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Oral cancer (OC) was reported to be the
sixth most common cancer in the world with a projected 400,000 newly diagnosed
cases and 223,000 mortalities in 2008. 1 Oral cancer is ranked first
among all cancer cases within males and third most common cancer among females
in various regions of India. Prevalence of tobacco associated oral squamous
cell carcinoma (OSCC) in North-East India is utmost among all the states which
is about 33% in total OC cases. 2 Oral leukoplakia is a common
pre-cancer lesion among tobacco user and it has an yearly incidence of
0.2-11.7% in different population of India, and a few of the leukoplakias
(2-12%) progress to cancer if not intervened at precancerous stage. Smoking,
smokeless tobacco products are the chief risk factors for oral cavity cancer. 3

Mitochondrial DNA (mtDNA) is particularly susceptible
to damage by environmental carcinogen as it does not contains introns, nor
protective histones and is exposed continuously to endogenous ROS. 4, 5,
6 Therefore mtDNA may serve as a potential sensor for cellular DNA
damages and could be a marker for cancer development. Although, alterations are
scattered all the way through the mitochondrial genome (16,569 bp) but the
mutations are more frequent in regulatory noncoding region (Displacement loop/D
loop) of the mtDNA. 6

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The regulatory D-loop is 1124 bp in size
(positional location 16024-576), is non coding region, act as a promoter for
both the heavy (H) and light (L) strands of the mtDNA, and contains essential
replication and transcription elements. The D-loop region is a hot spot for mtDNA
alteration which contains various sites viz: (a) Hypervariable region I (HVI)
at position 16024-16383 (b) Hypervariable region II (HVII) at position 57-372
contain a stretch of D310 region which has a mononucleotide repeat of Cytosine
(Cs) that ranges from nt 304-316 is highly polymorphic in human population. The
number of cytosine in 7 bp tract varies from 6-13 bp and is called C tract
region. The first stretch of Cs can vary from seven to nine in normal
individual. 7 Genetic variability in the D-loop has been suggested to
influence the function of the respiratory chain that is responsible for ROS
elevation and could contribute to cancer initiation through DNA damage. 8

Few scientific groups have documented
their reports on mutation in mitochondrial DNA from Northeast Indian population.
9, 10 Since there is limited data available about mutational hot
spot in the D-loop regions and their association with clinico-pathological
markers from Indian OC patients 11, 12, the aim of the present
study was to: (1) Assess mutational hot spot and their frequency in OC tissue
and (2) to assess the relation between these mutation and the
clinico-pathological characteristics of OC in study population.

Materials and Methods

and sample specimens

The study was carried out at the Institute
of Medical Sciences under Banaras Hindu University, Varanasi, India. The study
protocol of present work was approved by the Institutional Ethics Committee.
Informed consent was obtained from each individual who attended Department of
Surgical Oncology, SS Hospital, BHU, during the period from July 2012 to
January 2015 for treatment. Initially seventy oral cancer patients were
recruited for the study with previous consent. A total N=;70 paired tissue
samples (OC tissue and adjacent normal tissue) of oral cancer patients were
collected after surgical resection. Histopathology was performed before study
plan, which revealed that 25 normal adjacent tissue showed tumor pathology,
thus dropped-out. Therefore study included N=70 tumor specimen and N=45 normal
adjacent specimen. All tissues were collected, frozen in liquid nitrogen and
stored at -800 C until DNA/protein extraction. A part of fresh
tissue was used for Single Cell Gel Electrophoresis (SCGE).

Cell Gel Electrophoresis (SCGE)/COMET Assay

Tumor tissue minced in 1ml Hepes buffered
medium without serum containing 20mM EDTA/10% DMSO, allowed to settle and
removed. The SCGE was carried out under alkaline conditions, as described in
our previous study. 13

To prevent additional DNA damage, all
steps were conducted under yellow light or in the dark. Additionally, to avoid
probable position effects through electrophoresis, two parallel replicate
slides per sample were prepared and processed in different electrophoretic run.
Slides were examined at 250x magnification using a fluorescence microscope (Olympus -BX43 Fluorescence), equipped with an excitation filter
(515–560 nm) and a barrier filter (590 nm). To avoid the potential variability,
one well trained scorer performed all scorings of comets with automated scoring
software. As a measure of DNA damage, percent amount of tail and head was used.
It was calculated from the midpoint of the head and presented in micrometres.


Total DNA was extracted from tissues using
TRIzol® Reagent according to the manufacturers (Invitrogen , Carlsbad, CA)
protocol. The concentration and purity of DNA was measured with NanoDrop (Biospec Nano, Shimadzu Biotech,

Polymerase Chain Reaction (PCR)

Amplification of
D-loop segment was done using Fermentas high fidelity PCR enzyme Mix. The total
reaction volume was 25 µl containing 2.5 µl of 10X PCR buffer with MgCl2,
2.5 µl 10 mM dNTPs, 0.25 µl (5 Units/µl) of Taq polymerase, 1µl each of 10
pmol/µl primer (forward and reverse), 50-100 ng of genomic DNA and the volume
make up to 25 µl by adding nuclease-free water.

Primer for D-loop amplification

The D-loop
region of mtDNA was analyzed for mutations with the use of direct sequencing of
the products from PCR. The primer pairs L16190 (nucleotide position
16190-16209, 5′-CCCCATGCTTACAAGCAAGT-3′) and H602 (602-583,
5′-GCTTTGAGGAGGTAAGCTAC-3′) were utilized for amplification of 982 bp DNA
segment from the D-loop region of mtDNA. The PCR programme used for
amplification was: initial denaturation at 940 C for 3 min; 33 cycle
of denaturation at 940 C for 30 s; annealing at 550 C for
45 s; and elongation at 720 C for 60 s and final elongation at 720
C for 5 min. The amplified product was observed in 1.5% agarose gel.

DNA Sequencing

Entire D-loop region was sequenced using automated
DNA sequencer. To diminish the sequencing artifacts induced by PCR, products
from at least more than one amplicon were sequenced using forward and reverse
primers. Sequences were compared aligned with the human mtDNA sequence (GENBANK
accession # NC-012920.1) and the mitochondrial database MITOMAP
( Sequence variants which were found in both the case
and control at a particular location in the corresponding mtDNA were classified
as polymorphisms. If the nucleotide sequence at a particular location in the
tumor mtDNA differed from the corresponding control mtDNA, then it was identified
as a somatic mutation.


An Association between mtDNA mutations and
clinical features were analyzed using ?2 test and compare means with
the help of one way Anova. All statistical analysis was performed in SPSS
version 16.0 software. Relative risk was estimated in terms of Odds Ratio (OR)
and 95% confidence interval (95% CI). P values (two sided) below 0.05 (P

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