|Year : 2018 | Volume
| Issue : 4 | Page : 399-403
Nicotinic acetylcholine receptor gene polymorphism is not associated with tobacco-related oral squamous cell carcinoma
Deepa Rajesh1, SM Azeem Mohiyuddin2, Sharath Balakrishna1, A V Moideen Kutty1
1 Department of Cell Biology and Molecular Genetics, Sri Devaraj Urs Academy of Higher Education and Research, Tamaka, Kolar, Karnataka, India
2 Department of Otorhinolaryngology and Head and Neck Surgery, Sri Devaraj Urs Academy of Higher Education and Research, Tamaka, Kolar, Karnataka, India
|Date of Web Publication||28-Feb-2019|
A V Moideen Kutty
Department of Cell Biology and Molecular Genetics, Sri Devaraj Urs Academy of Higher Education and Research, Tamaka, Kolar, Karnataka
Source of Support: None, Conflict of Interest: None
BACKGROUND: Nicotinic acetylcholine receptor is implicated in carcinogenesis indirectly through increasing nicotine dependence and directly through its impact on cell-cycle regulation. Functional polymorphism in nicotinic acetylcholine receptor alpha-5 subunit gene (CHRNA5 c.1192G>A; rs16969968) is associated with nicotine dependence and risk of lung cancer. AIM: The aim of this study was to evaluate the association of CHRNA5 c.1192G>A polymorphism with the risk of oral squamous cell carcinoma (OSCC). SETTINGS AND DESIGN: This was a rural teaching hospital-based case-control study. MATERIALS AND METHODS: A total of 100 histopathologically confirmed cases of OSCC patients and 100 age- and gender-matched healthy individuals were genotyped for CHRNA5 c.1192G>A polymorphism by polymerase chain reaction-restriction fragment length polymorphism method. Allele and genotype frequencies among case and control groups were compared by Chi-squared test (Fisher's exact). RESULTS: The frequency of CHRNA5 c.1192A allele was 22% in OSCC patients and 26% in control individuals. The difference in the distribution of alleles and genotypes between case and control groups was not significant (P > 0.05). CONCLUSIONS: CHRNA5 c.1192G>A polymorphism is not associated with the risk of developing OSCC.
Keywords: CHRNA5 gene, genetic susceptibility, nicotinic acetylcholine receptor, oral squamous cell carcinoma
|How to cite this article:|
Rajesh D, Azeem Mohiyuddin S M, Balakrishna S, Kutty A V. Nicotinic acetylcholine receptor gene polymorphism is not associated with tobacco-related oral squamous cell carcinoma. Indian J Cancer 2018;55:399-403
|How to cite this URL:|
Rajesh D, Azeem Mohiyuddin S M, Balakrishna S, Kutty A V. Nicotinic acetylcholine receptor gene polymorphism is not associated with tobacco-related oral squamous cell carcinoma. Indian J Cancer [serial online] 2018 [cited 2019 May 25];55:399-403. Available from: http://www.indianjcancer.com/text.asp?2018/55/4/399/253291
| » Introduction|| |
Oral squamous cell carcinoma (OSCC) is the fifth most common cancer worldwide and accounts for about 30% of all cancers in India., The incidence of oral cancer is significantly high globally with an estimated 650,000 new cases and 350,000 cancer deaths each year. Oral cancer accounts for 50–70% of total cancer mortality and has the highest incidence among Asian countries. Despite the improvements in surgical and therapeutic approaches, the survival rates have not improved. In India, majority of the OSCCs involve buccal mucosa and lower alveolus and most of these patients usually present with locally advanced disease.
Major risk factors for OSCC include tobacco, alcohol consumption, infection with high-risk strains of human papilloma virus (HPV), and genetic factors. Infection with high-risk strains of HPV like 16 and 18 are observed in about 20–50% of OSCC patients across geographical regions. HPV proteins E6 and E7 are responsible for inactivation of tumor suppressor proteins p53 and pRb, respectively, and thus contribute to carcinogenesis. Tobacco may be consumed in the form of cigarette smoking, betel-quid chewing, or snuff. All these forms of tobacco contain nicotine which is a highly addictive psychoactive substance., Tobacco use is a complex, multistage behavior which includes initiation, trialing, regular use, addiction, cessation, and relapse., This multistage behavior is affected by both genetic and environmental factors. The risk of developing OSCC in tobacco chewers is 4.8 times higher compared to nontobacco chewers. Nicotine is an agonist for nicotinic acetylcholine receptors (nAChRs). Several polymorphisms in the genes that code for nAChRs have been reported.,, The major polymorphism linked with the risk of nicotine dependence is located in CHRNA5 gene which codes for cholinergic receptor nicotinic alpha-5 subunit (CHRNA5 c.1192G>A).,
An enormous body of literature shows that tobacco plays an important role in carcinogenesis. The role of nAChRs is well appreciated in connection with tobacco dependence. However, emerging evidences show that nAChRs have a direct role in carcinogenesis through their influence on cell proliferation, apoptosis inhibition, and angiogenesis. Furthermore, polymorphisms in genes that codes for nAChR proteins like CHRNA5 have been linked to susceptibility to lung cancer., CHRNA5 c.1192G>A (rs16969968) polymorphism results in missense variation at amino acid position 398 (aspartic acid to asparagine). CHRNA5 c.1192G>A polymorphism is associated with nicotine dependence and also lung cancer. There is a paucity of information on the association of CHRNA5 c.1192G>A polymorphism with OSCC, and therefore, this study was undertaken.
| » Materials and Methods|| |
The study was conducted by adopting case-control design. Biopsy-proven OSCC patients were included as cases, whereas age- and gender-matched healthy individuals without any history of cancer and habits were included as control. OSCC patients were recruited for a period of 3 years from 2014 to 2017 from the Department of Otorhinolaryngology of R.L. Jalappa Hospital and Research Centre attached to Sri Devaraj Urs Medical College, Kolar, Karnataka. The study was approved by Institutional Ethics Committee of Sri Devaraj Urs Medical College. Following informed consent, details on demographics (age, gender), habits (tobacco use, smoking, and alcohol), and family history of cancer were collected systematically using a study proforma. Clinical and histopathological details were collected by the head and neck surgeon and patients' medical records. Tumor staging was done according to the 7th edition of American Joint Committee on Cancer TNM (T—primary tumor staging, N—nodal status, M—metastasis) staging system for OSCC.
Tumor specimens collected from OSCC patients were stored at −80°C and blood samples collected from controls were stored at 4°C until processing. Tissue homogenate of the specimen was used for the genomic DNA isolation by previously published method with the following modification for tumor specimen. Tissue sample was minced and placed in a microcentrifuge tube containing cell lysis buffer to which sodium dodecyl sulfate and proteinase K were added. Contents of the tube were incubated at 37°C until complete digestion of the tissue. After the tissue digestion, NaCl and isopropyl alcohol were used for the precipitation of the DNA. The resultant DNA was washed with ethanol, dried, and dissolved in Tris–EDTA buffer. Peripheral blood was used for preparation of genomic DNA from controls. Quantity and purity of the DNA preparation were assessed by UV spectroscopy.
Genotyping of CHRNA5 c.1192G>A polymorphism
This was carried out by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method as previously described by Bierut et al. Genomic DNA was amplified by PCR on Bio-Rad C1000 Touch Thermal Cycler. The primer pairs used were 5′-CGCCTTTGGTCCGCAAGATA-3′ (forward) and 5′-TGCTGATGGGGGAAGTGGAG-3′ (reverse). About 25 μl of the reaction mixture included 1× assay buffer, 150 ng genomic DNA, 0.2 mM dNTP, 1 pmol of each primer, 1.5 mM MgCl2, and 1 unit Taq DNA polymerase (Bangalore Genei, India). The program comprised an initial denaturation at 95°C for 5 min followed by 30 cycles at 94°C for 1 min, 60°C for 30 s, and 72°C for 45 s; final extension involved 5 min at 72°C. The PCR product was analyzed on 1% agarose gel. The 435-bp amplicon was subjected to restriction digestion with 10 units of TaqI restriction enzyme (New England Biolabs, USA) at 65°C and analyzed on 2% agarose gel with ethidium bromide staining. “GG” genotype showed two bands of sizes 288 and 147 bp. “GA” genotype showed three bands of sizes 435, 288, and 147 bp. “TT” genotype showed one band of size 435 bp. Representative agarose gel picture is shown in [Figure 1]. About 10% of the samples were randomly selected and subjected to PCR-RFLP for confirmation and the results were 100% concordant. Sanger-sequenced samples were used as positive controls.
|Figure 1: Representative image of agarose gel electrophoresis showing PCR-RFLP pattern for CHRNA c.1192G > A polymorphism. Lanes: L (100 bp DNA ladder), 1 (PCR amplicon – 435 bp), 2 (“GG” genotype – 288 + 147 bp), 3 (“GA” genotype – 435 + 288 bp + 147 bp), 4 (“AA” genotype – 435 bp). bp: Base pair|
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The control population was tested for conformity to Hardy–Weinberg equilibrium using the web program by Rodriguez and coworkers. Statistical analysis was carried out using the web-based calculation available at www. OpenEpi.com (updated 2013/04/06). Allele and genotype frequencies of the two groups were compared by calculating P value from Chi-square test (Fisher's exact). P values < 0.05 were considered as statistically significant.
| » Results|| |
A total of 100 OSCC patients (79 females, 21 males) and 100 pair-matched healthy individuals were recruited for the study. The demographic, clinicopathological profile of the study participants are summarized in [Table 1]. The mean age of the OSCC patients was 53.8 ± 10.7 years and 54.0 ± 11.6 years for controls. All the OSCC patients included in this study were habituated to chewing carcinogenic substances, like tobacco, betel nut, and gutkha (betel leaf, arecanut, slaked lime, spices, and catechu packed in tins or pouches), and alcohol. The median duration of tobacco addiction was found to be 31 years. Seven percent of the patients were habituated to smoking along with chewing tobacco. About 6% of the patients were habituated to alcohol consumption along with tobacco chewing and gutkha. All the patients included were negative for HPV infection by PCR assay. Thus, chewable tobacco consumption was the major risk factor in the patient group.
|Table 1: Demographic and clinicopathological parameters of the study participants|
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In terms of histopathological grade, majority of the OSCC samples were well differentiated (71%) followed by moderate differentiation in 27% and poor differentiation in 2%. The most commonly affected site was found to be the buccal mucosa (65%), followed by lower alveolus, anterior two-third of tongue, retromolar trigone, and floor of mouth. About 11% patients had cancer of the lower gingivobuccal sulcus, which is an OSCC subtype unique to the Indian subcontinent. It involves buccal mucosa and lower alveolus. According to TNM classification, majority of the patients had locally advanced stage IVa tumor (59%) followed by stage III (21%) and stage II (20%). Type 2 diabetes mellitus was the major comorbidity seen in 11% of the patients.
The distribution of the genotypes of the CHRNA5 c.1192G>A polymorphism in the control population was in agreement with Hardy–Weinberg equilibrium (χ2 = 0.16). The profile of allele and genotype frequency is shown in [Table 2]. Minor allele frequency (1192A) was 22% among patients and 26% in the control group. The distribution of the CHRNA5 c.1192G>A alleles among case and control groups was compared by means of contingency table. P value for the distribution profile was 0.4 indicating that the two groups did not show any statistically significant difference. We also compared the distribution of genotypes in patient and control group. The major allele “G” was found to be present predominantly in homozygous condition (57% in case group and 54% control group). The frequency of the minor allele “A” in homozygous condition was found to be comparatively lower in patient group (1%) than in the control group (6%). The distribution of the genotypes in both the groups was compared by means of contingency table. P value was 0.19 indicating that the two groups did not show any statistically significant difference. The two groups were also compared by considering dominant, recessive, additive, and overdominant genetic models [Table 3]. P values were found to be 0.77, 0.1, 0.15, and 0.77, respectively. Therefore, statistically significant difference was not evident in any of the four models.
|Table 2: Distribution of allele and genotype frequencies of CHRNA5 c.1192G>A polymorphism in the study groups|
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|Table 3: Evaluation of association between CHRNA5 c.1192G>A polymorphism and OSCC risk under different genetic models|
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Multivariate logistic regression analysis of the genotype data was performed in the patient group to evaluate the impact of CHRNA5 c.1192G>A polymorphism on clinicopathological parameters like age, gender, stage, grade, and duration of tobacco usage [Table 4]. The genotype containing the risk allele (GA + AA) did not show any statistically significant association with age, gender, stage, grading as well as with the duration of tobacco chewing (P value > 0.05). The median duration of tobacco consumption in the patient group was found to be 31 years.
|Table 4: Univariate and multivariate analysis of association between clinicopathological parameters of OSCC and CHRNA5 c.1192G>A polymorphism|
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| » Discussion|| |
Tobacco is carcinogenic in both smokable and smokeless forms. However, most of the scientific interest has been focused on the smokable form as combustion of tobacco constituents further expands the repertoire of carcinogens. Smokeless tobacco is commonly consumed either in chewable or snuff form. Smokeless tobacco contains carcinogens like nicotine, nitrosamines, polycyclic aromatic hydrocarbons, metals, and aldehydes. Furthermore, tobacco carcinogens like nitrosamines, N′-nitrosonornicotine (NNN), 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosodiethylamine, and N-nitrosoanabasine have been found in saliva of individuals habituated to smokeless tobacco consumption. The carcinogenic potential of these substances is augmented by their ability to be absorbed by the oral epithelium.
Nicotine plays an important and a direct role in tumorigenesis through stimulation of the nAChR in the target cells. The direct tumorigenic role of nicotine is supported by studies in cell cultures, animal models, and human patients., nAChR is expressed ubiquitously in several tissues including the oral epithelium and is not restricted to the neuromuscular endplate. Chronic exposure of oral keratinocytes to nicotine has been shown to result in the upregulation of α5 subunit of nAChR with the dependent increase in signaling response to nicotine. In vitro studies have shown that nicotine and its metabolites like NNN and NNK are competent to stimulate cell proliferation, inhibition of apoptosis, and induction of tumorigenic transformation of HET-1A cells. Moreover, it is observed that nicotine-derived nitrosamines activate nAChR which further promotes cell proliferation and apoptotic inhibition. NNN and NNK have been shown to stimulate the activation of PI3k/Akt pathway-dependent cell proliferation and NFkB-dependent reduction in apoptosis, thus providing likely mechanism by which nicotine and its metabolites promote tumor development.,
The direct role of nAChR in carcinogenesis has motivated screening of polymorphisms in the corresponding genes for the elucidation of genetic predisposition. Several meta-analyses have shown that CHRNA5 c.1192G>A polymorphism predicts heavy smoking, delayed quitting, and earlier onset and higher likelihood to develop lung cancer.,,, CHRNA5 gene codes for the alpha-5 subunit of nAChR. Furthermore, a recent meta-analysis found that patients with “AA” genotype have a 1.6-fold increased risk for the development of lung cancer than those with “GG” genotype.
Most of the previous studies have focused on smokers except for a single study in which tobacco chewing was evaluated for association with CHRNA5 c.1192G>A polymorphism. CHRNA5 c.1192G>A polymorphism was found to be linked to frequency of tobacco chewing but not with OSCC risk. However, the impact of clinicopatholgical parameters on the genetic association was not undertaken. In this study, we have evaluated the association between CHRNA5 c.1192G>A polymorphism and clinicopathological parameters. We found that the “GA” and “AA” genotypes and “A” allele for CHRNA5 c.1192G>A polymorphism did not confer higher disease risk as compared to other “GG” genotype and “G” alleles, respectively. Our results indicate that CHRNA5 c.1192G>A polymorphism is unlikely to have a significant impact on the risk of OSCC. In addition, we did not notice any improvement in the association when the genotype frequencies were analyzed in various genetic models. Furthermore, multivariate analysis of the data in terms of clinicopathological parameters like stage and grade did not indicate any positive association. These observations compel us to assume that CHRNA5 gene c.1192G>A polymorphism is unlikely to have a significant impact on the risk of OSCC.
| » Conclusions|| |
We conclude that the results of our study indicate that CHRNA5 gene c.1192G>A polymorphism is not a risk factor for the development of OSCC. Complete sequencing of CHRNA5 gene may unravel novel variations that are associated with OSCC.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]