Expression of Human Papillomavirus DNA and p53 Polymorphisms through Polymerase Chain Reaction in Normal Mucosa and Oral Leukoplakia Individuals with Deleterious Oral Habits.

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.

Introduction

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%.[45]

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

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

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

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

Gel electrophoresis of human papillomavirus and p53 with 100bp ladder (human papillomavirus – 450bp, p53 -162 bp)

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 2

restriction fragment length polymorphism – p53 polymorphisms digested by Sma1 enzyme (Arginine/Arginine – 162bp, Arginine/Proline – 162,135,27bp, Proline/Proline -135, 27bp)

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

Results

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

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 (