PROTECTIVE GERMLINE VARIANTS (SNPs) ASSOCIATED WITH ORAL

CANCER IN PROLONGED TOBACCO CHEWERS OF NORTHEAST INDIA

Sharbadeb Kundu1, Akalesh Kumar Verma2, Vijayalakshmi Ramshankar3, Arvind Krishnamurthy4, Rajeev Kumar5, Ravi Kannan5, Sankar Kumar Ghosh1*
1 Department of Biotechnology, Assam (Central) University, Silchar, Assam, India,
2 Department of Zoology, Cotton College State University, Guwahati, Assam, India,
3 Department of Preventive Oncology (Research), Cancer Institute (W.I.A.), Chennai, India,
4 Department of Surgical Oncology, Cancer Institute (W.I.A.), Chennai, India,
5 Department of Molecular Oncology, Cachar Cancer Hospital & Research Centre, Silchar, Assam.

Abstract Results DFNA5 SND1

Chr 7
Smokeless tobacco is the single most important cause of high incidence of oral cancer in India. In Case Control
Northeast India, the incidence of tobacco-related oral cancer is reported to be about 33%. The
genetic susceptibility to a disease is influenced by germline Single Nucleotide Polymorphisms (SNPs)
located within the promoter or other regulatory regions of the associated genes. Therefore, in addition 16 132 8
to somatic alterations, the germline variations may also play an important role in influencing cancer
prognosis and disease outcome. On the other hand, sometimes the germline variants may act as a
protective marker for a disease. They play a crucial role in the regulation of different carcinogens
metabolizing signaling pathways. In this study, our objective is to identify the potential protective Figure 1 Distribution of 156 variants
germline variants that are associated with tobacco-related oral cancer in long-term tobacco chewers between cases and controls Figure 2 Representative LD plot for Chromosome 7 (r2<0.8)
of Northeast (NE) India. 170 targets from 75 genes have been selected from some recent GWAS and
candidate gene-based studies related to oral cancer to be screened in NE Indian population. The
targeted re-sequencing experiment has been carried out in Ion-PGM™ platform. Initially, the hg19
sequencing has been carried out on 40 case-control samples (20 patients and 20 healthy individuals rs2237306 (chr7:24,757,202)
24,809,244 24,737,974
with more than 15 years of tobacco chewing habit as discovery set). Furthermore, the result is
validated and then replicated in independent 84 case-control samples (30 patients and 54 healthy
individuals with more than 15 years of tobacco chewing habit as confirmation set) using the Figure 3 Chromosomal position and structure of DFNA5 gene (on reverse strand); No.1-14 are Exons
Sequenom iPLEX MassARRAY platform. Out of 1828 SNPs present in our targeted region, primarily
we have screened out 156 SNPs based on their recurrences in our discovery set samples. Finally, 39
SNPs have been confirmed to have the significant association with oral cancer, out of which, 14 SNPs
showed the protective effect. Out of these, in the confirmation set, we observed one protective non-
coding SNP, rs2237306 in DFNA5 genetic region associated significantly (OR=0.40, p-value=0.026)
with tobacco-related oral cancer. Further in silico analysis showed that this protective variant is involved  Responsible for autosomal dominant hearing loss
in the NMD pathway that triggers the degradation of nonsense DFNA5-transcripts. We also observed  Inversely correlated with estrogen receptor expression in Breast Cancer (Thompson and Weigel 1998)
that two major chemical component of tobacco, Benzo(a)pyrene and Tetrachlorodibenzodioxin, are  Down-regulation of DFNA5 contributes to acquired Etoposide resistance in melanoma cells: positively
involved in the increased expression of DFNA5 gene. At length, this approach in a larger scale would associated with etoposide-induced apoptotic pathway and caspase-3 activity (Lage et al. 2001)
further help in developing a tobacco-protective SNPs panel. In the current era of genome editing,  Positively involved in p53-induced apoptosis in response to DNA damage (Masuda et al. 2006)
understanding the role of the protective SNPs in different cell signaling pathways may lead us to new  mutDFNA5-induced cell death is mediated by the MAPK pathway, especially through the extracellular
therapeutic intervention. signal regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) branch (Van Rossom et al. 2015)
 There is a potential link between endoplasmic reticulum and mitochondria in DFNA5-induced cell death.
Introduction
Oral cancer,a subtype of Head and Neck carcinoma, is
the sixth most common cancer globally.
rs2237306
In India, about 90% of oral cancer is tobacco-related. (NC_000007.13:g.24757202C>A / ENST00000411476.2:c.346-209G>T)
In the Northeast Indian region, the incidence of
tobacco related oral cancer is reported to be about  Change in TF binding site; creates the binding site for c-Myb transcription factor
33% (Bhattacharjee et al. 2006).  Additional RNA binding site motif “GTTTG” (a known ESR: hnRNP-B) found
Northeastern people are used to have fermented foods,  Donor splice site is broken (The variation score for HSF = -31.77)
smoked meat, dried fish, diverse types and patterns of  Correlated with epigenetic transcriptional activation by methylation (H3K4me1,
tobacco habits, HPV and/or EBV infection. H3K4me3) and acetylation (H3K9Ac, H3K27Ac) in Dnd41 T-cell Leukemia cell line
The atypical food habits and lifestyles along with the
genetically predisposed factors might be the potential Benzo(a)pyrene
Tetrachlorodibenzodioxin
cause for higher oral cancer incidence in this region. Acetaminophen
Evidences of no oral cancer incidence among the Aflatoxin B1

prolonged tobacco chewers are there. But, the reasons Potassium chromate(VI)
Trichostatin A
are not clearly known. 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-
1H-imidazol-2-yl) benzamide

Recent GWAS and candidate gene-based works (6-(4-(2-piperidin-1-ylethoxy)phenyl))-3-

pyridin-4-ylpyrazolo(1,5-a)pyrimidine

related to oral cancer have identified 170 targets from Copper

Cyclosporine
75 genes significantly associated with the disease
globally as well as in other parts of Indian subcontinent. Source: Redrawn and language names edited by Roger Blench from a map published by Bishop’s House, Guwahati

Figure 4 Enrichment map of overrepresented GO categories Figure 5 Top interacting chemicals with DFNA5.
for DFNA5 & TP53 genes generated from BiNGO (p<0.001)
Methodologies Discussions
Wild DFNA5
Identification of Validation & Replication
Targeted Re-sequencing
Genome wide hotspot (Sequenom iPLEX
(Ion PGM™ Platform) INDEL in Transversion (C>G) at 6-bp
germline SNPs MassARRAY Platform) Intron 7 upstream of Exon 8
(Bischoff et al. 2004)

mutDFNA5
Workflow For Targeted Resequencing Skipping of Exon 8

 Deleterious Function of
mutDFNA5
Presence of  Problem in the p53-dependent
NO
rs2237306 (the pathway for suppressing
NMD Transcript cancer
Ion Reporter™ variant)
Samples AmpliSeq™ Designer Ion PGM™ Platform Torrent Suite
Exclusive Tobacco Chewers (Panel Size - 31.34 Kb ) Software
YES
(for more than 15 years)

Cancer Healthy Validation &

Patients Controls Activation of Nonsense Degradation of
Replication
Workflow For SNP genotyping in Sequenom Mediated mRNA Decay
aberrant transcript
(NMD) Pathway
(no mutDFNA5)

Amplification Primer Extension Detection and

Ratio Analysis Figure 6 Proposed model for probable mechanism of action (MOA) of the identified protective SNP

Conclusions
 Experimental verification for the proposed model is yet to be done.
 This approach in a larger scale (e.g. WES, WGS etc.) would further help in developing
ONE a tobacco-protective SNPs panel
Statistical analysis for identification of significantly associated variants
 Initial identification of significant variants (in Ion Reporter™ Software)
protective  In the current era of genome editing, this potential target might become a new
 Chromosome wise Single Marker based association study (in Haploview)
SNP therapeutic intervention
 Stratified association study (in PLINK) identified

Acknowledgement
Our humble acknowledgement goes to the Department of Biotechnology (DBT), Govt. of India for providing fund for the NER
Twinning project (BT/349/NE/TBP/2012) and Department of Science &Technology (DST), Govt. of India for providing fellowship

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* For any comments/ suggestions/ queries contact: drsankarghosh@gmail.com