Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 
  Users Online: 713 Home Print this page Email this page Small font sizeDefault font sizeIncrease font size  

 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 7  |  Issue : 2  |  Page : 134-138  

Expression of human papillomavirus dna and p53 polymorphisms through polymerase chain reaction in normal mucosa and oral leukoplakia individuals with deleterious oral habits


1 Department of Oral Pathology and Microbiology, GITAM Dental College, Visakhapatnam, Andhra Pradesh, India
2 Department of Orthodontics and Dentofacial Orthopedics, Kalinga Institute of Dental Sciences, Bhubaneswar, Odisha, India

Date of Submission28-Mar-2016
Date of Acceptance15-Mar-2017
Date of Web Publication8-May-2017

Correspondence Address:
Atmakuri Shanmukha Ramya
Department of Oral Pathology and Microbiology, GITAM Dental College, Visakhapatnam, Andhra Pradesh
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijabmr.IJABMR_57_16

Rights and Permissions
   Abstract 

Context: Oral leukoplakia (OL) is a well-recognized precancerous lesion with various etiological factors. Most commonly deleterious oral habits such as tobacco smoking and viral etiologies mainly human papillomavirus (HPV) play an important role. p53 polymorphisms mostly homozygous Arginine (Arg) allele has a greater risk of degradation by HPV. Hence, HPV infection and p53 polymorphisms may act as synergistic factors for increased the risk of malignant transformation in oral precancerous lesions. Aims: The aim of this study is to evaluate the risk of OL and its malignant transformation due to infection by HPV and p53 polymorphisms in the oral biopsy samples through polymerase chain reaction (PCR). Subjects and Methods: A total of 40 individuals were involved– 10 individuals were controls without deleterious habits, 15 were controls with deleterious habits, and 15 were with histologically confirmed OL individuals with deleterious habits. PCR and restriction fragment length polymorphism using sma1 enzyme were carried out to evaluate the expression of HPV and p53 polymorphisms. Statistical Analysis Used: Chi-square test, Fischer's exact t-test, and odds ratio. Results: (1) HPV DNA expression was higher in Leukoplakia individuals than controls. (2) p53 genotype with homozygous Arg was more in HPV-infected individuals. Conclusions: To conclude HPV infected OL cases were mostly with Arg/Arg type of p53 polymorphism.

Keywords: Human papillomavirus, Leukoplakia, p53, polymerase chain reaction-restriction fragment length polymorphism


How to cite this article:
Ramya AS, Majumdar S, Babu T M, Uppala D, Srinivas B, Rao AK. Expression of human papillomavirus dna and p53 polymorphisms through polymerase chain reaction in normal mucosa and oral leukoplakia individuals with deleterious oral habits. Int J App Basic Med Res 2017;7:134-8

How to cite this URL:
Ramya AS, Majumdar S, Babu T M, Uppala D, Srinivas B, Rao AK. Expression of human papillomavirus dna and p53 polymorphisms through polymerase chain reaction in normal mucosa and oral leukoplakia individuals with deleterious oral habits. Int J App Basic Med Res [serial online] 2017 [cited 2020 Apr 6];7:134-8. Available from: http://www.ijabmr.org/text.asp?2017/7/2/134/205820


   Introduction Top


Oral Leukoplakia (OL) is the most common potentially malignant disorder (PMD) of the oral cavity and has been considered to confer increased risk for the development of oral cancer.[1] Oral cancer usually involves multiple alterations in the genomic level which progressively gets accumulated during a protracted period, the overall effect of which surpasses the inherent reparative ability of the cell.[2] The sum total of these alterations are of diagnostic and prognostic relevance which are designated as “Precancerous changes.”[2]

About 70%–90% of PMDs mainly OL s are related to smoking and alcohol use, either alone or in combination.[3] Several other etiological factors apart from smoking include biological agents such as bacteria, fungi, and chiefly virus. Among all viruses, human papillomavirus (HPV) is the major cancer pathogens in humans which constitute around 23.5%.[4],[5]

HPV is a 55KD, nonenveloped, double-stranded, circular DNA virus that has been implicated in a variety of anogenital and aerodigestive diseases, ranging from common warts to laryngeal papilloma to cervical cancer.[6] More than 120 different subtypes of HPV have been identified which are divided into 3 major groups as super Group A (Alpha papillomavirus), super Group B (Beta papilloma virus), and the remaining group of HPVs are members of super Group E (Mu and Nu-papilloma viruses).[5] The alpha papillomavirus group are mucosotrophic HPVs which are further divided into two classes based on their risk to humans, that is, High Risk-HPV type (potentially oncogenic) and Low Risk-HPV type (nononcogenic). The E6 and E7 oncoproteins of high-risk HPV have the capacity to mediate carcinomatous transformation of infected keratinocytes by inactivating p53 and retinoblastoma (Rb) tumor suppressor pathways.[7]

p53 is a tumor suppressor gene which plays an important role in the maintenance of genomic integrities through induction of cell cycle arrest or apoptosis failing DNA damage. The loss of activity of the wild-type p53 (wtp53) protein can be achieved by two different mechanisms: Either by a mutation of the p53 gene or by binding to the HPV-encoded E6 protein.[8] A common polymorphism in p53 gene at exon 4 codon 72, encoding for either a proline (CCC/Pro) or arginine (CGC/Arg) amino acid, among which p53Arg homozygous genotype has been implicated to have more susceptibility to HPV-infected cancers.[8]

This study is to evaluate the risk of tobacco-associated OL and its malignant transformation due to p53 polymorphisms and infection by HPV in the oral biopsy samples through polymerase chain reaction (PCR).


   Subjects and Methods Top


Fifteen consecutive subjects with OL, Healthy individuals with deleterious habits and 10 healthy individuals without deleterious habits visiting the outpatient department of GITAM Dental College, satisfying the inclusion and exclusion criteria were included for the study [Table 1].
Table 1: Inclusion and exclusion criteria

Click here to view


An informed written consent was obtained before the study. The study protocol was approved by the Ethical committee of the institution.

  • Controls: Group 1: (n = 10) Healthy individuals without any deleterious oral habits
  • Study group: Group 2: (n = 15) Healthy individuals with deleterious oral habits
  • Group 3: (n = 15) Individuals who are clinically and histopathologically diagnosed as cases of OL with deleterious oral habits.


Tissue sample collection

A detailed case history was obtained from all individuals before the study. Incisional biopsy samples were collected from the Group 3 individuals. Samples from Group 1 and 2 were collected during routine dental surgical procedures. A part of the tissue was taken for Hematoxylin and Eosin staining to confirm clinical diagnosis of Leukoplakia. Another part of the tissue was placed in TBS buffer and was stored at −20°C for DNA extraction.

HPV DNA isolation and detection

Genomic DNA was extracted using the Qiagen kit method. The presence of HPV in the controls and OL individuals was detected by PCR, using primers (MY09 and MY11) from the consensus L1 region [Table 2].
Table 2: Primers used for polymerase chain reaction amplification

Click here to view


The PCR was run by an initial denaturation at 94°C for 5 m followed by 35 cycles of 940°C for 1 m, annealing at 51°C for 1 m, extension at 72°C for 1 m followed by final extension at 72°C for 8 m and holding the temperature at 10°C for 1 min. The amplified DNA fragments were observed in 2% agarose gel at 450 base pair [Figure 1].{Figure 1}

p53 gene polymorphism at codon 72, exon 4

Extraction of genomic DNA, for p53 detection, was carried out by the same method as done for HPV DNA isolation. A portion of each reaction product was electrophoresed on 2% agarose gel to check for the quality of the desired PCR product at 162 base pairs. After PCR amplification, 2.5 μL PCR products were digested with Sma I enzyme and incubated at 37°C for overnight and then subjected to agarose gel electrophoresis. In CG (Pro/Arg) genotype, one DNA was cut at 135 and 27 and the other was left uncut. In GG (Arg/Arg) genotype, both DNA were left uncut. The gel pattern was visualized under UV light after ethidium bromide staining [Figure 2].
Figure 1: Gel electrophoresis of human papillomavirus and p53 with 100bp ladder (human papillomavirus – 450bp, p53 -162 bp)

Click here to view


Chi-square test and Fischer's exact t-test were performed to analyze the results.


   Results Top


A total of 40 individuals were included in the study. The demographic data, the status of HPV expression, and type of p53 polymorphisms of all the individuals involved in the study are included in [Table 3].
Table 3: Demographic data of all the three groups

Click here to view


Human papillomavirus analysis

HPV was detected in 1 out of 25 individuals of Group 2 and 3 out of 15 individuals of Group 3. In the present study, HPV was seen mostly associated with sites exposed to microtrauma i-e buccal mucosa in Leukoplakia individuals, but these associations were statistically nonsignificant. Among 3 HPV infected OL patients, 1 was smokeless tobacco chewer and 2 were smoke tobacco chewers. There was no significant association between HPV infection and risk of OL (odds ratio = 6, 95% confidence interval = 0.56–63.9, P->0.13). The correlation between HPV infection and the degree of dysplasia was also found to be nonsignificant (>P = 0.28).

p53 polymorphisms analysis

No significant association between codon 72 polymorphism and OL was observed but, there was a significant correlation between the presence of Arg/Arg type of p53 polymorphism and HPV-positive cases of Leukoplakia (<P = 0.05).


   Discussion Top


In India, it has been reported that 30%–80% malignancies of oral cavity arise from premalignant lesions such as Leukoplakia. The major risk factor for the development of premalignant lesions and malignant lesions is tobacco consumption. Tobacco is being in use as both smoke and smokeless forms (khaini and betel quid).[9] Tobacco-specific nitrosamines are the strong carcinogens associated with tobacco smoke.[10] Apart from deleterious habits, accumulative evidence indicates that individual susceptibility to precancer also depends on genetic predisposition and viral infections mainly HPV.[11]

The present study was based on study of the expression of HPV and p53 polymorphisms in individuals with a mean age of 45 years in all the 3 groups considered. This study reported that OL was more frequent in buccal mucosa followed by the palate. Mitra et al.[10] and Sarnath et al.[11] gave the reasons for the varying sites of occurrence of Leukoplakia which include continuous contact of irritants, variations in the degree of keratinization and permeability of the oral mucosa, contribute to the modulating effects of tobacco toxins.[12] According to the present study mostly males with smoke tobacco habits were more susceptible for OL which can be due to the synergistic effect of elevated temperature in the oral cavity of males thereby making the epithelium more susceptible to the genotoxic effect of tobacco products.[13]

Mucosotropic or epitheliotropic viruses mostly HPVs are capable of causing lesions in both oral mucosa as well as in cervical mucosa because of a similar type of epithelium. Hence, it is important to investigate the relationship between HPV and its contribution to premalignancy.[14]

According to Terai et al.[15] and Califano et al.[6] the prevalence of HPV infection shows extensive variation in healthy mucosa ranging from 12% to 81.1%, but the prevalence of HPV in the present study were– Group B-6.7% and Group C-20%, respectively. This low prevalence of HPV in the oral cavity without any lesions was due to the salivary immune response which includes IgA, cystatins, and proteolytic enzymes that protect the oral mucosa.[3] Other studies by Giovannelli et al.,[16] Gichki et al.,[17] Miller et al.[18] and Sikka et al.[19] the prevalence of HPV ranges from 17 to 40.8% in OPMD, which was in accordance with the present study. Martinez et al.[20] reported that increased frequency of HPV in OPMDs than in the oral carcinomas was due to the “hit and run” theory, according to which viral genome does not need to be present to maintain cell transformation once genetic damage has been inflicted at an early stage.[20] The site prevalence of HPV according to Mravak-Stipetic et al.[21] and the present study was mostly in areas more exposed to micro-trauma.

Apart from the viral basis of etiology, carcinogenesis also has a genetic basis of occurrence. The link between viral contribution and genetic factors is due to the effect of viral proteins on tumor suppressor genes-a major step in viral carcinogenesis.

Most frequently effected tumor suppressor gene by the viral proteins is p53 which is located at locus 17p13.1, the name was due to its molecular mass: Which was in the 53 KD fractions of cell proteins. A p53 has many polymorphisms due to exonic and intronic sequence variations. Most commonly occurring polymorphisms is ate exon 72 which include homozygous Arg allele, homozygous Pro allele and heterozygous Pro and Arg alleles. According to Mousami et al. Arg/Arg polymorphism of p53 codon has more risk of OL. The frequencies of p53 polymorphisms in this study were in correlation with findings of Sikka et al.[19] Mitra et al.[10] reported that individuals with an Arg form of p53 were less efficient in processing tobacco-associated DNA damage signals and hence were more susceptible to develop OL.

An interesting observation in the present study was a significant correlation between frequency of expression of HPV and frequency of the homozygous Arg type of p53 polymorphism, thereby indicating a higher risk of Arg/Arg type of p53 allele to HPV-mediated degradation which could be an important risk factor for malignant transformation of HPV infected OPMDs. Hou et al.,[22] Storey et al.[23] and Nagpal et al.[24] reported similar findings regarding HPV and p53 polymorphisms.

To summarize, although a majority of premalignant lesions occur in individuals with deleterious habits, only some of these lesions are transforming into malignancy. Hence, identification of these high-risk premalignant lesions with increased susceptibility to malignancy and their early detection may help in downstaging of oral cancer and can have a better prognosis. Thus, detection of HPV and type of p53 polymorphism can be highly effective in this scenario as cancer predictive biomarkers in view of reducing global head and neck cancer burden.


   Conclusion Top


So to conclude, this study focuses on the role of HPV in the premalignant lesion and also its role in the degradation of p53. Although the sample size in the present study was small, there was a significant result correlating the presence of HPV and Arg/Arg genotype of p53 in individuals with Leukoplakia. However, larger sample size involving wider cross-section of the population should be included to achieve an affirmative result. This study may provide a stepping stone for the future research in which HPV and p53 polymorphisms may be considered as important markers for suggesting the malignant transformation which further can play a major role in treatment modalities and prognosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Lee JJ, Hong WK, Hittelman WN, Mao L, Lotan R, Shin DM, et al. Predicting cancer development in oral leukoplakia: Ten years of translational research. Clin Cancer Res 2000;6:1702-10.  Back to cited text no. 1
    
2.
Farah CS, Woo SB, Zain RB, Sklavounou A, McCullough MJ, Lingen M. Oral cancer and oral potentially malignant disorders. Int J Dent 2014;2014:853479.  Back to cited text no. 2
    
3.
Feller L, Lemmer J. Oral leukoplakia as it relates to HPV infection: A review. Int J Dent 2012;2012:540561.  Back to cited text no. 3
    
4.
Butel JS. Viral carcinogenesis: Revelation of molecular mechanisms and etiology of human disease. Carcinogenesis 2000;21:405-26.  Back to cited text no. 4
    
5.
Sailan AT. HPV and p16 in head and neck cancer. Infect Agent Cancer 2009;4:1-6.  Back to cited text no. 5
    
6.
Ha PK, Califano JA. The role of human papillomavirus in oral carcinogenesis. Crit Rev Oral Biol Med 2004;15:188-96.  Back to cited text no. 6
    
7.
Vidal L, Gillison ML. Human papillomavirus in HNSCC: Recognition of a distinct disease type. Hematol Oncol Clin North Am 2008;22:1125-42, vii.  Back to cited text no. 7
    
8.
Koh JY, Cho NP, Kong G, Lee JD, Yoon K. p53 mutations and human papillomavirus DNA in oral squamous cell carcinoma: Correlation with apoptosis. Br J Cancer 1998;78:354-9.  Back to cited text no. 8
    
9.
Pfeifer GP, Denissenko MF, Olivier M, Tretyakova N, Hecht SS, Hainaut P. Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers. Oncogene 2002;21:7435-51.  Back to cited text no. 9
    
10.
Mitra S, Sikdar N, Misra C, Gupta S, Paul RR, Roy B, et al. Risk assessment of p53 genotypes and haplotypes in tobacco-associated leukoplakia and oral cancer patients from Eastern Idia. Int J Cancer 2005;117:786-93.  Back to cited text no. 10
    
11.
Saranath D, Tandle AT, Teni TR, Dedhia PM, Borges AM, Parikh D, et al. p53 inactivation in chewing tobacco-induced oral cancers and leukoplakias from India. Oral Oncol 1999;35:242-50.  Back to cited text no. 11
    
12.
Freitas MD, Blanco-Carrión A, Gándara-Vila P, Antúnez-López J, García-García A, Gándara Rey JM. Clinicopathologic aspects of oral leukoplakia in smokers and nonsmokers. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:199-203.  Back to cited text no. 12
    
13.
Garg KN, Raj V, Chandra S. Trends in frequency and duration of tobacco habit in relation to potentially malignant lesion: A 3 years retrospective study. J Oral Maxillofac Pathol 2013;17:201-6.  Back to cited text no. 13
[PUBMED]  [Full text]  
14.
Ankit H. Bharti, Kiran Chotaliya, Y. S. Marfatia. An update on oral human papillomavirus infection. Indian J Sex Transm Dis 2013;34:77-82.  Back to cited text no. 14
    
15.
Terai M, Hashimoto K, Yoda K, Sata T. High prevalence of human papillomaviruses in the normal oral cavity of adults. Oral Microbiol Immunol 1999;14:201-5.  Back to cited text no. 15
    
16.
Giovannelli L, Campisi G, Lama A, Giambalvo O, Osborn J, Margiotta V. Human Papillomavirus DNA in Oral Mucosal Lesions. J Infect Dis 2002;185:833-6.  Back to cited text no. 16
    
17.
Gichki AS, Buajeeb W, Doungudomdacha S, Khovidhunkit SO. Detection of human papillomavirus in normal oral cavity in a group of Pakistani subjects using real-time PCR. Asian Pac J Cancer Prev 2012;13:2299-304.  Back to cited text no. 17
    
18.
Miller CS, Johnstone BM. Human papillomavirus as a risk factor for oral squamous cell carcinoma: A meta-analysis, 1982-1997. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:622-35.  Back to cited text no. 18
    
19.
Sikka S, Sikka P. Association of human papilloma virus 16 infection and p53 polymorphism among tobacco using oral leukoplakia patients: A clinicopathologic and genotypic study. Int J Prev Med 2014;5:430-8.  Back to cited text no. 19
    
20.
Martinez SL, Gomez GE, Trapero JC, Rodriguez PC, Martinez AB, Moreno- Lopez LA et al. Genotypic Determination by PCR-RFLP of Human Papillomavirus in Normal Oral Mucosa, Oral Leukoplakia and Oral Squamous Cell Carcinoma Samples in Madrid (Spain). Anticancer Res 2008;28:3733-42.  Back to cited text no. 20
    
21.
Mravak-Stipetic M, Sabol I, Kranjcic J, Kneževic M, Grce M. Human papillomavirus in the lesions of the oral mucosa according to topography. PLoS One 2013;8:e69736.  Back to cited text no. 21
    
22.
Hou J, Gu Y, Hou W, Wu S, Lou Y, Yang W, et al. P53 codon 72 polymorphism, human papillomavirus infection, and their interaction to oral carcinoma susceptibility. BMC Genet 2015;16:1-9.  Back to cited text no. 22
    
23.
Storey A, Thomas M, Kalita A, Harwood C, Gardiol D, Mantovani F, et al. Role of a p53 polymorphism in the development of human papilloma-virus-associated cancer. Nature 1998;393:229-34.  Back to cited text no. 23
    
24.
Nagpal JK, Patnaik S, Das BR. Prevalence of high-risk human papilloma virus types and its association with p53 codon 72 polymorphism in tobacco addicted oral squamous cell carcinoma (OSCC) patients of Eastern India. Int J Cancer 2002;97:649-53.  Back to cited text no. 24
    


    Figures

  [Figure 2], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


This article has been cited by
1 Mucosal human papillomavirus detection and TP53 immunohistochemical expression in non-melanoma skin cancer in Tunisian patients
Ines Ben Ayed,Haifa Tounsi,Amira Jaballah,Monia Ardhaoui,Afifa Maaloul,Thalja Lassili,Najla Mezghani,Sonia Abdelhak,Samir Boubaker
Journal of Cutaneous Pathology. 2019;
[Pubmed] | [DOI]
2 Effect of TP53 rs1042522 on the susceptibility of patients to oral squamous cell carcinoma and oral leukoplakia: a meta-analysis
Zhen Sun,Wei Gao,Jiang-Tao Cui
BMC Oral Health. 2018; 18(1)
[Pubmed] | [DOI]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
   Subjects and Methods
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed1739    
    Printed16    
    Emailed0    
    PDF Downloaded232    
    Comments [Add]    
    Cited by others 2    

Recommend this journal