Mutation Research 729 (2012) 2434

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Mutation Research/Fundamental and Molecular

Mechanisms of Mutagenesis
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Associated risk of XRCC1 and XPD cross talk and life style factors in progression
of head and neck cancer in north Indian population
Anil Kumar a , Mohan Chand Pant b , Hirdya Shanker Singh c , Shashi Khandelwal a,
a
b
c

CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, India

C.S.M.Medical University, Lucknow, India
Ch. Charan Singh University, Meerut, India

a r t i c l e

i n f o

Article history:
Received 14 June 2011
Received in revised form 4 September 2011
Accepted 8 September 2011
Available online 16 September 2011
Keywords:
XRCC1
XPD
Polymorphism
Head and neck cancer

a b s t r a c t
Effective DNA repair machinery ensures maintenance of genomic integrity. Environmental insults, ageing
and replication errors necessitate the need for proper DNA repair systems. Any alteration in DNA repair
efcacy would play a dominant role in progression of squamous cell carcinoma of head and neck (SCCHN).
Genotypes of XRCC1 geneArg194Trp, Arg280His, Arg399Gln and XPD Lys751Gln, by PCR-RFLP were
studied in 278 SCCHN patients and an equal number of matched healthy controls residing in north India.
In XRCC1 polymorphisms, Arg194Trp and Arg399Gln variants showed a reduced risk, whereas, XPD
Lys751Gln variants exhibited 2-fold increase in SCCHN risk. With XRCC1-Arg280His variants, there was
no association with SCCHN risk. Arg399Gln of XRCC1 appears to have a protective role in people those
consume alcohol, while XPD Lys751Gln variants indicated 2-fold increased risk of SCCHN in all the
co-variate groups.
Comparison of genegene interaction among XRCC1 Arg280His and XPD Lys751Gln suggested
enhanced risk of SCCHN by 2.3-fold in group one and 6.1-fold in group two. In dichotomized groups
of this combination, the risk was 2.4 times. Haplotype analysis revealed the frequency of CGGG
and CAGG to be signicantly associated with an increased risk of SCCHN. On the contrary, TGAA
signicantly diminished the risk. CART analysis results showed that the terminal node that contains
homozygous mutants of XPD Lys751Gln and XRCC1 Arg194Trp, wild type of XRCC1 Arg399Gln and
homozygous mutant of XRCC1 Arg280His, represent the highest risk group.
Our results demonstrate high degree of genegene interaction involving DNA repair genes of NER
and BER pathways, namely XRCC1 and XPD. This study amply demonstrates positive association of XPD
Arg751Gln polymorphism with an increased risk of SCCHN. Further, XRCC1 Arg280His variant though dormant individually, may also contribute to the development of cancer in combination with XPD Arg751Gln.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Squamous cell carcinoma of head and neck (SCCHN) is the sixth
most common cancer in the world [1] and in India it accounts for
about 21% of the total cancer cases. The major head and neck sites
include oral cavity, tongue, larynx and pharynx. Globally, SCCHN is
on the increase in young adults probably because there has been
a rise in tobacco smoking, chewing and alcohol consumption. In
India, smoking is common in urban, whereas chewing tobacco is
more prevalent in rural population (National Sample Survey Organization, 1998). Since SCCHN is also reported in cases with no
history of smoking, chewing tobacco or alcoholism, the possibility of genetic variability may exist. The occurrence of head and

Corresponding author. Tel.: +91 522 2627586x316; fax: +91 522 2628471.
E-mail address: skhandelwal.itrc@gmail.com (S. Khandelwal).
0027-5107/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrfmmm.2011.09.001

neck cancer in patients below 30 years further strengthens this

hypothesis.
Multiple genetic and environmental factors are involved
in pathological processes underlying SCCHN. Individuals differ
widely in their capacity to repair DNA damage on exposure to
exogenous sources like tobacco smoke, smokeless tobacco, alcohol, ultraviolet radiation and to endogenous reactions involving
oxidants [2].
Cancer of oral cavity, especially related to tongue and buccal mucosa is quite widespread in north India, particularly in
Uttar Pradesh. High consumption of tobacco in the form of surti,
pan masala, gutkha and alcohol could possibly attribute to cancer incidences at these sites [3]. These carcinogenic products
generate free radicals and the sustained oxidative burden may
induce various types of DNA damage [4,5], leading to apoptosis or to un-regulated (proliferative) cell growth resulting in
carcinoma.

A. Kumar et al. / Mutation Research 729 (2012) 2434

The DNA repair system, which is fundamental to maintenance

of genomic integrity is constantly challenged by replication errors,
environmental insults and cumulative effects of ageing. Hence,
their altered/compromised efciency of could increase the cancer
risk [6]. Polymorphism in potentially functional variants of DNA
repair genes, in regions coding for integrating domain of repair
enzymes, may alter the activity of its protein product [7]. Single
nucleotide polymorphism (SNP) in DNA repair genes is very common and recent studies show a signicant association of SNP with
cancer.
DNA repair pathway involves direct reversal pathway, excision
repair pathway and post-replication/bypass pathway. The excision
repair pathway includes base excision repair (BER), nucleotide excision repair (NER) and mismatch repair. In BER pathway, XRCC1
(X-ray repair cross-complementing group) located on chromosome
19q13.2, consisting of 17 exons encoding 633 amino acids, is one
of the key members of this pathway. It coordinates as a scaffold
protein with DNA ligase III [8,9] and DNA polymerase- [10] in
detection and protection of DNA strand breaks and subsequent
targeting of BER complex to the damaged site. Three common polymorphisms that lead to amino acid substitutions in XRCC1 gene
have been described in codon 194 (exon 6, base C to T, amino acid
Arg to Trp), codon 280 (exon 9, base G to A, amino acid Arg to His)
and codon 399 (exon 10, base G to A, amino acid Arg to Gln).
The repair of bulky lesions such as pyrimidine dimers, larger
chemical adducts and cross-links are carried out in NER pathway.
The genes involved in this pathway are XPC, XPD, XPF and DNA
polymerase [11]. The gene ERCC2/XPD, which is co-localized on
chromosome19q13.2-q13.3 along with XRCC1 is a well characterized helicase that plays a crucial role in the unwinding of DNA. It
removes photoproducts from UV radiation and bulky adducts from
a number of chemicals, cross-links and oxidative DNA adducts, with
the help of twenty proteins and several multi-protein complexes
[12]. XPD, a highly polymorphic gene has been extensively studied in relation to cancer risk and codon 751 (exon 23, base A to C,
amino acid Lys to Gln) and has been considered very crucial, since
studies have shown its association in causing alterations in DNA
repair efciency. Subjects with 751 Gln allele are reported to have
sub-optimal DNA repair capacity for benzo(a)pyrene adducts and
UVDNA damage [13,14].
Polymorphism of DNA repair genes XRCC1-Arg194Trp, XRCC1Arg280His, XRCC1-Arg399Gln and XPD-Lys751Gln has been found
to be very critical. There is evidence that polymorphic variants
are functionally signicant, since studies have demonstrated their
association with alterations in DNA repair efciency [15,16]. In
addition to the role of single gene interaction with DNA repair
capacity, multiple genegene interactions have gained prominence. Additive and/or synergistic impacts have been illustrated
in a number of cancer incidences, such as oral, lung, gallbladder
and oesophageal [1719].
In the present study geneenvironment and genegene association of XRCC1 Arg194Trp, XRCC1 Arg280His, XRCC1 Arg399Gln and
XPD Lys751Gln were analyzed by multiple modes for ascertaining
interactive factors responsible for the risk of SCCHN.
2. Materials and methods
2.1. Subjects
The case-control study involved 278 male SCCHN patients and 278 geographically and racially matched healthy male controls, including visitors of Radiotherapy
Department of C.S.M. Medical University, Lucknow, but not relatives of SCCHN
patients. None of the control individuals had a personal or a family history of
malignancy. All controls were matched for sex, age and habits. The study subjects belonging to Indo-European linguistic group, resided in Lucknow city or
neighbouring places in north India [20]. The investigation was initiated, following
approval of the respective Human Ethical Committees of C.S.M. Medical University, Lucknow and of Indian Institute of Toxicology Research, Lucknow. The protocol

25

conrmed to provisions of declaration of Helsinki in 1995. Informed consent was

obtained from the study subjects for inclusion in the study, and subject anonymity
was ensured prior to collection of blood samples. All study subjects completed a
questionnaire covering medical, residential and occupational history. Information
concerning dietary habits, family history disease, smoking, tobacco chewing and
alcohol consumption was also obtained from the questionnaire. The test patients
had pathologically conrmed squamous cell carcinoma of head and neck.

2.2. Genotyping
One millilitres blood was collected in sodium citrate (3.4%, pH 7.6) coated vials
from all the study subjects. Genomic DNA was isolated from blood samples using
QIAamp DNA mini kit (Qiagen Inc.,USA) following the manufacturers protocol.
The quantity and purity of DNA samples were measured by Nanodrop
Spectrophotometer (ND 1000V.3.3) and stored at 20 C till further use. Gene polymorphism of XRCC1 Arg194Trp, XRCC1 Arg280His, XRCC1 Arg399Gln and XPD
Lys751Gln was studied using PCR-RFLP.
The nal volume of each PCR reaction mixture was 25 l and each reaction tube
contained 12.5 l Dream Taq Master Mix (Fermentas Life Sciences), 0.6 l of each
primer (10 pmole), 50 ng genomic DNA and 9.3 l of nuclease free water. All PCR
reactions were performed on a peltier based thermal cycler (G-storm, Labmate India)
and amplicons were visualized by electrophoresis on 1.5% agarose gel containing
0.5 g/ml ethidium bromide. The RFLP digestion was carried out at 37 C overnight
and resolved on 2.5% agarose gel to identify the band pattern. The sequence of
PCR amplied bands was conrmed commercially by a Genomics Service Provider
(Xcelris Labs Ltd, Ahmedabad, India).
XRCC1-Arg194Trp polymorphism was determined by using the following
primers: sense 5 -GTT CCG TGT GAA GGA GGA GGA-3 ; antisense 5 -CGA GTC TAG
GTC TCA ACC CTA CTC ACT-3 . The cycling conditions were: initial denaturation at
94 C for 5 min, followed by 34 cycles of denaturation for 50 s, annealing at 56 C for
30 s and elongation at 72 C for 50 s. The nal extension was at 72 C for 7 min.
Ten micro litres of 138 bp PCR product was digested with 10 units of PvuII (Fermentas Life Sciences). The Trp/Trp genotype gave the digested products of 75 and
63 bp fragments, Arg/Trp-138, 75 and 63 bp and 138 bp undigested showed Arg/Arg
(Fig. 1A and B).
XRCC1-Arg280His polymorphism was determined by using the following
primers: sense 5 -CCA GTG GGT GCT AAC CTA ATC-3 ; antisense 5 -CAC TCA GCA
CCA GTA CCA CA-3 . The cycling conditions were: initial denaturation at 94 C for
5 min, followed by 35 cycles of denaturation for 50 s, annealing at 58 C for 30 s and
elongation at 72 C for 50 s. The nal extension was at 72 C for 7 min.
Ten micro litres of 201 bp PCR product was digested with 10 units of RsaI (Fermentas Life Sciences). Digested product of XRCCI codon 280 Arg/Arg, Arg/His and
His/His genotype had band patterns of 145 and 56, 201, 145 and 56 and 201 bp,
respectively (Fig. 2A and B).
XRCC1-Arg399Gln polymorphism was determined by using the following
primers: sense 5 -TCT CCC TTG GTC TCC AAC CT-3 ; antisense 5 -AGT AGT CTG CTG
GCT GGC TCT GG-3 . The cycling conditions were: initial denaturation at 94 C for
5 min, followed by 35 cycles of denaturation for 50 s, annealing at 60 C for 30 s,
elongation at 72 C for 50 s followed by nal extension step at 72 C for 7 min.
Ten micro litres of 402 bp PCR product was digested with 10 units of MspI (Fermentas Life Sciences). Digested products of XRCC1 codon 399 Arg/Arg, Arg/Gln and
Gln/Gln genotype had band patterns of 269 and 133, 402, 269 and 133 and 402 bp,
respectively (Fig. 3A and B).
XPD-Lys751Gln polymorphism was determined by using the following primers:
sense 5 -CCC CCT CTC CCT TTC CTC TG-3 ; antisense 5 -AAC GGC CAG GCA AGA C-3 .
The cycling conditions were: initial denaturation at 94 C for 5 min, followed by 35
cycles of denaturation for 50 s, annealing at 57 C for 30 s and elongation at 72 C for
50 s. The nal extension was at 72 C for 7 min.
Ten micro litres of 184 bp PCR product was digested with 10 units of MboII
(Fermentas Life Sciences). The Gln/Gln genotype was cut into 112 and 72 bp, Lys/Gln
into 184, 112 and 72 bp fragments and Lys/Lys remained as the undigested 184 bp
(Fig. 4A and B).

2.3. Statistical analysis

Considering the minor allele frequency of XRCC1 Arg194Trp, XRCC1 Arg280His,
XRCC1 Arg399Gln and XPD Lys751Gln and sample size of the study, the data was
analyzed using QUANTO 1.1. Our case control study (278 SCCHN cases and 278
controls) was adequate to give a power of 80%. Genotype frequencies of XRCC1
Arg194Trp, XRCC1 Arg280His, XRCC1 Arg399Gln and XPD Lys751Gln among controls were determined for Hardy Weinberg equilibrium (HWE) using standard Chi
square test. The haplotype analysis (haplotype frequency estimation and pair-wise
linkage disequilibrium between the SNPs were carried out using Haploview software (www.broad.mit.edu/mpg/haploview). Odds ratios (ORs) and 95% condence
intervals (95%CI) were estimated to ascertain association of individual genotype
with SCCHN risk. Multiple logistic regression analysis was performed to calculate
adjusted ORs for subsequent analysis of potential risk factors like age, smoking,
tobacco chewing and alcohol consumption. All statistical analyses were performed
with SPSS software package (version 12.0 for windows; SPSS Chicago, IL). The

26

A. Kumar et al. / Mutation Research 729 (2012) 2434

Fig. 1. (A) Representative PCR-RFLP analysis of XRCC1 Arg194Trp polymorphism. M = 50 bp DNA ladder lane 1,3,4,5,6-Arg/Arg homozygous wild genotype, lane 2-Arg/Trp
heterozygous genotype, lane 7-Trp/Trp homozygous mutant genotype. (B) DNA sequence histogram revealing indicated sequence changes by arrow. In 2nd histogram, red
line for thymine and blue line for cytosine observed in codon 194 site (marked as N), indicating heterozygous genotype. (For interpretation of the references to color in this
gure legend, the reader is referred to the web version of the article.)

false-positive report probability for statistically signicant results was also calculated [21].
Classication and regression tree (CART) analysis was carried out employing
CART software (version 6.0, Salford Systems, San Diego, California) [22,23] to explore
higher order of genegene interactions in modulating head and neck cancer risk. In
CART, a tree based model was created by recursive partitioning of data, to identify
effect modications between variables that are less visible by traditional logistic
regression. The algorithm splitted the study samples into a number of homogenous subgroups based on risk factors and the nal model was a tree structure with
terminal nodes, dening a range of risk subgroups.

3. Results
The median age of SCCHN cases was 52 years and 50 years for
the control group. The mean age difference was not statistically
signicant in SCCHN and controls. Out of 278 SCCHN cases, 155
were of oral cancer, 72 of larynx and 51 of pharynx.
The distribution of age, gender, smoking, tobacco chewing and
alcohol consumption among controls and cases with SCCHN are

shown in Table 1. The genotypic distribution in two DNA repair

genes for both cases and controls is shown in Table 2. The genotypic frequencies of XRCC1 Arg194Trp, XRCC1 Arg280His, XRCC1
Arg399Gln and XPD Lys751Gln in controls were in agreement with
Hardy Weinberg equilibrium (p > 0.05) and their distribution is in
agreement with those found in the north Indian population [24].
The lowered frequency of wild type (XRCC1, codon 194 and 399)
than that of heterozygous genotype agrees well with the report of
Kiran et al. [25]. The frequencies of minor allele of XRCC1 194Trp,
XRCC1 280His, XRCC1 399Gln and XPD 751Gln among controls
were 0.33, 0.28, 0.40 and 0.34, respectively.
Odds ratio was estimated to show the association between
genetic variants of XRCC1 and XPD genes and the risk of SCCHN
has been depicted in Table 2. There was a signicant variation
between distribution of polymorphic variants between cases and
controls.
In XRCC1 Arg194Trp polymorphism, genotype Arg/Trp was
associated with 29% reduced risk of SCCHN with OR of 0.71

Fig. 2. (A) Representative PCR-RFLP analysis of XRCC1 Arg280His polymorphism. M = 50 bp DNA ladder lane 1-His/His homozygyous mutant genotype, lane 28-Arg/Arg
homozygous wild genotype, lane 9-Arg/His heterozygous genotype. (B) DNA sequence histogram revealing the indicated sequence changes by arrow. In 2nd histogram, black
line for guanine and green line for adenine observed in codon 280 site (marked as N), indicating heterozygous genotype. (For interpretation of the references to color in this
gure legend, the reader is referred to the web version of the article.)

A. Kumar et al. / Mutation Research 729 (2012) 2434

27

Fig. 3. (A) Representative PCR-RFLP analysis of XRCC1 Arg399Gln polymorphism. M = 50bp DNA ladder lane 1,3,4,5-Arg/Gln heterozygous genotype, lane 2-Gln/Gln homozygous mutant genotype, lane 6-Arg/Arg homozygous wild genotype. (B) DNA sequence histogram revealing the indicated sequence changes (arrow). In 2nd histogram, green
line for adenine and black line for guanine observed in codon 399 site (marked as N), indicating the heterozygous genotype. (For interpretation of the references to color in
this gure legend, the reader is referred to the web version of the article.)

Table 1
Demographic variables, risk estimates and distribution of SCCHN cases by tumor
sites.
Variable

Control n (%)

Cases n (%)

Subjects
Age (mean)
Range
Smokers
Non-smokers
Tobacco Chewers
Non-tobacco Chewers
Alcohol users
Non-alcohol users

278
50
(2570)
157 (57)
121 (44)
144 (52)
134(48)
124 (45)
154 (55)

278
52
(2575)
222 (80)
56 (20)
153 (55)
125 (45)
134 (48)
144 (52)

Site of tumor
Oral Cavity
Larynx
Pharynx

155
72
51

(95%CI = 0.501.07), Trp/Trp appeared to reduce the risk with

OR of 0.74 (95%CI = 0.752.36), but remained statistically nonsignicant. On comparing the variant containing genotypes
(Arg/Arg + Trp/Trp) with homozygous wild-type genotype, OR of

0.72 (95%CI = 0.511.00) indicated considerable protection (28%)

by XRCC1 Arg194Trp polymorphism, which was conrmed by trend
test (p = 0.028).
XRCC1 Arg280His polymorphism on the other hand, did not
show any association with SCCHN and the nding was also conrmed by trend test (p = 0.12).
In XRCC1 Arg399Gln polymorphism, genotypes Arg/Gln and
Gln/Gln were associated with reduced risk of 38% and 50%
in SCCHN with OR of 0.62 (95%CI = 0.430.88) and OR of 0.50
(95%CI = 0.309.35), respectively. On combining the two variant
genotypes (Arg/Gln + Gln/Gln) with homozygous wild type, an OR
of 0.60 (95%CI = 0.430.84) indicated signicant protection, which
was further established by trend test (p = 0.003).
The variants of XPD Lys751Gln gene were associated with an
increased risk of SCCHN with OR value 1.59 (95%CI = 1.012.31)
and OR of 2.19 (95%CI = 1.363.55), respectively as shown in
Table 2. When both the variants (Arg/Gln + Gln/Gln) were compared with homozygous wild-type genotype, a signicant OR of
1.75 (95%CI = 1.242.47) was observed and was conrmed by trend
test (p = 0.001).
The above data indicate that except for XPD, none of the other
three SNPs, individually, contribute to cancer risk.

Fig. 4. (A) Representative PCR-RFLP analysis of XPD Lys751Gln polymorphism. M = 50 bp DNA ladder lane 1,2,5-Gln/Gln homozygous mutant genotype, lane 6-Lys/Gln
heterozygous genotype, lane 3,4-Lys/Lys homozygous wild genotype. (B) DNA sequence histogram revealing the indicated sequence changes (arrow). In 2nd histogram, blue
line for cytosine and green line for adenine observed in codon 751 site (marked as N), indicating the heterozygous genotype. (For interpretation of the references to color in
this gure legend, the reader is referred to the web version of the article.)

28

A. Kumar et al. / Mutation Research 729 (2012) 2434

Table 2
Distribution of XRCC1 and XPD genotypes among Controls (n = 278) and SCCHN Cases (n = 278).
Control

Control n (%)

Case n (%)

OR

95%CI

p-value

XRCC1 codon 194

Arg/Arg
Arg/Trp
Trp/Trp
Arg/Trp + Trp/Trp
ptrend

121
131
26
157

144
111
23
134

1.00a
0.71
0.74
0.72

(0.501.07)
(0.752.36)
(0.511.00)

0.04*
0.35
0.03*
0.028*

XRCC1 codon 280

Arg/Arg
Arg/His
His/His
Arg/His + His/His
ptrend

142
116
20
136

129
123
26
149

1.00a
1.17
1.43
1.21

(0.821.65)
(0.772.67)
(0.861.68)

0.43
0.27
0.31
0.116

XRCC1 codon 399

Arg/Arg
Arg/Gln
Gln/Gln
Arg/Gln + Gln/Gln
ptrend

98
144
36
180

128
124
26
150

1.00a
0.66
0.55
0.64

(0.460.94)
(0.310.97)
(0.450.90)

0.02*
0.04*
0.01*
0.003*

XPD codon 751

Lys/Lys
Lys/Gln
Gln/Gln
Lys/Gln + Gln/Gln
ptrend

129
110
39
149

92
125
61
186

1.00a
1.59
2.19
1.75

(1.012.31)
(1.363.55)
(1.242.47)

0.013*
0.002*
0.002*
0.002*

a
*

Reference group; OR = crude odds ratio; 95%CI: 95% condence interval.

p < 0.05 is considered statistically signicant.

3.1. Geneenvironment interaction: inuence of age, smoking,

tobacco chewing and alcohol consumption on DNA repair gene
polymorphism

Data analysis of the geneenvironment interaction showed that

the various life style factors exhibited mild inuence on XPD genotypes.

No signicant inuence of age was seen between the ORs for

SCCHN cases vs. healthy controls, when adjusted for smoking,
tobacco chewing and alcohol.
Stratied analysis to estimate interaction between the three
genotypes of XRCC1 i.e. Arg194Trp, Arg280His, Arg399Gln and XPD
Lys751Gln and smoking, smokeless tobacco chewing and alcohol
drinking was performed. The XRCC1 Arg194Trp and Arg280His
genotypes of XRCC1 displayed no statistical signicance in all the
covariates monitored in our study (Tables 3 and 4).
Arg399Gln of XRCC1 appears to be associated with reduced risk
of SCCHN (48%) in the case of alcohol consumption (Table 5) with
an OR of 0.52 (95%CI = 0.300.88, p = 0.01).
Table 6 summarizes the effect of life style factors on various
genotypes of XPD Lys751Gln gene. The frequency of variant genotypes of XPD Lys751Gln was much higher in SCCHN as compared to
controls. Lys751Gln of XPD seems to be associated with increased
risk (2-fold) in all the covariant groups.

3.2. Genegene interaction

To understand the combined effect of all four SNPs, within the
same chromosome (Chr 19), on development of SCCHN, we analyzed genegene interaction of two SNPs, Haplotype analysis of
three polymorphic variants of XRCC1 (Arg194Trp, Arg280His and
Arg399Gln) and also in combination with XPD Arg751Gln variant
and CART analysis for higher order of genegene interactions.
3.2.1. Association of DNA repair genes polymorphisms and
SCCHN risk
To determine interaction of DNA repair genes polymorphism
and the risk of developing SCCHN, ORs were analyzed by SPSS
(Table 7). XRCC1 Arg194Trp + Arg399Gln with an OR of 0.4
(95%CI = 0.290.68, p = 0.001) displayed a reduced SCCHN risk in
group one. In group two, although interaction of the two variants of XRCC1 showed further reduced cancer risk (OR = 0.14,

Table 3
Interaction of XRCC1 Arg194Trp variants with smoking, tobacco chewing, alcohol consumption and SCCHN risk.
Genotype

Non-habituate

Habituate
a

Control (n)

Cases (n)

OR

95%CI

p-value

Control (n)

Cases (n)

ORa

95%Cl

p-value

Smokers
Arg/Arg
Arg/Trp + Trp/Trp

49
72

29
27

1.0b
0.63

(0.341.90)

0.19

72
85

115
10

1.0b
0.79

(0.521.19)

0.30

Tobacco chewers
Arg/Arg
Arg/Trp + Trp/Trp

65
69

68
57

1.0b
0.79

(0.772.04)

0.38

56
88

76
77

1.0b
0.65

(0.411.02)

0.06

Alcoholics
Arg/Arg
Arg/Trp + Trp/Trp

68
86

73
71

1.0b
0.77

(0.491.21)

0.30

53
71

71
53

1.0b
0.66

(0.411.08)

0.11

*p < 0.05 is considered statistically signicant.

a
OR = age adjusted odds; 95%CI: 95% condence interval.
b
Reference group.

A. Kumar et al. / Mutation Research 729 (2012) 2434

29

Table 4
Interaction of XRCC1 Arg280His variants with smoking, tobacco-chewing, alcohol consumption and SCCHN risk.
Genotype

Non-habituate

Habituate

Control (n)

Cases (n)

ORa

95%CI

p-value

Control (n)

ORa

Smokers
Arg/Arg
Arg/His + His/His

95%Cl

p-value

65
51

27
26

1.0b
1.25

(0.662.34)

0.52

77
80

102
120

1.0b
1.13

Tobaccco chewers
Arg/Arg
Arg/His + His/His

(0.751.70)

0.68

76
58

64
61

1.0b
1.25

(0.772.04)

0.39

66
80

64
88

1.0b
1.16

(0.741.84)

0.56

Alcoholics
Arg/Arg
Arg/His + His/His

91
63

82
64

1.0b
1.13

0.711.78)

0.64

51
73

47
85

1.0b
1.26

(0.762.09)

0.37

Cases (n)

ORa

95%Cl

p-value

Cases (n)

*p < 0.05 is considered statistically signicant.

a
OR = age adjusted odds; 95%CI: 95% condence interval.
b
Reference group.
Table 5
Interaction of XRCC1 Arg399Gln variants with smoking, tobacco-chewing, alcohol consumption and SCCHN risk.
Genotype

Non-habituate
Control (n)

Habituate
Cases (n)

ORa

95%CI

p-value

Control (n)

Smokers
Arg/Arg
Arg/Arg + Gln/Gln

43
78

28
28

1.0b
0.55

(0.291.04)

0.07

55
102

100
122

1.0b
0.67

(0.380.90)

0.06

Tobacco Chewers
Arg/Arg
Arg/Arg + Gln/Gln

40
101

48
81

1.0b
0.67

(0.401.12)

0.15

58
79

80
73

1.0b
0.67

(0.421.06)

0.10

Alcoholics
Arg/Arg
Arg/Arg + Gln/Gln

55
99

60
84

1.0b
0.78

(0.481.24)

0.34

43
81

68
66

1.0b
0.52

(0.300.88)

0.01*

*
a
b

p < 0.05 is considered statistically signicant.

OR = age adjusted odds; 95%CI: 95% condence interval.
Reference group.

95%CI = 0.020.96), the combination remained statistically nonsignicant. The XRCC1 Arg280His and XPD Lys751Gln showed a
signicant increased risk of 2.3 folds (OR = 2.31, 95%CI = 1.363.14,
p = 0.002) in group one and 6.1-fold increased risk in group two
(OR = 6.13, 95%CI = 1.0845.05, p = 0.028). In dichotomized group
of this combination, the risk remained 2.4 folds (OR = 2.42,
95%CI = 1.434.10, p = 0.001).
3.2.2. Haplotype analysis
Eight combinations of haplotypes constructed by maximumlikelihood method for XRCC1 gene alone, the haplotypes
TGA (OR = 0.55, 95%CI = 0.340.89) and TAA (OR = 0.51,
95%CI = 0.270.97) were related to reduced risk of head and neck
cancer (Table 8A). In another set of fteen combinations of the

haplotypes of XRCC1 Arg194Trp, Arg280His, Arg399Gln and XPD,

the frequency of CGGG (OR = 1.77, 95%CI = 1.142.74) and
CAGG (OR = 1.83, 95%CI = 1.041.66) haplotypes were signicantly associated with an increased risk of SCCHN, while TGAA
(OR = 0.50, 95%CI = 0.431.01) signicantly reduced the SCCHN risk
(Table 8B).
3.2.3. CART analysis
CART analysis was applied to explore high order genegene
interactions and the tree structure so generated, included all investigated genetic variants of the DNA repair pathway. The nal tree
structure contained eleven terminal nodes representing a range of
low to high-risk subgroups by different combinations of genotypes
(Fig. 5).

Table 6
Interaction of XPD Lys751Gln genotype with smoking, tobacco-chewing, alcohol consumption and SCCHN risk.
Genotype

Non-habituate

Habituate
a

Cases (n)

ORa

95%Cl

p-value

76
81

69
153

1.0b
2.08

(1.363.17)

0.001*

0.26

68
81

44
109

1.0b
2.20

(1.383.57)

0.001*

0.77

58
103

41
176

1.0b
1.99

(1.23.31)

Control (n)

Cases (n)

OR

95%CI

p-value

Smokers
Lys/Lys
Lys/Gln + Gln/Gln

53
68

23
33

1.0b
1.16

(0.612.19)

0.75

Tobacco chewers
Lys/Lys
Lys/Gln + Gln/Gln

61
73

48
77

1.0b
1.34

(0.822.20)

Alcoholics
Lys/Lys
Lys/Gln + Gln/Gln

71
77

51
39

1.0b
1.56

(0.982.48)

*
a
b

p < 0.05 is considered statistically signicant.

OR = age adjusted odds; 95%CI: 95% condence interval
Reference group.

Control (n)

0.01*

30

A. Kumar et al. / Mutation Research 729 (2012) 2434

Table 7
Genegene interaction and head and neck cancer risk estimates.
Genotype

Cases (n = 278)

ORa

95%CI

p-value

72
56
2

1.0b
0.80

(0.491.32)

0.45

0.98

72
58

1.0b
0.83

(0.511.36)

0.53

62
42
1

1.0b
0.40
0.14

(0.290.68)
(0.020.96)

0.001*
0.08
0.40

62
43

1.0b
1.32

(0.772.25)

0.34

53
60
2

41
63
4

1.0b
1.36
2.58

(0.792.32)
(0.3821.53)

0.28
0.40

53
61

41
69

1.0b
1.46

(0.862.49)

0.18

50
48
2

1.0b
0.81

(0.481.36)

0.50

0.045*

50
50

1.0b
0.75

(0.441.28)

0.34

70
50
2

40
66
7

1.0b
2.31
6.13

(1.363.14)
(1.0845.05)

0.002*
0.028*

70
52

40
72

1.0b
2.42

(1.434.10)

0.001*

40
58
7

41
65
6

1.0b
1.09
0.84

(0.631.91)
(0.272.60)

0.78
1.00
0.85

40
65

41
71

1.0b
1.07

(0.621.84)

0.89

Control (n = 278)

XRCC1 Arg194Trp and XRCC1 Arg280His

0
63
1
61
2
0
ptrend
Dichotomized groups
63
0
12
61
XRCC1 Arg194Trp and XRCC1 Arg399Gln
0
44
1
75
2
5
ptrend
Dichotomized groups
0
44
12
80
XRCC1 Arg194Trp and XPD Lys751Gln
0
1
2
ptrend
Dichotomized groups
0
12

XRCC1 Arg280His and XRCC1 Arg399Gln

0
51
1
68
0
2
ptrend
Dichotomized groups
0
51
12
68
XRCC1 Arg280His and XPD Lys751Gln
0
1
2
ptrend
Dichotomized groups0
12
XRCC1 Arg399Gln and XPD Lys751Gln
0
1
2
ptrend
Dichotomized groups
0
12
*
a
b

p < 0.05 is considered statistically signicant.

OR = adjusted for age, smoking, tobacco chewing and alcohol; 95%CI: 95% condence interval.
Reference group.

The rst split was XPD Lys751Gln, separating individuals

into AA and AC + CC genotypes. Subsequent splits were XRCC1
Arg399Gln, XRCC1 Arg194Trp and XRCC1 Arg280His. To calculate
ORs as dened by 11 terminal nodes, we chose terminal node 1

individuals with homozygous wild type of XPD Lys751Gln and

homozygous mutants of XRCC1 Arg399Gln and XRCC1 Arg194Trp,
as the reference group. This node exhibited low risk of SCCHN with
24% case rate. The ORs of terminal nodes ranged from 1.79 (node

Table 8A
Frequencies of XRCC1 Arg194Trp(C > T)-XRCC1 Arg280His(G > A)-XRCC1 Arg399Gln(G > A) haplotype.
Haplotype

Control (n)

Cases (n)

ORa

95%CI

p-value

CGG
CGA
CAG
TGG
TGA
TAG
CAA
TAA

156
111
77
78
55
25
26
28

175
99
98
72
34
34
27
16

1.0b
0.8
1.14
0.82
0.55
1.21
0.93
0.51

(0.561.12)
(0.791.64)
(0.561.21)
(0.340.89)
(0.702.11)
(0.521.65)
(0.270.97)

0.22
0.51
0.33
0.01*
0.57
0.88
0.05*

*
a
b

p < 0.05 is considered statistically signicant.

OR = adjusted for age, smoking, tobacco chewing and alcohol; 95%CI: 95% condence interval.
Reference group.

A. Kumar et al. / Mutation Research 729 (2012) 2434

31

Table 8B
Frequencies of XRCC1 Arg194Trp(C > T)-XRCC1 Arg280His(G > A)-XRCC1 Arg399Gln(G > A)-XPD Lys751Gln(A > C) haplotype.
Haplotype

Control (n)

Case (n)

ORa

95%CI

p-value

CGGA
CGGG
CGAA
TGGA
CAGA
CGAG
CAGG
TGAA
TGGG
TGAG
TAAA
TAGA
CAAG
TAGG
CAAA

97
58
72
61
48
38
29
38
18
18
24
15
14
10
14

86
91
54
49
50
42
47
17
23
19
11
18
18
16
10

1.0b
1.77
0.85
0.91
1.17
1.25
1.83
0.50
1.44
1.19
0.52
1.35
1.45
1.80
0.81

(1.142.74)
(0.521.18)
(0.731.23)
(0.851.39)
(0.861.45)
(1.041.66)
(0.431.01)
(0.871.63)
(0.771.55)
(0.401.12)
(0.821.64)
(0.851.69)
(0.931.84)
(0.541.46)

0.01*
0.47
0.68
0.52
0.41
0.03*
0.03*
0.29
0.63
0.09
0.42
0.33
0.17
0.62

*
a
b

p < 0.05 is considered statistically signicant.

OR = adjusted for age, smoking, tobacco chewing and alcohol; 95%CI: 95% condence interval.
Reference group.

N=556
SCCHN 278(50.0%)
Control 278 (50.0%)

XPDLys751Gln
XPD 751 (W)
SCCHN 80 (38.5%)
Control 128 (61.5%)

XPD 751(M)
SCCHN 198 (56.9%)
Control 150 (43.1%)

XRCC1 Arg399Gln
XRCC399(M)
SCCHN 39 (30.7%)
Control 88 (69.3%)

XRCC1 Arg194Trp

XRCC1Arg280His

XRCC1 Arg194Trp
Node 1
XRCC194 (M)
SCCHN 17 (23.9%)
Control 54 (76.1%)

XRCC194 (M)
SCCHN 95 (52.5%)
Control 86 (47.5%)

XRCC 399 (W)

SCCHN 41 (50.6%)
Control 40 (49.4%)

XRCC 194(W)
SCCHN 22 (39.3%)
Control 34 (60.7%)

Node 4
XRCC280 (W)
SCCHN 21 (40.4%)
Control 31 (59.6%)

XRCC1 Arg399Gln

Node 5
XRCC280 (M)
SCCHN 20 (69.0%)
Control 9 (31.0%)

XRCC1Arg280His
Node 2
XRCC280 (W)
SCCHN 13 (36.1%)
Control 23 (63.9%)

Node 3
XRCC280 (M)
SCCHN 9 (45.0%)
Control 11 (55.0%)

XXRCC 194 (W)

SCCHN 103 (61.7%)
Control 64 (38.3%)

XRCC399 (W)
SCCHN 44 (60.3%)
Control 29 (39.7%)

XRCC 399 (M )
SCCHN 51 (47.2%)
Control 57 (52.8%)

XPDLys751Gln
Node 6
XPD751 (M)
SCCHN 35 (43.2%)
Control 46 (56.8%)

Node 7
XPD751 (W)
SCCHN 16 (59.3%)
Control 11 (40.7%)

XRCC1 Arg399Gln
Node 10
XRCC399 (M)
SCCHN 50 (60.2%)
Control 33 (39.8%)

Node 11
XRCC399 (W)
SCCHN 53 (63.1%)
Control 31 (36.9%)

XRCC1Arg280His
Node 8
XRCC280 (W)
SCCHN 13 (44.8%)
Control 16 (55.2%)

Node 9
XRCC280 (M)
SCCHN 31(70.5%)
Control 13 (29.5%)

Fig. 5. CART analysis displaying genetic variants of DNA repair genes in modulating SCCHN risk W = common homozygous genotype, M = heterozygous + variant homozygous
genotype.

Table 9A
Risk estimates of CART terminal nodes.
Node

Genotype of individuals in each node

Control (n)

Cases (n)

Case ratea

ORb (95%CI)

p-value

Node 1
Node 2
Node 3
Node 4
Node 5
Node 6
Node 7
Node 8
Node 9
Node 10
Node 11

XPD(W) + XRCC1 399(M) + XRCC1 194(M)

XPD(W) + XRCC1 399(M) + XRCC1 194(W) + XRCC1 280(W)
XPD(W) + XRCC1 399(M) + XRCC1 194(W) + XRCC1 280(M)
XPD(W) + XRCC1 399(W) + XRCC1 280(W)
XPD(W) + XRCC1 399(W) + XRCC1 280(M)
XPD(M) + XRCC1 194(M) + XRCC1 399 (M) + XPD(M)
XPD(M) + XRCC1 194(M) + XRCC1 399(M) + XPD(W)
XPD(M) + XRCC1 194(M) + XRCC1 399 (W) + XRCC1 280 (W)
XPD(M) + XRCC1 194(M) + XRCC1 399(W) + XRCC1 280(M)
XPD(M) + XRCC1 194(W) + XRCC1 399 (M)
XPD(M) + XRCC1 194(M) + XRCC1 399(W)

54
23
11
31
9
46
11
16
13
33
31

17
13
9
21
20
35
16
13
31
50
53

24
36
45
40
69
43
59
45
71
40
63

1.0c
1.79(0.694.69)
3.75(1.2811.13)
2.15(0.925.09)
7.06(2.4720.74)
2.42(1.145.18)
4.62(1.6413.26)
2.58(0.947.11)
7.56(3.0119.45)
4.81(2.2610.34)
5.43(2.5311.70)

0.255
0.010*
0.075
0.001*
0.016*
0.002*
0.054*
0.001*
0.001*
0.001*

*
a
b
c

p < 0.05 is considered statistically signicant.

Case rate is percentage of SCCHN cases among all individuals in each terminal node [case/(case + control)] 100.
OR = adjusted for age, smoking, tobacco chewing and alcohol; 95%CI: 95% condence interval.
Reference group.

32

A. Kumar et al. / Mutation Research 729 (2012) 2434

Table 9B
Combined analysis of the effects of genetic variants in DNA repair pathway on SCCHN risk based on the results of CART analysis.
Risk Groupa
Low
Medium
High
a
b
c
*

Control (n)
86
115
77

Cases (n)

ORb (95%CI)

p-value

30
94
154

1.0c
2.34 (1.433.84)
5.73 (3.499.41)

0.001*
0.001*

Low risk case ratio < 40%; Medium risk case ratio 4060%; High risk case ratio > 60%.
OR = odds ratio adjusted for age, smoking, tobacco chewing and alcohol; 95%CI: 95% condence interval.
Reference group.
p < 0.05 is considered statistically signicant.

2) to 7.56 (node 9) (Table 9A). The highest risk group were individuals in terminal node 9 that contains homozygous mutants of XPD
Lys751Gln and XRCC1 Arg194Trp, wild type of XRCC1 Arg399Gln
and homozygous mutant of XRCC1 Arg280His.
OR estimates were generated for 3 different risk groups based
on the case ratio of all eleven CART terminal nodes (Table 9B). The
medium risk (case ratio between 4060%) and high-risk groups
(case ratio > 60%) when compared to the low risk group (case
ratio < 40%) were associated with higher SCCHN risk of 2.3 folds
(OR = 2.34, 95%CI = 1.433.84, p = 0.001) and 5.7 folds (OR = 5.73,
95%CI = 3.499.41, p = 0.001), respectively.
3.3. Predictions of deleterious and damaging effect of
non-synonymous SNPs
To predict whether amino acid substitution may have an impact
on protein function, analysis of structural parameters, functional
annotations and evolutionary information analysis was performed
using sorting intolerant from tolerant (SIFT) algorithm. All 4 nonsynonymous SNPs (XRCC1 Arg194Trp, XRCC1 Arg280His, XRCC1
Arg399Gln, and XPD Lys751Gln) in this investigation were submitted to SIFT program to check their tolerance index. Out of 4
non-synonymous SNPs, 3 were classied as tolerant, having scores
between 0.110.85, while rs1799782 (XRCC1 Arg194Trp) having
a score of 0.00, means that it may have low condence damaging
effects. (The low condence has been dened as protein alignment
not having enough sequence diversity. Because the position articially appears to be conserved, an amino acid may incorrectly
predict to be damaging). By PolyPhen software the two variants
XRCC1 Arg399Gln, and XPD Lys751Gln were shown to have PSIC
score difference of 0.186 and 0.278, and were predicted to be
benign, meaning that they lack any phenotypic effects. The other
two variants XRCC1 Arg194Trp and XRCC1 Arg280His, having a PSIC
score difference of 2.654 and 1.694, were predicted to be damaging.
4. Discussion
In the present study, inuence of life style factors on the variant genotypes of two DNA repair genes XRCC1 and XPD and their
association with risk of head and neck cancer were examined. Our
results indicated potential contributory role of XPD Lys751Gln in
the development of SCCHN, demonstrating an increase in risk by
the Gln allele. However, XRCC1, had a protective role.
The XRCC1 gene polymorphisms have been found to be associated with various cancers: gallbladder, gastric, lung, esophageal,
breast, oral and head and neck [20,2629]. The protein encoded
by the human XRCC1 gene is a scaffolding protein that directly
associates DNA ligase I, DNA polymerase and poly(ADPribose)
polymerase (PARP) in a complex to facilitate the processes in single strand break and base excision repair pathway. Of the two
BRCA1 carboxyl terminus (BRCT) domains (BRCT I and BRCT
II) located in central and C terminals of XRCC1, respectively
BRCTI has been of considerable interest since Arg399Gln polymorphism is located in that domain [10,30]. Arg Gln substitution
produces signicant conformational changes at BRCT I, that affect

DNA repair protein-protein interactions. Arg399Gln polymorphism

may therefore, modulate DNA repair activity, which can further
cause genomic instability and may lead to cancer. Several studies have assessed the functional signicance of polymorphisms in
this DNA repair gene. Lunn et al. [31] demonstrated that a change
in 399 codon of XRCC1 (Arg to Gln) altered the XRCC1 phenotype
resulting in decient DNA repair. They found that Gln/Gln genotype
was associated with increased placental aatoxin B1-DNA adducts
and glycophorin A mutations in erythrocytes. Higher risk of ionization radiation sensitivity and tobacco related DNA adducts has
been shown in carriers of XRCC1 399Gln allele [3234].
Two more XRCC1 polymorphisms i.e. Arg194Trp and Arg280His
have signicant impact in the development of several cancers.
XRCC1 Arg194Trp SNP, occurs in the region of nuclear antigenbinding region of proliferating cell and is suggested to enhance
DNA repair capability [35]. The XRCC1 Arg280His polymorphism
is present in linker region that separates DNA polymerase interacting domain from poly(ADPribose) polymerase (PARP)domain
[10]. The XRCC1 Arg194Trp has been shown to be associated
with decreased sensitivity to bleomycin and benzo[a]pyrene-diolepoxide (BPDE), whereas XRCC1 Arg280His displayed increased
sensitivity [36].
In our case-control study, we observed that risk of SCCHN was
protected by XRCC1 Arg194Trp and Arg399Gln variants, while
XRCC1 Arg280His did not exhibit signicant change. Our data of
XRCC1 Arg194Trp polymorphism is in accordance with breast and
prostrate cancer studies [36,37]. XRCC1 Arg194Trp is associated
with certain cancers, for e.g. in oral cancer of south Indian population [29], hepatocellular, gallbladder, lung and bladder cancer
in north Indian population [18,19,38,39], gastric cancer in Chinese
population [40] and no association in breast and head and neck
cancer [4143]. Furthermore, lower risk was reported in salivary
gland, breast and prostate cancer [36,37,44].
Our study shows no association of XRCC1 Arg280Gln polymorphism with SCCHN and this has been conrmed by several authors,
for oral, gastric and head and neck cancers [29,40,43,45]. Nevertheless, there are reports elucidating the role of XRCC1 Arg280His
polymorphism in cancer progression, such as in hepatocellular,
head and neck and bladder cancers [17,46,47]. Recently, Langsenlehner et al. [48] has reported its protective inuence in prostate
cancer.
Likewise, XRCC1 Arg399Gln SNP has also been shown to be
involved in various cancers. A protective inuence by this variant
as shown by us, has also been reported in lung, oesophageal and
bladder cancers [18,26,49,50]. However, a positive association was
reported in breast, rectal, oral, esophageal and in head and neck
cancer [36,5153], whereas, there was no association in gallbladder
and head and neck cancers [42,54,55].
XPD, another well-studied gene with two functions, is known
to impact many types of diseases and cancers. It is a DNA helicase (ATP-dependent helicase activity), which forms the basal
component of TFIIH and is involved in transcription and in NER
pathway. XPD unwinds the double helix during the process of
DNA repair and transcription [56]. The hereditary defects in XPD
can result in three distinct human disorders: the fault in gene

A. Kumar et al. / Mutation Research 729 (2012) 2434

will result in highly cancer prone skin disease xeroderma pigmentosum (XP), alteration in its transcription process will produce
trichothiodystrophy (TTD) disorder and cockayne syndrome [57].
Positive association of XPD Arg751Gln polymorphism with
SCCHN is similarly reported by several researchers in head and
neck, esophageal, lung, rectal and bladder cancers [26,52,54,58].
A few reports have provided evidence of either protection or no
association of cancer with XPD Arg751Gln SNP [59,60].
Gene environment interaction was also investigated considering the impact of life style factors, like smoking, tobacco chewing
and alcohol consumption on cancer development and progression. On further analysis of 278 SCCHN cases, we observed a very
high incidence of cancer in smokers in comparison to tobacco
chewers and alcohol users as shown in Table 1. Tobacco smoke
contains more than 4000 chemicals, out of which 250 are toxic and
more than 55 are potent carcinogens, including tobacco specic
nitrosamines and BPDE. These chemicals can affect DNA repair processes by providing a strong free radical generating environment,
which could lead to oxidation of DNA to form DNA adducts and
single strand breaks. In the case of alcohol, acetaldehyde, which is
the main metabolite of alcohol could be responsible for increased
risk of cancer. Alcohol is also known to enhance free radical generation, activate proto-oncogenes, cause abnormal DNA methylation
as well as suppress immune system [61].
In this investigation, we observed that carriers of XPD 751Gln
allele in smokers, tobacco chewers and alcoholics are associated
with a 2.7-fold increased risk of SCCHN. Further, interaction of
all the three variants of XRCC1 genotypes with smoking, tobacco
chewing and alcohol consumption did not further modulate the
risk of SCCHN. Smoking and tobacco chewing increase the risk of
oral leukoplakia [62] while, smoking and drinking increase the risk
of head and neck cancer with respect to XRCC1 and XPD polymorphism [45,52,63].
There is increasing evidence regarding the combined impact of
commonly occurring SNPs on cancer risk, which is supported by
polygenic models in many cancers such as head and neck, lung, gastric, gallbladder, breast and prostate [18,19,37,63,64]. Genegene
interaction revealed that XRCC1 Arg194Trp variant independently
had a protective inuence on the development of SCCHN and
in combination with XRCC1 Arg399Gln, further lowered the risk.
On the other hand, interaction with XPD Arg751Gln and XRCC1
Arg280His variant, increased the risk 6.1 folds, suggesting that
cross talk between various DNA repair genes might modulate susceptibility towards SCCHN. Though, no associated effects of some
common polymorphisms have been reported and are also of no
importance in cancer risk, but in combination with other SNPs, may
confer an increased risk of cancer as demonstrated by us in the case
of XRCC1 Arg280His and XRCC1 Arg194Trp variants. To the best of
our knowledge, we are the rst to report the increase in SCCHN risk
by genegene cross talk in north Indian population.
To increase power of allelic association of SNPs on the
same chromosome, haplotype analysis of XRCC1 revealed that
194Trp280Arg399Gln (TGA), 194Trp280His399Gln (TAA)
and 194Trp280Arg399Gln751Lys (TGAA) haplotypes were
observed to have a protective effect in SCCHN, a pattern similar
to that reported in gallbladder cancer (194Arg399Gln, CA) [50].
Although, CGG and CAG haplotypes of XRCC1 show no risk, but in
combination with XPD, increase the risk by 1.8 times. On the other
hand, TGA and TAA haplotypes of XRCC1 become non-signicant in
combination with XPD.
Since head and neck cancer is a multifactorial disease with complex interplay of genetic and environmental factors; we, therefore,
performed CART analysis to further dene high order genegene
interactions of DNA repair pathway in SCCHN development. With
this analytic approach, subgroups of individuals with a range of
SCCHN cancer risk prole were identied based on combinations

33

of DNA repair gene polymorphisms and each terminal node consisted of specic groups of risk genotypes. The result however,
should be interpreted with caution because of the limited number
of subjects in some of the CART terminals. From the tree structure,
we observed that XPD Arg751Gln SNP was the best polymorphic
signature for distinguishing between cases and controls and was
consistent throughout with all single and pooled gene analysis.
The divergence in results of different researchers on XRCC1
and XPD polymorphism may be linked to various life style factors,
dietary habits, the most important being the ethnicity. As cancer
is not a disease related to single gene, genetic make of an individual and the cross talk among various genes could contribute
signicantly to the development of cancer.
In summary, our results illustrate high degree of genegene
interaction involving DNA repair genes of NER and BER pathways,
namely XRCC1 and XPD. The salient feature of this investigation is
the positive association of XPD Arg751Gln polymorphism with an
enhanced risk of SCCHN. Further, XRCC1 Arg280His variant though
dormant individually, may also contribute to the development of
cancer in combination with XPD Arg751Gln.
Conict of interest
None
Acknowledgements
The authors are grateful to Director, CSIR-Indian Institute of
Toxicology Research, Lucknow for his keen interest and support
in carrying out the study. AK is thankful to UGC, New Delhi for providing Senior Research Fellowship. Financial support of CSIR-IITR:
Supra Institutional Project (SIP-08) is gratefully acknowledged.
CSIR-IITR Communication Number: 2955
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