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
CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, India
b
C.S.M.Medical University, Lucknow, India
c
Ch. Charan Singh University, Meerut, India

a r t i c l e i n f o a b s t r a c t

Article history: Effective DNA repair machinery ensures maintenance of genomic integrity. Environmental insults, ageing
Received 14 June 2011 and replication errors necessitate the need for proper DNA repair systems. Any alteration in DNA repair
Received in revised form 4 September 2011 efcacy would play a dominant role in progression of squamous cell carcinoma of head and neck (SCCHN).
Accepted 8 September 2011
Genotypes of XRCC1 geneArg194Trp, Arg280His, Arg399Gln and XPD Lys751Gln, by PCR-RFLP were
Available online 16 September 2011
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
Keywords:
Lys751Gln variants exhibited 2-fold increase in SCCHN risk. With XRCC1-Arg280His variants, there was
XRCC1
XPD
no association with SCCHN risk. Arg399Gln of XRCC1 appears to have a protective role in people those
Polymorphism consume alcohol, while XPD Lys751Gln variants indicated 2-fold increased risk of SCCHN in all the
Head and neck cancer 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 dor-
mant individually, may also contribute to the development of cancer in combination with XPD Arg751Gln.

2011 Elsevier B.V. All rights reserved.

1. Introduction neck cancer in patients below 30 years further strengthens this

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 Orga-
nization, 1998). Since SCCHN is also reported in cases with no
history of smoking, chewing tobacco or alcoholism, the possibil-
ity 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).
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, alco-
hol, ultraviolet radiation and to endogenous reactions involving
oxidants [2].
Cancer of oral cavity, especially related to tongue and buc-
cal 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 can-
cer 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 apopto-
sis or to un-regulated (proliferative) cell growth resulting in
carcinoma.

0027-5107/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrfmmm.2011.09.001
 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 com-
mon 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 exci-
sion 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 poly-
morphisms 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 character-
ized 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 stud-
ied 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, XRCC1-
Arg280His, 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 promi-
nence. 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 associa-
tion 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 geographi-
cally 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 sub-
jects 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 Univer-
sity, 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 poly-
morphism 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 (Fer-
mentas 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 (Fer-
mentas 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 (Fer-
mentas 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 con-
trols 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 soft-
ware (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 calcu-
lated [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 homoge-
nous 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 geno-
typic 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 homozy-
gous 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 0.72 (95%CI = 0.511.00) indicated considerable protection (28%)

Demographic variables, risk estimates and distribution of SCCHN cases by tumor
by XRCC1 Arg194Trp polymorphism, which was conrmed by trend
sites.
test (p = 0.028).
Variable Control n (%) Cases n (%) XRCC1 Arg280His polymorphism on the other hand, did not
Subjects 278 278 show any association with SCCHN and the nding was also con-
Age (mean) 50 52 rmed by trend test (p = 0.12).
Range (2570) (2575) In XRCC1 Arg399Gln polymorphism, genotypes Arg/Gln and
Smokers 157 (57) 222 (80)
Gln/Gln were associated with reduced risk of 38% and 50%
Non-smokers 121 (44) 56 (20)
Tobacco Chewers 144 (52) 153 (55) in SCCHN with OR of 0.62 (95%CI = 0.430.88) and OR of 0.50
Non-tobacco Chewers 134(48) 125 (45) (95%CI = 0.309.35), respectively. On combining the two variant
Alcohol users 124 (45) 134 (48) genotypes (Arg/Gln + Gln/Gln) with homozygous wild type, an OR
Non-alcohol users 154 (55) 144 (52)
of 0.60 (95%CI = 0.430.84) indicated signicant protection, which
Site of tumor was further established by trend test (p = 0.003).
Oral Cavity 155 The variants of XPD Lys751Gln gene were associated with an
Larynx 72
increased risk of SCCHN with OR value 1.59 (95%CI = 1.012.31)
Pharynx 51
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 com-
pared with homozygous wild-type genotype, a signicant OR of
(95%CI = 0.501.07), Trp/Trp appeared to reduce the risk with 1.75 (95%CI = 1.242.47) was observed and was conrmed by trend
OR of 0.74 (95%CI = 0.752.36), but remained statistically non- test (p = 0.001).
signicant. On comparing the variant containing genotypes The above data indicate that except for XPD, none of the other
(Arg/Arg + Trp/Trp) with homozygous wild-type genotype, OR of 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 121 144 1.00a
Arg/Trp 131 111 0.71 (0.501.07) 0.04*
Trp/Trp 26 23 0.74 (0.752.36) 0.35
Arg/Trp + Trp/Trp 157 134 0.72 (0.511.00) 0.03*
ptrend 0.028*

XRCC1 codon 280

Arg/Arg 142 129 1.00a
Arg/His 116 123 1.17 (0.821.65) 0.43
His/His 20 26 1.43 (0.772.67) 0.27
Arg/His + His/His 136 149 1.21 (0.861.68) 0.31
ptrend 0.116

XRCC1 codon 399

Arg/Arg 98 128 1.00a
Arg/Gln 144 124 0.66 (0.460.94) 0.02*
Gln/Gln 36 26 0.55 (0.310.97) 0.04*
Arg/Gln + Gln/Gln 180 150 0.64 (0.450.90) 0.01*
ptrend 0.003*

XPD codon 751

Lys/Lys 129 92 1.00a
Lys/Gln 110 125 1.59 (1.012.31) 0.013*
Gln/Gln 39 61 2.19 (1.363.55) 0.002*
Lys/Gln + Gln/Gln 149 186 1.75 (1.242.47) 0.002*
ptrend 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, Data analysis of the geneenvironment interaction showed that
tobacco chewing and alcohol consumption on DNA repair gene the various life style factors exhibited mild inuence on XPD geno-
polymorphism types.

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 geno-
types 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 ana-
lyzed 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 vari-
ants 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 49 29 1.0b 72 115 1.0b
Arg/Trp + Trp/Trp 72 27 0.63 (0.341.90) 0.19 85 10 0.79 (0.521.19) 0.30

Tobacco chewers
Arg/Arg 65 68 1.0b 56 76 1.0b
Arg/Trp + Trp/Trp 69 57 0.79 (0.772.04) 0.38 88 77 0.65 (0.411.02) 0.06

Alcoholics
Arg/Arg 68 73 1.0b 53 71 1.0b
Arg/Trp + Trp/Trp 86 71 0.77 (0.491.21) 0.30 71 53 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) Cases (n) ORa 95%Cl p-value

Smokers
Arg/Arg 65 27 1.0b 77 102 1.0b
Arg/His + His/His 51 26 1.25 (0.662.34) 0.52 80 120 1.13 (0.751.70) 0.68

Tobaccco chewers
Arg/Arg 76 64 1.0b 66 64 1.0b
Arg/His + His/His 58 61 1.25 (0.772.04) 0.39 80 88 1.16 (0.741.84) 0.56

Alcoholics
Arg/Arg 91 82 1.0b 51 47 1.0b
Arg/His + His/His 63 64 1.13 0.711.78) 0.64 73 85 1.26 (0.762.09) 0.37

*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 Habituate

Control (n) Cases (n) ORa 95%CI p-value Control (n) Cases (n) ORa 95%Cl p-value

Smokers
Arg/Arg 43 28 1.0b 55 100 1.0b
Arg/Arg + Gln/Gln 78 28 0.55 (0.291.04) 0.07 102 122 0.67 (0.380.90) 0.06

Tobacco Chewers
Arg/Arg 40 48 1.0b 58 80 1.0b
Arg/Arg + Gln/Gln 101 81 0.67 (0.401.12) 0.15 79 73 0.67 (0.421.06) 0.10

Alcoholics
Arg/Arg 55 60 1.0b 43 68 1.0b
Arg/Arg + Gln/Gln 99 84 0.78 (0.481.24) 0.34 81 66 0.52 (0.300.88) 0.01*
*
p < 0.05 is considered statistically signicant.
a
OR = age adjusted odds; 95%CI: 95% condence interval.
b
Reference group.

95%CI = 0.020.96), the combination remained statistically non-
signicant. 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 maximum-
likelihood 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 signi-
cantly 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 inves-
tigated 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
Control (n) Cases (n) OR 95%CI p-value Control (n) Cases (n) ORa 95%Cl p-value

Smokers
Lys/Lys 53 23 1.0b 76 69 1.0b
Lys/Gln + Gln/Gln 68 33 1.16 (0.612.19) 0.75 81 153 2.08 (1.363.17) 0.001*

Tobacco chewers
Lys/Lys 61 48 1.0b 68 44 1.0b
Lys/Gln + Gln/Gln 73 77 1.34 (0.822.20) 0.26 81 109 2.20 (1.383.57) 0.001*

Alcoholics
Lys/Lys 71 51 1.0b 58 41 1.0b
Lys/Gln + Gln/Gln 77 39 1.56 (0.982.48) 0.77 103 176 1.99 (1.23.31) 0.01*
*
p < 0.05 is considered statistically signicant.
a
OR = age adjusted odds; 95%CI: 95% condence interval
b
Reference group.
30 A. Kumar et al. / Mutation Research 729 (2012) 2434

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

Genotype Control (n = 278) Cases (n = 278) ORa 95%CI p-value

XRCC1 Arg194Trp and XRCC1 Arg280His

0 63 72 1.0b
1 61 56 0.80 (0.491.32) 0.45
2 0 2
ptrend 0.98
Dichotomized groups
0 63 72 1.0b
12 61 58 0.83 (0.511.36) 0.53

XRCC1 Arg194Trp and XRCC1 Arg399Gln

0 44 62 1.0b
1 75 42 0.40 (0.290.68) 0.001*
2 5 1 0.14 (0.020.96) 0.08
ptrend 0.40
Dichotomized groups
0 44 62 1.0b
12 80 43 1.32 (0.772.25) 0.34

XRCC1 Arg194Trp and XPD Lys751Gln

0 53 41 1.0b
1 60 63 1.36 (0.792.32) 0.28
2 2 4 2.58 (0.3821.53) 0.40
ptrend
Dichotomized groups
0 53 41 1.0b
12 61 69 1.46 (0.862.49) 0.18

XRCC1 Arg280His and XRCC1 Arg399Gln

0 51 50 1.0b
1 68 48 0.81 (0.481.36) 0.50
2 0 2
ptrend 0.045*
Dichotomized groups
0 51 50 1.0b
12 68 50 0.75 (0.441.28) 0.34

XRCC1 Arg280His and XPD Lys751Gln

0 70 40 1.0b
1 50 66 2.31 (1.363.14) 0.002*
2 2 7 6.13 (1.0845.05) 0.028*
ptrend
Dichotomized groups0 70 40 1.0b
12 52 72 2.42 (1.434.10) 0.001*

XRCC1 Arg399Gln and XPD Lys751Gln

0 40 41 1.0b
1 58 65 1.09 (0.631.91) 0.78
2 7 6 0.84 (0.272.60) 1.00
ptrend 0.85
Dichotomized groups
0 40 41 1.0b
12 65 71 1.07 (0.621.84) 0.89
*
p < 0.05 is considered statistically signicant.
a
OR = adjusted for age, smoking, tobacco chewing and alcohol; 95%CI: 95% condence interval.
b
Reference group.

The rst split was XPD Lys751Gln, separating individuals individuals with homozygous wild type of XPD Lys751Gln and
into AA and AC + CC genotypes. Subsequent splits were XRCC1 homozygous mutants of XRCC1 Arg399Gln and XRCC1 Arg194Trp,
Arg399Gln, XRCC1 Arg194Trp and XRCC1 Arg280His. To calculate as the reference group. This node exhibited low risk of SCCHN with
ORs as dened by 11 terminal nodes, we chose terminal node 1 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 156 175 1.0b

CGA 111 99 0.8 (0.561.12) 0.22
CAG 77 98 1.14 (0.791.64) 0.51
TGG 78 72 0.82 (0.561.21) 0.33
TGA 55 34 0.55 (0.340.89) 0.01*
TAG 25 34 1.21 (0.702.11) 0.57
CAA 26 27 0.93 (0.521.65) 0.88
TAA 28 16 0.51 (0.270.97) 0.05*
*
p < 0.05 is considered statistically signicant.
a
OR = adjusted for age, smoking, tobacco chewing and alcohol; 95%CI: 95% condence interval.
b
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 97 86 1.0b
CGGG 58 91 1.77 (1.142.74) 0.01*
CGAA 72 54 0.85 (0.521.18) 0.47
TGGA 61 49 0.91 (0.731.23) 0.68
CAGA 48 50 1.17 (0.851.39) 0.52
CGAG 38 42 1.25 (0.861.45) 0.41
CAGG 29 47 1.83 (1.041.66) 0.03*
TGAA 38 17 0.50 (0.431.01) 0.03*
TGGG 18 23 1.44 (0.871.63) 0.29
TGAG 18 19 1.19 (0.771.55) 0.63
TAAA 24 11 0.52 (0.401.12) 0.09
TAGA 15 18 1.35 (0.821.64) 0.42
CAAG 14 18 1.45 (0.851.69) 0.33
TAGG 10 16 1.80 (0.931.84) 0.17
CAAA 14 10 0.81 (0.541.46) 0.62
*
p < 0.05 is considered statistically signicant.
a
OR = adjusted for age, smoking, tobacco chewing and alcohol; 95%CI: 95% condence interval.
b
Reference group.

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

XPDLys751Gln
XPD 751 (W) XPD 751(M)
SCCHN 80 (38.5%) SCCHN 198 (56.9%)
Control 128 (61.5%) Control 150 (43.1%)

XRCC1 Arg399Gln XRCC1 Arg194Trp

XRCC399(M) XRCC 399 (W) XRCC194 (M) XXRCC 194 (W)
SCCHN 39 (30.7%) SCCHN 41 (50.6%) SCCHN 95 (52.5%) SCCHN 103 (61.7%)
Control 88 (69.3%) Control 40 (49.4%) Control 86 (47.5%) Control 64 (38.3%)

XRCC1 Arg194Trp XRCC1Arg280His XRCC1 Arg399Gln XRCC1 Arg399Gln

Node 1 Node 5 Node 10

XRCC 194(W) Node 4 XRCC 399 (M ) XRCC399 (W) Node 11
XRCC194 (M) XRCC280 (M) XRCC399 (M)
SCCHN 22 (39.3%) XRCC280 (W) SCCHN 51 (47.2%) SCCHN 44 (60.3%) XRCC399 (W)
SCCHN 17 (23.9%) SCCHN 20 (69.0%) SCCHN 50 (60.2%)
Control 34 (60.7%) SCCHN 21 (40.4%) Control 57 (52.8%) Control 29 (39.7%) SCCHN 53 (63.1%)
Control 54 (76.1%) Control 9 (31.0%) Control 33 (39.8%)
Control 31 (59.6%) Control 31 (36.9%)

XRCC1Arg280His XPDLys751Gln XRCC1Arg280His

Node 2 Node 3 Node 6 Node 7 Node 8 Node 9
XRCC280 (W) XRCC280 (M) XPD751 (M) XPD751 (W) XRCC280 (W) XRCC280 (M)
SCCHN 13 (36.1%) SCCHN 9 (45.0%) SCCHN 35 (43.2%) SCCHN 16 (59.3%) SCCHN 13 (44.8%) SCCHN 31(70.5%)
Control 23 (63.9%) Control 11 (55.0%) Control 46 (56.8%) Control 11 (40.7%) Control 16 (55.2%) 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 XPD(W) + XRCC1 399(M) + XRCC1 194(M) 54 17 24 1.0c

Node 2 XPD(W) + XRCC1 399(M) + XRCC1 194(W) + XRCC1 280(W) 23 13 36 1.79(0.694.69) 0.255
Node 3 XPD(W) + XRCC1 399(M) + XRCC1 194(W) + XRCC1 280(M) 11 9 45 3.75(1.2811.13) 0.010*
Node 4 XPD(W) + XRCC1 399(W) + XRCC1 280(W) 31 21 40 2.15(0.925.09) 0.075
Node 5 XPD(W) + XRCC1 399(W) + XRCC1 280(M) 9 20 69 7.06(2.4720.74) 0.001*
Node 6 XPD(M) + XRCC1 194(M) + XRCC1 399 (M) + XPD(M) 46 35 43 2.42(1.145.18) 0.016*
Node 7 XPD(M) + XRCC1 194(M) + XRCC1 399(M) + XPD(W) 11 16 59 4.62(1.6413.26) 0.002*
Node 8 XPD(M) + XRCC1 194(M) + XRCC1 399 (W) + XRCC1 280 (W) 16 13 45 2.58(0.947.11) 0.054*
Node 9 XPD(M) + XRCC1 194(M) + XRCC1 399(W) + XRCC1 280(M) 13 31 71 7.56(3.0119.45) 0.001*
Node 10 XPD(M) + XRCC1 194(W) + XRCC1 399 (M) 33 50 40 4.81(2.2610.34) 0.001*
Node 11 XPD(M) + XRCC1 194(M) + XRCC1 399(W) 31 53 63 5.43(2.5311.70) 0.001*
*
p < 0.05 is considered statistically signicant.
a
Case rate is percentage of SCCHN cases among all individuals in each terminal node [case/(case + control)] 100.
b
OR = adjusted for age, smoking, tobacco chewing and alcohol; 95%CI: 95% condence interval.
c
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 Control (n)
Low 86
Medium 115
High 77
a
Low risk case ratio < 40%; Medium risk case ratio 4060%; High risk case ratio > 60%.
b
OR = odds ratio adjusted for age, smoking, tobacco chewing and alcohol; 95%CI: 95% condence interval.
c
Reference group.
*
p < 0.05 is considered statistically signicant.
2) to 7.56 (node 9) (Table 9A). The highest risk group were individ-
uals 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 non-
synonymous SNPs (XRCC1 Arg194Trp, XRCC1 Arg280His, XRCC1
Arg399Gln, and XPD Lys751Gln) in this investigation were sub-
mitted 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 arti-
cially 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 vari-
ant 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 asso-
ciated 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 sin-
gle 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 polymor-
phism is located in that domain [10,30]. Arg Gln substitution
produces signicant conformational changes at BRCT I, that affect
Cases (n) ORb (95%CI) p-value
30 1.0c
94 2.34 (1.433.84) 0.001*
154 5.73 (3.499.41) 0.001*
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 stud-
ies 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 ion-
ization 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 antigen-
binding 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 inter-
acting 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-diol-
epoxide (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 pop-
ulation [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 polymor-
phism with SCCHN and this has been conrmed by several authors,
for oral, gastric and head and neck cancers [29,40,43,45]. Never-
theless, 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, Langsen-
lehner 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 heli-
case (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 pigmen-
tosum (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 consider-
ing the impact of life style factors, like smoking, tobacco chewing
and alcohol consumption on cancer development and progres-
sion. 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 pro-
cesses 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 gener-
ation, 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 polymor-
phism [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, gas-
tric, 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 sus-
ceptibility 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 com-
plex 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 con-
sisted 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 indi-
vidual 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 pro-
viding 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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