|Year : 2022 | Volume
| Issue : 4 | Page : 518-523
Comparative evaluation of XPD and XPG gene polymorphism in oral squamous cell carcinoma and tobacco chewers: An observational study
Prabhanshu Shrivastava, Suchitra Gosavi, Dipak Ghatge, Aishwarya Naik, Archana Marlapalle, Abhinandh Krishna
Departments of Oral Pathology, Government Dental College and Hospital, Nagpur, Maharashtra, India
|Date of Submission||29-May-2021|
|Date of Decision||22-Aug-2022|
|Date of Acceptance||08-Sep-2022|
|Date of Web Publication||22-Dec-2022|
Department of Oral Pathology, Government Dental College and Hospital, Nagpur, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: The study indicated that XPD and XPG gene polymorphism is associated with the development of oral potentially malignant disorders (OPMDs) and oral squamous cell carcinoma. Xeroderma pigmentosa (XP) is a part of the complex DNA repair system. Xeroderma pigmentosum group G (XPG) and xeroderma pigmentosum group D (XPD) gene function in the nucleotide excision repair (NER) pathway. XPG and/or XPD gene alterations can cause defective DNA repair efficiency which ultimately leads to genomic instability and carcinogenesis. Thus, this study helps in early detection of OPMDs among individuals who have not yet developed any oral lesions and also helps in the management of oral squamous cell carcinoma as if XP gene polymorphism is known.
Aims and Objectives: The aim of the study was to evaluate the expression of XPD and XPG gene polymorphism in oral squamous cell carcinoma cases. The study also had the objective to evaluate and compare the expression of XPG and XPD gene polymorphism in oral squamous cell carcinoma (OSCC) cases, tobacco chewers without any oral lesions, and normal healthy individuals without any habit.
Materials and Method: A total of 150 subjects were included in the study and genotyped for the expression of XPD (AC) and XPG (GC) gene polymorphism using polymerase chain reaction (PCR) and agarose gel electrophoresis method.
Results: XPD genotype for the study shows that most of the cases of OSCC show heterozygous (AC) genotype (64%), whereas in tobacco chewers without any oral lesions wild (AA) genotype (54%) is more common than other types. XPG genotype for the study shows that wild (GG) type is the most dominant genotype both in OSCC cases (78%) and tobacco chewers without any oral lesion (56%).
Conclusion: The study shows the association of XPD and XPG gene polymorphism with the risk of developing OPMDs and oral cancer.
Keywords: NER, oral cancer, oral squamous cell carcinoma, XPD and XPG gene polymorphism
|How to cite this article:|
Shrivastava P, Gosavi S, Ghatge D, Naik A, Marlapalle A, Krishna A. Comparative evaluation of XPD and XPG gene polymorphism in oral squamous cell carcinoma and tobacco chewers: An observational study. J Oral Maxillofac Pathol 2022;26:518-23
|How to cite this URL:|
Shrivastava P, Gosavi S, Ghatge D, Naik A, Marlapalle A, Krishna A. Comparative evaluation of XPD and XPG gene polymorphism in oral squamous cell carcinoma and tobacco chewers: An observational study. J Oral Maxillofac Pathol [serial online] 2022 [cited 2023 Feb 3];26:518-23. Available from: https://www.jomfp.in/text.asp?2022/26/4/518/364794
| Introduction|| |
Oral squamous cell carcinoma (OSCC), a form of head and neck cancer, is the most common malignant epithelial neoplasm that affects the oral cavity. India has the largest number of oral cancer cases and one-third of the total burden of oral cancer globally. In spite of the advancements in diagnostic and treatment modalities, the five-year survival rate has not improved in over the past 50 years.
The major cause of this poor prognosis is delayed diagnosis. Usually, the patients are diagnosed at an advanced stage of the disease with nodal metastasis. At least 50% of patients with locally advanced head and neck cancer develop regional or distant relapses. Cervical lymph-nodes status is the single most important prognostic indicator for the survival of patients with oral cancer. The development of nodal metastases halves the five-year survival rate.
The aetiology of oral squamous cell carcinoma is multifactorial. They include extrinsic and intrinsic factors, including genetic alterations, genetic predisposition, environmental influences such as tobacco, alcohol, chronic inflammation, viral infections, diet and nutrition, fungal infections, dental factors, trauma, age, and immuno-suppression. Aetiological factors like to bacco and alcohol consumption are well established; however 15% to 20% of oral cancer develops in patients without any habit. More than 90% of OSCC has shown association with tobacco habit. The disproportionately higher incidence of OSCC in relation to other malignancies in India may be due to the use of tobacco in various forms, consumption of alcohol, low socioeconomic condition related to poor hygiene, poor diet, and rampant viral infections.
The individual's genetic susceptibility is an important aspect of oral carcinogenesis. Due to the presence of multiple types of gene polymorphism, different persons metabolize various carcinogens in different ways. The inherent instability of genomic DNA as well as endogenously produced reactive oxygen species (ROS) and a wide range of exogenous agents such as radiation and poisons, make it very prone to damage. DNA damage has an important role in tumorigenesis. DNA repair genes are essential to maintain the integrity of the genome. DNA repair genes are required for maintaining genomic integrity. The DNA repair mechanism is a multi-pathway system that includes nucleotide excision repair (NER), double-strand breaks repair (DSBR), base excision repair (BER), mismatch repair, and homologous recombinant repair (HRR). NER is one of these mechanisms, and it works in four steps to eliminate helix distorting DNA lesions and structures from the genome. These four steps are lesion identification, protein binding, oligonucleotide excision, and DNA fragment synthesis. There are at least eight core functional genes in the NER pathway. These include excision repair cross complementing group 1 (ERCC1) and xeroderma pigmentosum group (XP) A–G., In coordinating DNA repair, cell cycle arrest, and apoptosis, the XP gene plays an important role and hence contributes to the prevention of carcinogenesis through the maintenance of genome integrity and the exclusion of abnormal cells.
It is found that gene mutation in NER pathway can lead to several human diseases. Xeroderma pigmentosum group G (XPG) and xeroderma pigmentosum group D (XPD) genes function in the NER pathway. Any alteration in XPG and/or XPD genes can impair DNA repair efficiency which can further cause genomic instability and carcinogenesis. XPG and/or XPD gene may harbour single nucleotide polymorphisms (SNPs) and many of those SNPs have been reported to alter the risk of different cancers including colorectal., lung.,, gastric., and laryngeal cancers.
Polymorphisms of XPD and XPG genes may have synergistic effects on the development of OSCC. It would also be important to elucidate possible association of the polymorphisms of the XP genes with altered functions and a cancer risk.
The carcinogens in tobacco causes DNA adducts. Genetic polymorphisms can affect metabolic activation of the carcinogens and will determine the DNA adduct levels. These DNA adducts if not repaired can accumulate and lead to malignancy.
Currently, new and highly sensitive detection methods such as molecular analysis and immunohistochemistry have been developed.
The polymerase chain reaction (PCR) is one of such method used to analyse tiny amounts of genetic material with precision and reliability.
The purpose of the present study was to utilise the precision offered by PCR to evaluate and analyse the polymorphism of XPD and XPG gene in individuals with or without cancer risk.
| Materials and Methods|| |
The study was evaluated on 150 subjects including 50 subjects from each group (group 1 included histopathologically confirmed cases of OSCC, group 2 included tobacco chewers without any oral lesions and group 3 consisted of normal healthy subjects without any oral lesions), who were registered in the department of oral pathology and microbiology, government dental college and hospital, Nagpur, Maharashtra. An informed consent was obtained from all the subjects.
Venous blood samples were collected in EDTA tube and stored at −80°, till DNA extraction. Genomic DNA extraction from blood was carried out.
Human blood genomic DNA isolation
Human genomic DNA was isolated using geneOmbio's Spin Column based Blood DNA extraction kit. Isolation of genomic DNA from blood samples was performed using reference protocol (GTPL/PRO/78) following the Spin-column based nucleic acid extraction. PCR primers used for the amplification of XPD A/C and XPG G/C polymorphisms were same as used by Sanyal et al.
PCR mix was prepared for all the DNA samples. Final volume of each reaction was 25.0 μL. Genomic DNA was added later to each tube:(PCR reagents used: Taq Polymerase Recombinant from Thermo Fisher Scientific).
Human genes were amplified using standard PCR reaction. The primer pairs, XPD and XPG forward and reverse were used in a PCR reaction with an annealing temperature of 55°C and 58°C, respectively. Reaction was performed in 2720 Thermal Cycler (Thermo Fisher, USA). Upon completion of the thermal cycling program, the PCR products were checked on 2% Agarose by Agarose Gel Electrophoresis and amplicon size was compared using reference ladder. Two percent and 3% agarose gel spiked with LabSafe Nucleic Acid staining dye (G-Biosciences, USA) was prepared by using Agarose (Seakem) in 0.5X TBE buffer. Five microlitre of PCR product was mixed with 1 μL of 6X Gel tracking dye. PCR products of XPD and XPG genes were digested by the PstI and NlaIII restriction enzymes, respectively. Gel images were recorded using BIO-RAD GelDocXR gel documentation system.
All data were compiled on a Microsoft Office Excel Sheet (v 2019, Microsoft Redmond Campus, Redmond, Washington, United States). Data was subjected to statistical analysis using Statistical package for social sciences (SPSS v 26.0, IBM). Descriptive statistics like frequencies and percentage for categorical data, Mean & SD for numerical data was depicted. Comparison of frequencies of categories of variables with groups was done using Chi-squared test. Inter-group comparison (two groups) was done using t-test. For all the statistical tests, P < 0.05 was considered to be statistically significant, keeping α error at 5% and β error at 20%, thus giving a power to the study as 80%.
| Results|| |
The study shows that in case of OSCC, the prevalence of male subjects (80%) is more than of the females (20%) and male predominance (92%) is also seen in subjects with tobacco chewers without any oral lesions, than females (8%). The study indicated that the predominant site of involvement of the lesion is the mandible and their surrounding tissues. On classifying the OSCC on the basis of histopathological grading, the maximum number of cases of well differentiated squamous cell carcinoma were found (82%).
XPD genotype for the study shows that most of the cases of OSCC show heterozygous (AC) genotype (64%), whereas in tobacco chewers without any oral lesions wild (AA) genotype (54%) is more common than other types. The Chi-squared value for the intergroup comparison of the XPD polymorphism was 151.231 and the P value 0.000**. From these values, it is clear that there was a statistically highly significant difference seen for the frequencies between the groups (P < 0.01) with higher frequency for AC in group 1, AA in group 2, NP in group 3 [Figure 1].
|Figure 1: Agarose (2% w/v) gel electrophoresis of amplification products from XPD gene. The samples showed amplification product of 273bp. Samples in well No 1-13 are serially 12879-12891. Lane M – 100 to 500 bp DNA ladder, Lane N – Negative Control sample|
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XPG genotype for the study shows that wild (GG) type is the most dominant genotype both in OSCC cases (78%) and tobacco chewers without any oral lesion (56%). The Chi-squared value for the intergroup comparison of the XPG genotype was 149.492 and P value was 0.000**. These values suggest that there was a statistically highly significant difference seen for the frequencies between the groups (P < 0.01) with higher frequency for W in group 1, W & H in group 2, N in group 3 [Figure 2].
|Figure 2: Agarose (3% w/v) gel electrophoresis of amplification products from XPG gene. The samples showed amplification product of 159 bp. Samples in well no. 1–13 are serially 12879-12891. Lane M – 100 to 500 bp DNA ladder, Lane N – Negative Control sample|
Click here to view
| Discussion|| |
In the present study, the mean age of the patients with OSCC was 50.02 ± 9.931 (age range 32–68) years with male predilection, and the site most commonly involved was the mandible and their surrounding tissues. This finding is in accordance with a study by Shenoi et al. (2012).
Distribution of XPD genotype/polymorphism in all three study groups
This study indicated that cases of OSCC show XPD gene polymorphism with AC polymorphism being the most common, whereas in tobacco chewers without any oral lesions AA polymorphism was seen most commonly compared to AC and CC polymorphism. In case of normal healthy individual, no polymorphism (94%) was seen. Therefore, all these findings are similar to the study performed by Wang et al. and the meta-analysis by Flores-Obando RE et al. and Zhang et al., and the case–control study by Avci, Iplik, Aydemir et al., and Nigam et al. All of these studies suggested that polymorphism in the XPD gene could play a role in the development of OSCC [Table 1].
Distribution of XPG genotype/polymorphism in all three study groups
OSCC and tobacco chewers without any lesion show polymorphism, with wild (GG) type being most common followed by heterozygous (AC) type and mutant (CC) type. About 98% of the normal healthy individuals show no polymorphism. The XPG gene harboured SNPs and many of the SNPs alter the risk of different cancers such as colorectal, lung, and laryngeal cancer as reported from the study done by Nigam et al. [Table 2].
The expression of XPG gene for OSCC and tobacco chewers without any lesion shows polymorphism, with wild (GG) type being the most common followed by heterozygous (AC) type and mutant (CC) type. Thus, tobacco chewers without any oral lesions and with XPG gene polymorphism are at potential risk of developing premalignant lesion that may lead to the development of oral cancer. Therefore, both XPG and XPD are claimed as potential biomarkers in terms of cancers. Our findings are in accordance with the findings of the study done by Nigam et al., Sameer, and Zhou et al., where they found the association of XPD and XPG genes in carcinogenesis.
Thus, this study can aid in early detection of OPMDs among individuals with habits who have yet not developed any lesions, but, are potentially at major risk of developing the premalignant lesions or oral cancer.
Limitations and future scope of the study
This study comprised of a smaller sample size, so studies with a much larger sample size are advocated. These findings are rather suggestive than conclusive and need a larger sample size with diverse population groups. Also, there is no definite genotype and polymorphism correlation in the literature.
Further studies could be done for determining the relationship of XPD and XPG gene polymorphism with other OPMDs and oral cancer involving multiple groups and correlating their results.
Clinical significance of the present study is that for the management or treatment of oral cancer, if the risk factors, such as XP gene polymorphism, is known then the targeted therapy for these patients can be instituted or developed in the near future.
| Conclusion|| |
This study highlighted that XPD and XPG gene polymorphisms are significant both in OSCC and tobacco chewers without any oral lesion. Both XPD and XPG gene polymorphisms are associated with increased risk of cancer development as these genes play a significant role in DNA repair. Thus, tobacco chewers are at increased risk of developing oral cancer. Early diagnosis of cases with potential risk for cancer development can reduce the mortality rate due to cancer and will help the physician in giving proper treatment. To conclude, the present study shows the association of XPD and XPG gene polymorphism with the risk of developing oral potentially malignant disorders and oral cancer. The study needs a larger population size for any conclusive results.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]