table 1], [Table 2], [Table 3], [Table 4]