Oral and Maxillofacial Surgery

https://doi.org/10.1007/s10006-020-00929-5

ORIGINAL ARTICLE

Genetic polymorphism of tumor necrosis factor alpha (TNF-α)

and tumor necrosis factor beta (TNF-β) genes and risk of oral
pre-cancer and cancer in North Indian population
Shalini Gupta 1 & Kumud Nigam 2 & Ratnesh Kumar Srivastav 1 & Md. Kaleem Ahmad 3 & Abbas Ali Mahdi 3 &
Somali Sanyal 2

Received: 23 August 2020 / Accepted: 2 December 2020

# The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Objective There are inconclusive data connecting single-nucleotide polymorphisms (SNPs) of TNF-α (rs361525) and TNF-β
(rs909253) to potential malignant oral disorder (PMOD) such as lichen planus and oral fibrosis. Here, we have investigated the
risk of oral squamous cell carcinoma as well as oral pre-cancerous lesions in North Indian population with the polymorphism of
the TNFα/ β genes.
Material and methods A total 500 patients with oral pre-cancer and OSCC and 500 healthy volunteers were genotypes for the
TNF-α (-238) G/A (rs361525) and TNF-β (252) A/G (rs909253) gene polymorphism. Genotypes were identified by polymerase
chain reaction (PCR) restriction fragment length polymorphism (RFLP). Genotype frequencies were evaluated by Chi-square test.
Results Compared to the GG genotype, the GA genotype of TNF-α (G238A) polymorphism (rs361525) has been found to
significantly increase the risk of oral disease (OR = 1.99) and especially the risk of lichen planus and OSCC (OR = 2.805 and
5.790, respectively). Similarly, the risk of oral disease was also more in the heterozygote (AG) than the common allele homo-
zygote (AA) of TNF-β (A252G) polymorphism (rs909253) (OR = 1.483).
Conclusion We conclude that the SNPs rs361525 and rs909253 were significantly associated with oral pre-cancer and OSCC.

Keywords Oral pre-cancer . OSCC . Cytokines . Tumor necrosis factor-α . β . Fibrosis . Lichen planus . Gene polymorphism

Introduction the cancers diagnosed in this gender. Oral pre-cancerous lesion, a

benign morphologically altered tissue that has a greater than
Oral cancer represents an important problem worldwide. The normal risk of malignant transformation, is also very common.
incidence and prevalence rates for these tumors are double in Leukoplakia, lichen planus, and oral submucous fibrosis are early
men than in women. Cancers of the oral cavity rank as the eighth indicators of damage to the oral mucosa with a transformation
most common cancer among men, being responsible for 3% of rate of 2–12% to frank malignancies [1]. These lesions are
termed as potential malignant oral disorder (PMOD). Many en-
vironmental factors like tobacco, smoking, and alcohol con-
Shalini Gupta and Kumud Nigam contributed equally to this work. sumption and genetic factors (oncogenes and suppressor genes)
are implicated in the development of oral cancer [2] which may
* Shalini Gupta sometimes predispose from a clinically evident PMOD, the most
sgmds2002@yahoo.co.in
common being the oral leukoplakia [3]. Tobacco and alcohol
* Somali Sanyal usage are strong risk factors with the development of oral cancer
ssanyal@lko.amity.edu
[4]. Oral diseases are highly prevalent in Indian subcontinent,
1
Department of Oral Pathology & Microbiology, King George’s and oral cancer ranked 3rd in Indian scenario [4].
Medical University, Lucknow, India Several salivary tumor markers are found to be significant-
2
Amity Institute of Biotechnology, Amity University, Lucknow, UP, ly increased in the saliva of oral cancer patients [5, 6] such as
India 8-OHdG and MDA biomarkers.
3
Department of Biochemistry, King George’s Medical University, Few biomarkers are needed to predict the evolution of oral
Lucknow, India cancer from an PMOD and also to screen the high-risk
 Oral Maxillofac Surg

Table 1 Primers and restriction

enzymes used for genotyping of Gene Primer sequence Annealing temp. Restriction
TNF-α (238) G/A and TNF-β (°C) enzyme
(252) A/G polymorphisms
TNF-α (238) F-AAACAGACCACAGACCTGGTC- 61 BamHI
G/A R-CTCACACTCCCCATCCTCCCG
GATC-
TNF-β (252) F-TGACTCTCCATCTGTCAGT- 57 NcoI
A/G R-AGAGAGAGAGACAGTGAGC-

patients with smoking and tobacco habits. It is important that
the combined early detection and the effective treatment strat-
egies could help in the prevention and the good prognostic
outcomes of oral cancer [7].
Several factors related to angiogenesis, inflammation, and
thrombosis have been associated with oral cancer [2, 4, 8].
Previous studies have shown that polymorphism in extracellu-
lar matrix MMP1 (1607GG) is highly associated with colorec-
tal cancer, renal cancer, and head and neck carcinoma [9–11].
The vascular endothelial growth factor (VEGF) family of pro-
teins known to involve in vasculogenesis and angiogenesis is
the prime requirement for tumor growth. Polymorphisms of
VEGF-C such as rs7664413 (− 33 nt upstream of the VEGF-
C gene) and rs2046463 (downstream 5008 nt) have been shown
to be strongly associated with OSCC growth [12]. Similarly,
polymorphism of VEGF-A is also known to regulate the malig-
nancy [13]. Polymorphisms of different inflammatory genes
such as IL-2 (−330A > C) [14], COX-2 gene −1195 G > A
(rs689466), +837 T > C (rs5275), and − 765 G > C (rs20417)
have also been shown to strongly correlated with oral cancer
progression [15]. Predisposition of individuals to different car-
cinogens is also associated with genetic polymorphisms, as
ALDH2 (aldehyde dehydrogenase-2) Rs671G > A is strongly
correlated with cancer progression [16]. Sensitivity toward to-
bacco consumption also varies with genetic polymorphism in
genes such as CYP1A1, GSTM1, and GSTT1 [17].
Susceptibility of pre-cancer oral diseases to genetic polymor-
phism is also well documented as SNPs in GSTM1 (null) [18],
CCND1 (G870A) [19], XPD (codon 751) [20], and MMP3 (-
1171; promotor region) [21] are well correlated across the ma-
jority of populations (Asians, Caucasians, Brazilians, and
others). Risk associated with a SNP in p53 (codon 72) was
reported in Indian populations only [22].
The progression of cytokines as biomarkers for detecting
malignancies has helped in realizing the importance of
inflammation-mediated carcinogenesis in PMODs and in oral
cancer. Inflammatory genes as tumor necrosis factor alpha
(TNF-α) and beta (TNF-β) are, respectively, encoded by
TNF-α and TNF-β genes [23]. TNF-α gene is located on
chromosome 6p21.231 in the polymorphic region of MHC
III. The promoter polymorphisms of TNF-α have been inten-
sively studied as a potential determinant of disease suscepti-
bility [24, 25]. There is also an increasing evidence that
TNF-α may promote the development and spread of cancer
[26–28]. Commonly described variants of TNF-α gene poly-
morphisms consist of G to A transitions in the promoter region
at positions _238 and _308 [24]. G to A transition at position
238 in promoter region is important because it results into
higher expression of TNF-α protein [29]. So far, TNF-α pro-
moter polymorphism has been related to numerous cancers,
such as bladder cancer [30], renal cell carcinoma [31], non-
small cell lung carcinoma [32], cervical cancer [33], and
breast carcinoma [34]. Importance of this polymorphism in
oral cancer has been shown in western population [35], but
significance in Indian population is still unknown. TNF-β is a
proinflammatory cytokine produced by lymphocytes and is

Fig. 1 2% agarose gel analysis of TNF-α (G238A) polymorphism. Lane 1 50 bp ladder; lane 2, 6,7 AA genotype 155 bp; line 3,4 AG genotype
155,130,25 bp; lane 5 GG genotype 130,25 bp
Oral Maxillofac Surg
Fig. 2 2% agarose gel electrophoresis analysis of TNF-β (A252G) gene polymorphism. Lane 2,4,5,7,8,9 AG genotype 237,180,57 bp; lane
3,6,10,11,12,13,14 AA genotype (237 bp); lane 1 50 bp ladder
structurally related to TNF-α [35]. The two factors have 30%
amino acid sequence identity, are recognized by the same
widely distributed cellular receptors, and, therefore, share
many of their functions [35]. The TNF-β gene is adjacent to
the TNF-α gene within a 7 kb locus in the major histocompat-
ibility complex [35]. An A/G polymorphism located at posi-
tion 252 within the first intron of the TNF-β gene affects
expression of both genes and concentration of TNF-α and
TNF- β proteins in plasma [36, 37]. The less common allele
B1 (252G) has been correlated with a higher TNF-β expres-
sion both at the mRNA and the protein level [37]. On the other
hand, the common allele B2 (252A) is associated with in-
creased TNF-α gene expression [36]. The TNF-β (A252G)
polymorphism has been associated with a risk of development
of breast, esophageal, gastric, and colorectal cancer [38–40].
The reported allele frequency of the high expression B1 ho-
mozygotes is about 16% in Europeans and 13% in Asians [38,
41]. Interestingly, when the combined TNF-α and TNF-β
polymorphisms were examined together in patients with
breast or esophageal cancer, certain genotypes were found to
be significantly overrepresented and were associated with an
increased risk of cancer development [38, 42]. Although the
role of TNF-alpha (-308 G/A) and TNF-beta (252 G/A) poly-
morphism in oral cancer was reported earlier, the study was
based upon European population [35]. There is one report of
the study of TNF α polymorphism in Indian population. Singh
et al. [43] showed TNF-α (-238) G/A is highly but TNF-α (-
308) G/A is not associated with incidence of oral cancer.
Singh et al. had not analyzed the impact of TNF-β (+252A/
G) or TNF-α (-238) G/A on oral cancer or pre-cancer lesions.
There was one study in Taiwan where TNF-α (-308) G/A
polymorphism was found associated with oral submucous fi-
brosis [44]. Another study conducted on Saudi Arabian pop-
ulation the polymorphism TNF-α (-308 G/A), and TNF-β
(+252A/G) was found significantly associated with oral lichen
planus [45]. These information strongly indicate that there is
no thorough study of TNF polymorphism in connection with
oral pre-cancer and cancer condition in Indian population.
Habit of taking pan masala, gutka, areca nut, betel nut, and
tobacco by North Indian population makes them highly vul-
nerable to oral diseases such as oral submucous fibrosis, oral
lichen planus, oral leukoplakia, and oral cancer. In this study,
the possible correlation of TNF-α (-308 G/A) and TNF-β
(+252A/G) with OPMD and OSCC has been evaluated in
North Indian population.
Materials and methods
Study subjects
The study was undertaken on a total of 500 patients
with previously treated and pathologically confirmed
oral pre-cancer and OSCC who were registered at
Department of Oral Pathology and Microbiology, King
George’s Medical University and 500 healthy controls.
This study was approved by the Institutional Ethics
Committee of the King George’s Medical University,
Lucknow. An informed written consent was obtained
from all subjects. Ethical clearance was obtained from
institutional ethical committee. Venous blood samples
were collected in EDTA tubes and stored at − 80 °C,
until DNA extraction. Genomic DNA extraction from
blood samples was carried out by salting out method
[46].
Genotyping by RFLP
Genotypes for polymorphisms in TNF-α (-238) G/A and
TNF-β (252) A/G were detected using PCR—RFLP
technique. PCR products are generated by using 10 ng
of genomic DNA in 10 μl volume reactions containing
20 mMTris- HCl, 50 mMKCl, 2.0 mM MgCl2, 0.11
mMeachdNTP, 0.3 mM each primer (Table 1), and 0.3
 Oral Maxillofac Surg

Table 2 Demographic parameters of patient and controls and their Statistical analysis
association with risk of oral pre-cancer and OSCC

Demographic character Cases Control p value The significance in this study was evaluated by Chi-square
n = 500 (%) n = 500 (%) test. Odds ratio (OR) was calculated as an estimate of relative
risk of having disease according to the relative frequency of
Male 300 (60) 350 (70) Ref
different genotypes among the cases as well as the controls. P
Female 200 (40) 150 (30) 0.001*
value was considered significant at < 0.05. Odd ratio (OR) is
Age distribution
given with 95% confidence interval (CI).
< 20–40 320 (64) 262 (52) Ref
< 40–70 180 (36) 238 (47) 0.0003*
Habitual risk
Results
Areca nut/pan masala 160 (32) 330 (66) -
Alcohol consumption 80 (16) 20 (04) - In this study, we have evaluated 500 individuals of oral pre-
Smoking 120 (24) 60 (12) - cancer and OSCC (300 males and 200 females) and their age
Tobacco chewing 140 (28) 90 (18) - ranging from 20 to 70 years. Oral pre-cancer and OSCC cases
Type of pre-oral cancer and OSCC were further categorized as oral submucous fibrosis (n = 200),
Leukoplakia 100 (20) - - lichen planus (n = 100), leukoplakia (n = 100), and OSCC (n =
O.S.M.F 200 (40) - - 100). TNM staging and grades of OSCC patients are detailed
Lichen planus 100 (20) - - in Table 2. Alcohol consumption, smoking, masala, and to-
OSCC 100 (20) - - bacco chewing all are found to increase the risk of oral pre-
TNM staging of OSCC - cancer as well as OSCC (Table 2).
Tumor Stage - - Genotype frequencies among the patients with oral disease
I 36 (36) and control are shown in Table 3. Compared to GG genotype,
II 32 (32) the frequency of GA genotype for TNF-α G238A polymor-
III 20 (20) phism was significantly more in cases than in control.
IV 12 (12) Similarly, the frequency of allele A for the same polymor-
Tumor T Status - - phism is significantly higher in cases than in control
≤ T2 62 (62) (Table 3). The genotype GA and allele A both confer high
> T2 38 (38) risk for developing oral diseases (OR = 1.99 and 1.88, respec-
Lymph node - - tively). Similarly, the risk of oral disease was also more in the
N0 74 (74) heterozygote (AG) than the common allele homozygote (AA)
N1 + N2 26 (26) of TNF-β (A252G) polymorphism (OR = 1.483).
Metastasis - - Risk of disease with different genotypes of TNF-α
M0 58 (58) (G238A) and TNF-β (A252G) polymorphisms is further eval-
M1 42 (42) uated in different disease categories of oral diseases including
Cell-differentiated grade - - oral submucous fibrosis, lichen planus, Leukoplakia, and
Grade 1 52 (52) OSCC (Table 4). Compared to the GG genotype, the GA
> Grade 1 48 (48) genotype of TNF-α (G238A) polymorphism has been found
to significantly increase the risk of lichen planus and OSCC
*
Significant value (OR = 2.805 and 5.790, respectively). The risk of lichen
planus and OSCC is also high with allele A compared to allele
U Taq DNApolymerase (Invitrogen, Paisley, UK). PCR G of TNF-α (G238A) polymorphism (Table 4). For the
primer used to genotype the TNF-α and TNF-β poly- TNF-β (A252G) polymorphism compared to A allele, the G
morphisms is detailed in Table 1. PCR products were allele was associated with lower risk of OSMF and
digested with the BamHI enzyme to screen the TNF Leukoplakia (OR = 0.45 and 0.63, respectively) and higher
alpha (-238G/A) polymorphism (Fig. 1) and NcoI en- risk of OSCC (OR = 2.81). Compared to the AA genotype, the
zyme to screen for the TNF-β (252) A/G polymorphism AG genotype of TNF-β (A252G) polymorphism increased the
(Fig. 2) that recognized and cut wild-type, homozygous, risk of lichen planus and OSCC (OR = 2.65 and 3.77, respec-
heterozygous sequences at 37 °C for overnight. The tively). However, the GG genotype is associated with lower
digested PCR products were resolved on a 2% agarose risk of OSMF (OR = 0.42) while compared to AA genotype of
gel and stained with ethidium bromide for visualization TNF-β (A252G) polymorphism (Table 4).
under UV light. Most of the assays were carried out In a stratified analysis, we have correlated the TNF-α
including samples with known genotypes as controls. G238A and TNF-β A252G genotypes with the risk of oral
Oral Maxillofac Surg

Table 3 Distribution of different genotypes and alleles from TNF-α (G238A), TNF-β (A252G) polymorphisms among cases of oral diseases and
controls

TNF-α (G238A) genotypes Cases Controls p value TNF-β(A252G) genotypes Cases Controls p value
n = 500 (%) n = 500 (%) n = 500 (%) n = 50 (%)

GG 380 (76) 432 (86) Ref AA 248 (50) 276 (56) Ref
GA 116 (23) 66 (13) 0.0001* AG 216 (43) 162 (32) 0.004*
1.99 1.48
(1.43–2.78) (1.13–1.93)
AA 04 (01) 02 (01) 0.57 GG 36 (07) 62 (12) 0.06
2.27 0.64
(0.41–12.48) (0.41–1.00)
G 876 (88) 930 (93) Ref A 712 (71) 714 (71) Ref
A 124 (12) 70 (07) 0.0001* G 288 (29) 286 (29) 0.96
1.88 1.01
(1.38–2.55) (0.83–1.22)
*
Significant value

disease among the smokers, masala/tobacco chewers, alco- for TNF-α (G238A) polymorphism. The genotype GA for
hol consumers, and areca nut/pan masala chewers (Table 5). the same TNF-α polymorphism was also found to lower the
Among the smokers, compared to GG genotype, the risk of risk of oral diseases among the areca nut/pan masala
oral disease was lower in the individual with AA genotype chewers. In this group of patients, the AG genotype and G

Table 4 Distribution of different genotypes and allele from TNF-α (G238A), TNF-β (A252G) polymorphisms among the subjects of oral submucous
fibrosis, lichen planus, leukoplakia, OSCC, and controls

Genotypes OSMF p value Lichen planus p value Leukoplakia p value OSCC p value Controls
n (%) n (%) n (%) n (%) n (%)

TNF-α (G238A) genotypes

GG 176 Ref 70 Ref 82 Ref 52 Ref 432
(88) (70) (82) (52) (86)
GA 22 0.52 30 (30) 0.0001* 18 0.27 46 0.0001* 66
(11) 0.81 2.80 (18) 1.43 (46) 5.79 (13)
(0.48–1.36) (1.70–4.62) (0.81–2.54) (3.60–9.30)
AA 02 0.71 00 0.56 00 0.53 02 0.09 02
(01) 2.45 (0.34–17.57) (00) 1.22 (00) 1.04 (02) 8.30 (01)
(0.05–25.84) (0.04–22.05) (1.14–60.25)
G 374 Ref 170 Ref 182 Ref 150 Ref 930
(94) (85) (91) (75) (93)
A 26 0.82 30 0.0003* 18 0.39 50 0.0001* 70
(06)0.92 (15) 2.34 (09) 1.31 (25) 4.42 (07)
(0.57–1.47) (1.48–3.70) (0.76–2.25) (2.96–6.61)
TNF-β (A252G) genotypes
AA 126 Ref 36 Ref 58 Ref 28 Ref 276 (56)
(63) (36) (58) (28)
AG 62 0.38 56 0.0001* 36 0.90 62 0.0001* 162 (32)
(31) 0.83 (56) 2.65 (36) 1.05 (62) 3.77
(0.58–1.20) (1.67–4.20) (0.66–1.67) (2.31–6.13)
GG 12 0.01* 08 0.97 06 0.11 10 0.33 62 (12)
(06) 0.42 (0.22–0.81) (08) 0.98 (06) 0.46 (10) 1.59
(0.43–2.23) (0.19–1.11) (0.73–3.44)
A 338 Ref 122 Ref 158 Ref 94 Ref 714 (71)
(85) (61) (79) (47)
G 62 0.0001* 78 0.004* 42 0.03* 106 0.0001* 286 (29)
(15) 0.45 (39) 1.59 (21) 0.66 (53) 2.81
(0.33–0.62) (1.16–2.18) (0.45–0.95) (2.06–3.83)

*Significant value
 Oral Maxillofac Surg

Table 5 Risk of oral diseases with different genotypes of TNF-α (G238A), TNF-β (A252G) polymorphism among tobacco smokers, chewers, alcohol
consumer, and areca nut/pan masala chewers

Cases Controls p value Odds 95% CI

Ratio

Smokers n (%) n (%)

TNF-α (G238A)
GG 64 (53) 38 (63) Ref 1 1
GA 56 (47) 16 (27) 0.05 2.07 1.04–4.12
AA 00 (00) 06 (10) 0.009* 0.04 0.002–0.83
G 184 (77) 92 (77) Ref 1 1
A 56 (23) 28 (23) 1.00 1.00 0.59–1.67
TNF-β (A252G)
AA 56 (47) 38 (63) Ref 1 1
AG 62 (52) 22 (37) 0.06 1.91 0.77–4.71
GG 02 (01) 00 (00) 0.67 3.40 0.15–73.00
A 146 (61) 72 (60) Ref 1 1
G 94 (39) 48 (40) 0.96 0.96 0.61–1.51
Tobacco chewers n (%) n (%)
Genotypes TNF-α (G238A)
GG 82 (59) 52 (58) Ref 1 1
GA 56 (40) 34 (38) 0.98 1.04 0.60–1.81
AA 02 (01) 04 (04) 0.34 0.31 0.05–1.79
G 220 (79) 138 (77) Ref 1 1
A 60 (21) 42 (23) 0.71 0.89 0.57–1.40
TNF-β (A252G)
AA 50 (36) 48 (53) Ref 1 1
AG 86 (61) 40 (44) 0.01* 2.06 1.19–3.56
GG 04 (03) 02 (01) 0.74 1.92 0.33–10.97
A 168 (60) 114 (63) Ref 1 1
G 112 (40) 66 (37) 0.53 1.15 0.78–1.69
Alcohol consumers n (%) n (%)
Genotypes TNF-α (G238A)
GG 52 (65) 12 (60) Ref 1 1
GA 28 (35) 08 (40) 0.87 0.80 0.29–2.20
AA 00 (00) 00 (00) - - -
G 132 (82) 32 (80) Ref 1 1
A 28 (18) 08 (20) 0.89 0.84 0.35–2.03
TNF-β (A252G)
AA 34 (43) 12 (60) Ref 1 1
AG 44 (55) 08 (40) 0.28 1.94 0.71- 5.28
GG 02 (02) 00 (00) 0.40 1.81 0.08–40.42
A 92 (58) 22 (55) Ref 1 1
G 68 (42) 18 (45) 0.91 0.90 0.44–1.81
Areca nut/pan masala chewers n (%) n (%)

TNF-α (G238A)
GG 82 (51) 108 (33) Ref 1 1
GA 52 (33) 174 (53) 0.0001* 0.39 0.25-0.60
AA 26 (16) 48 (14) 0.29 0.71 0.40–1.24
G 216 (68) 390 (59) Ref 1 1
A 104 (32) 270 (41) 0.01* 0.69 0.52–0.92
Oral Maxillofac Surg

Table 5 (continued)

Cases Controls p value Odds 95% CI

Ratio

TNF-β (A252G)
AA 78 (49) 98 (30) Ref 1 1
AG 54 (34) 182 (55) 0.0001* 0.37 0.24–0.57
GG 28 (17) 50 (15) 0.26 0.70 0.40–1.22
A 210 (66) 378 (57) Ref 1 1
G 110 (34) 282 (43) 0.01* 0.70 0.53–0.92
*
Significant value

allele of TNF-β A252G polymorphism were also associated
with reduced risk of oral diseases. However, the AG geno-
type of TNF-β A252G polymorphism was found to increase
the risk of oral diseases among the masala/tobacco chewers.
Interaction between alcohol consumption and TNF-α
G238A and TNF-β A252G genotypes is not found to mod-
ulate the risk of diseases (Table 5).
Influence of genotypes with the disease outcome/clinical
parameters of OSCC patients is analyzed and documented
on Table 6. None of the genotypes from TNF-α G238A and
TNF-β A252G polymorphisms is found to be associated
with the tumor stage, grade, or metastasis. However, the risk
for developing larger sized tumor (tumor T status) was sig-
nificantly lower with A and G allele for TNF-α G238A and
TNF-β A252G polymorphisms, respectively. The A allele
of TNF-α G238A polymorphism also reduces the risk of
lymph node metastasis among the patients of OSCC
(Table 6).
Since each person can express TNFα as well as TNFβ, it
was important to analyze impact of combined polymorphism
on disease progression. Four different genotype combinations
of TNFα: TNFβ are analyzed, namely GG: AA, GA: AA,
GG:AG, and GA:AG (Table 7). We found that the combine
genotype GG:AA was more frequent in healthy control sub-
jects where as other combined genotypes where more frequent
in oral disease patients. Whenever TNFα G238A hetero ge-
notype is present, it plays dominant role than TNFβ G252A
homo or hetero genotype. Comparison between TNFα GA
and TNFβ AA genotype was not possible as there was no
healthy control available with this genotypes, although trend
shows more diseased type phenotype. More samples are need-
ed for comparison.
Discussion
The present study indicates association of TNF-α G238A and
TNF-β A252G polymorphisms with the risk of oral diseases
including pre-oral cancer lesions and OSCC. Boyle et al. [47]
establish the fact that 71% of pre-oral cancer or cancer salivary
sample shows tumor-specific p53 mutation.
Heterozygotes from both the polymorphism were as-
sociated with increased risk for development of lichen
planus as well as OSCC. However, among the smokers
and areca nut/pan masala chewers, these polymorphisms
seem to reduce the risk of OSCC. Similarly, variant
alleles for both the polymorphisms (A and G, respective-
ly, for TNF-α G238A and TNF-β A252G polymor-
phisms) were found to reduce the risk of developing
larger sized tumor in patients with OSCC. These results
indicate that susceptibility of person to pan masala, areca
nut, and smoking is independent of tumor or pre-lesions
growth.
TNF-α gene is located on chromosome 6p21.231, and its
promoter polymorphisms have been explored as a potential
determinant of disease susceptibility [24, 25]. TNF-α not
only alters disease susceptibility but may also promote the
development and spread of cancer [26–28]. Commonly de-
scribed variants of TNF-α gene polymorphisms consist of G
to A transitions in the promoter region at positions _238 and
_308 [24]. So far, many studies have reported the associa-
tion of TNF-α promoter polymorphism with different can-
cers, including bladder cancer [29], renal cell carcinoma
[30], non-small cell lung carcinoma [31], cervical cancer
[28, 32], and breast carcinoma [33]. Some previous studies
reported that the TNF-α (-238) G/A gene polymorphism
was significantly associated with oral cancer and non-
small cell lung carcinoma (NSCLC) [48, 49]. Our report
confirms these previous findings. Although TNF-α level is
necessary to maintain cell in-setting and to prevent certain
diseases through regulating immune response [50] howev-
er, excessive TNF-α is associated with autoimmune dis-
eases and malignant tumors.
The G allele of the 252A/G polymorphism of TNF-β gene
has repeatedly been correlated with a higher expression of
TNF-β gene both at the level of mRNA and the protein [36].
The G allele has been detected to be significantly increased in
patients with osteosarcoma, as well as breast, colorectal, and
 Oral Maxillofac Surg

Table 6 Association of different

genotypes for TNF-α (G238A) TNF-α (G238A) and TNF-β (A252G) genotypes/alleles
and TNF-β (A252G) polymor-
phism with tumor stage, tumor T Tumor stage III + IV I + II p value Odds 95% CI
Status, lymph node, metastasis, n (%) n (%) ratio
and grade in OSCC patients.
TNF-α (G238A)
GG 41 (54) 11 (46) Ref 1 1
GA + AA 35 (46) 13 (54) 0.64 0.72 0.28–1.81
TNF-β (A252G)
AA 23 (30) 05 (21) Ref 1 1
AG + GG 53 (70) 19 (79) 0.52 0.60 0.20–1.82
Tumor T Status T3 + T4 T1 + T2
n (%) n (%)
TNF-α (G238A)
GG 31 (55) 21 (48) Ref 1 1
GA + AA 25 (45) 23 (52) 0.57 0.73 0.33–1.62
TNF-β (A252G)
AA 14 (25) 14 (32) Ref 1 1
AG + GG 42 (75) 30 (68) 0.59 1.40 0.58–3.36
Lymph node N1 + N2 + N3 N0
n (%) n (%)
TNF-α (G238A)
GG 36 (55) 16 (47) Ref 1 1
GA + AA 30 (45) 18 (53) 0.61 0.74 0.32–1.69
TNF-β (A252G)
AA 21 (32) 07 (21) Ref 1 1
AG + GG 45 (68) 27 (79) 0.34 0.55 0.20–1.48
Metastasis M1 M0
n (%) n (%)
TNF-α (G238A)
GG 13 (46) 39 (54) Ref 1 1
GA + AA 15 (54) 33 (46) 0.63 1.36 0.56–3.27
TNF-β (A252G)
AA 08 (29) 20 (28) Ref 1 1
AG + GG 20 (71) 52 (72) 0.93 0.96 0.36–2.53
Cell-differentiated grade > Grade 1 Grade 1
n (%) n (%)
TNF-α (G238A)
GG 31 (55) 21 (48) Ref 1 1
GA + AA 25 (45) 23 (52) 0.57 0.73 0.33–1.62
TNF-β (A252G)
AA 18 (32) 10 (23) Ref 1 1
AG + GG 38 (68) 34 (77) 0.41 0.62 0.25–1.52
*
Significant value

bladder cancer [32, 51–53]. Similar to that, we have also ob-
served a significantly higher frequency of the G allele of
TNF-β 252A/G polymorphism among the subjects of OSCC
and lichen planus. It seems that TNF-β contributes in certain
malignancies due to its high expression level which is
imparted by allele G of 252A/G polymorphism of TNF-β
gene.
This study not only confirmed previous finding [35] but
also shows the importance of TNF-α and TNF-β in the growth
of pre-oral cancer lesions and OSCC in North Indian popula-
tion. This also points that susceptibility to carcinogen and
growth of pre-cancer or OSCC lesions are differently regulat-
ed by polymorphic genes of TNF-α and TNF-β. This study
indicates that the susceptibility of different pre-oral cancer
Oral Maxillofac Surg

Table 7 Distribution of different combined genotypes of TNF-α (G238A), TNF-β (A252G) polymorphisms among the subjects of oral submucous
fibrosis, lichen planus, leukoplakia, OSCC, and controls

Genotypes OSMF p value Lichen planus p- value Leukoplakia p value OSCC p value Controls
(TNFα: TNFβ) n = 200 n = 100 n = 100 (%) n = 100 n = 500
(%) (%) (%) (%)

GG: AA 104 Ref 27 Ref 44 Ref 11 Ref 290

(52) (27) (44) (11) (56.31)
GA: AA 22 - 9 (09) - 14 - 15 - 0
(11) (14) (15) (0)
GG: AG 62 0.013* 25 0.151 33 0.0012* 36 0.002* 120
(31) (25) (33) (36) (23.30)
GA: AG 0 - 21 0.0001* 03 0.0001* 26 0.0001* 42
(0) (21) (03) (26) (8.10)
*
Significant value

lesions to these polymorphisms is not the same too. We do not
claim that analysis of these polymorphisms would predict the
OSCC of pre-oral lesions as the cohorts in our study are quite
heterogeneous. Further, subcategorization of different pre-oral
cancer lesions is needed to strongly determine the correlation
between TNF-α/ TNF-β polymorphism and predisposition of
North Indian population to the OSCC. The study confirmed
that TNFα genotype has stronger impact compare to TNF.β
on oral disease progression.
In conclusion, two common functional polymorphisms in
TNF-αand TNF-β genes seem to increase the risk of develop-
ment of OSCC as well as pre-oral cancer lesions in North
Indian population. The detection of such genetic predisposi-
tion may be of considerable importance for safeguarding the
life and health status of certain individuals at risk in the gen-
eral population through early prevention measures. Future
studies with larger sample sizes and validation at protein level
are needed. .Investigation to find correlation among different
polymorphism is required to understand the interplay of
TNFα and TNFβ.
Results of association between TNF-α, TNF-β polymor-
phisms, and OSCC as well as pre-oral cancer lesions are con-
tradictory. However, due to small sample size in different
disease group, our findings are rather suggestive than conclu-
sive and warn larger studies.
Availability of data and material All the data and material are presented
in this article.
Funding This study was supported by Council of Science & Technology
U.P. (UPCST), India.
Compliance with ethical standards
Conflict of interest Authors declare that they have no conflict of
interest.
Code availability Not applicable.
Ethics approval Ethical clearance was obtained from ethical committee
of King George’s Medical University. The study was performed in ac-
cordance with the Declaration of Helsinki.
Consent to participate An informed written consent was obtained from
all subjects involved in this study.
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