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ORIGINAL PAPER
Association between single genetic polymorphisms of MDR1 geneand gastric cancer susceptibility in Chinese
Wen Qiao • Tao Wang • Li Zhang •
Qing Tang • Dan Wang • Hongkun Sun
Received: 19 May 2013 / Accepted: 14 June 2013 / Published online: 26 June 2013
� Springer Science+Business Media New York 2013
Abstract Gastric cancer is a common cancer worldwide.
The multidrug resistance 1 gene (MDR1) is one of the most
important candidate genes for influencing gastric cancer
susceptibility. This study aimed to analyze the association
between genetic variants of MDR1 gene and the suscepti-
bility to gastric cancer in Chinese Han population. A total
of 365 gastric cancer patients and 367 cancer-free controls
were enrolled in this study. The single genetic polymor-
phisms (SNPs) of MDR1 gene were genotyped by the
created restriction site-polymerase chain reaction method.
Our data suggested that the allele and genotype frequencies
of c.159G [ T and c.1564A [ T were statistically differ-
ent between gastric cancer patients and cancer-free con-
trols. Association analyses indicated that these two SNPs
were statistically associated with the increased risk of
gastric cancer (for c.159G [ T, TT versus (vs.) GG: OR
2.34, 95 % CI 1.31–4.19; TT vs. GT/GG: OR 2.32, 95 %
CI 1.32–4.08; T vs. G: OR 1.27, 95 % CI 1.01–1.59; for
c.1564A [ T, TT vs. AA: OR 2.27, 95 % CI 1.31–3.93; TT
vs. AT/AA: OR 2.21, 95 % CI 1.30–3.75; T vs. A: OR
1.30, 95 % CI 1.04–1.62). The allele-T of both these two
SNPs may contribute to the susceptibility to gastric cancer
in Chinese Han population. The c.159G [ T and
c.1564A [ T genetic variants might be used as molecular
markers for detecting gastric cancer susceptibility.
Keywords Gastric cancer � MDR1 gene �Single nucleotide polymorphisms � Susceptibility
Introduction
Gastric cancer is one of the most common malignancies
and leading cause of cancer-related deaths worldwide
[1–4]. It is a global health problem in the world. Over the
last few decades, the incidence and mortality rate of gastric
cancer have been decreased. However, in China, gastric
cancer still remains one of the leading cause of cancer-
related deaths [5–8]. There is evidence that genetic factors
play key roles in the pathogenesis of gastric cancer.
Recently, several reports suggest that the multidrug resis-
tance 1 gene (MDR1) is one of the most important candi-
date genes for influencing gastric cancer susceptibility
[9–19]. The MDR1 gene encodes P-glycoprotein (Pgp), a
transmembrane efflux transporter conferring resistance to
natural cytotoxic drugs and potentially toxic xenobiotics
[20–22]. Previous studies indicated that the single genetic
polymorphisms (SNPs) of MDR1 gene could impact on the
expression and function of Pgp, thus influencing the sus-
ceptibility to various diseases including gastric cancer
[9–17, 22–25]. The potential associations of SNPs in
MDR1 gene, such as C3435T, with the risk of gastric
cancer have been assessed [9–13, 15, 18]. The C3435T
SNP was found to be associated with altered Pgp function
and decreased tissue protein expression and activity
[26, 27]. However, up to date, there are no similar studies
that have reported association of MDR1 c.159G [ T and
c.1564A [ T SNPs with gastric cancer risk factors. Thus,
this study aims to investigate the distribution of these two
SNPs and to evaluate the potential associations with the
susceptibility to gastric cancer.
W. Qiao (&) � T. Wang � L. Zhang � Q. Tang �D. Wang � H. Sun
Department of Gastroenterology, The First Affiliated Hospital,
Medical School of Xi’an Jiaotong University, No. 277 Yanta
West Road, Xi’an 710061, Shaanxi Province,
People’s Republic of China
e-mail: [email protected]
123
Med Oncol (2013) 30:643
DOI 10.1007/s12032-013-0643-3
Materials and methods
Subjects
A total of 732 subjects were recruited from the First
Affiliated Hospital, Medical School of Xi’an Jiaotong
University, consisting of 365 gastric cancer patients with a
pathology-confirmed diagnosis and 367 healthy age-mat-
ched subjects who had no history of any gastric diseases as
controls. Table 1 shows the demographic clinical charac-
teristics of subjects. The protocol of this study was
approved by the Ethics Committee of the First Affiliated
Hospital, Medical School of Xi’an Jiaotong University.
The written informed consent form was obtained from each
subject.
PCR amplification
Genomic DNA was isolated from peripheral venous blood,
using the standard extraction method, and then stored at
-80 �C [28]. The specific polymerase chain reaction
(PCR) primers were designed by Primer Premier 5.0 soft-
ware according to MDR1 gene DNA and mRNA reference
sequences (GenBank IDs: NG_011513.1 and NM_000927.4).
Table 2 shows the primers sequences, annealing temperature,
fragment region, and size. For PCRs , 50 ng template DNA
was amplified in 20 lL of reaction mixture containing
19 buffer (Tris–HCl 100 mmol/L, pH 8.3; KCl 500 mmol/L),
0.25 lmol/L primers, 2.0 mmol/L MgCl2, 0.25 mmol/L
dNTPs, and 0.5U Taq DNA polymerase (Promega, Madison,
WI, USA). The PCR protocol was performed on 94 �C for
5 min, followed by 32 cycles of 94 �C for 32 s, annealing at
the corresponding temperature (shown in Table 2) for 32 s
and 72 �C for 32 s, and a final extension at 72 �C for 8 min.
The amplified PCR products were separated by electropho-
resis on agarose gel and then observed under UV light.
Genotyping
The c.159G [ T and c.1564A [ T SNPs of MDR1 gene
were genotyped through the created restriction site-poly-
merase chain reaction (CRS-PCR) method with one of the
primers including a nucleotide mismatch, which enables
the use of selected restriction enzymes for discriminating
sequence variations [29–33]. According to the supplier’s
manual, aliquots of 5 lL amplified PCR products were
digested with 2U selected restriction enzyme (MBI Fer-
mentas, St. Leon-Rot, Germany, Table 2) at 37 �C for
10 h. The digested products were separated by 2.5 %
agarose gel electrophoresis and observed under UV light.
10 % of random samples were re-analyzed by DNA
sequencing method (ABI3730xl DNA Analyzer, Applied
Biosystems, Foster City, CA, USA) to make sure concor-
dance with the genotyping results from CRS-PCR.
Table 1 Clinical characteristics
of gastric cancer cases and
cancer-free controls
Characteristics Cases (n) Controls (n) v2 value P value
Number 365 49.86 367 50.14
Gender (n) 0.3445 0.5573
Male 197 53.97 206 56.13
Female 168 46.03 161 43.87
Age (years) 0.7936 0.3730
Mean ± SD 61.55 ± 11.82 62.74 ± 13.21
\60 169 46.30 182 49.59
C60 196 53.70 185 50.41
Tobacco smoking 1.4339 0.2311
Yes 210 57.53 195 53.13
No 155 42.47 172 46.87
Alcohol drinking 2.7491 0.0973
Yes 222 60.82 201 54.77
No 143 39.18 166 45.23
H. pyori infection (n) 1.6132 0.2040
Yes 209 57.26 193 52.59
No 156 42.74 174 47.41
Family history of gastric cancer (n) 1.0674 0.3015
Yes 169 46.30 156 42.51
No 196 53.70 211 57.49
Page 2 of 6 Med Oncol (2013) 30:643
123
Statistical analysis
The Hardy–Weinberg equilibrium in genotype distributions
and clinical characteristics were evaluated by the chi-
squared (v2) test. P value \0.05 was considered as statis-
tically significant. All statistical analyses were performed
by SPSS software (Windows version release 15.0; SPSS
Inc., Chicago, IL, USA).
Results
General characteristics
In this case–control study, we enrolled 732 Chinese sub-
jects with Han nationality. There were no significant dif-
ferences between gastric cancer patients and cancer-free
controls in regard to gender, age, tobacco smoking, alcohol
drinking, H. pyori infection, and family history of gastric
cancer (all P values [0.05, Table 1).
MDR1 SNPs identification
Through CRS-PCR and DNA sequencing methods, we
investigated the c.159G [ T and c.1564A [ T SNPs of
MDR1 gene. Results from sequence analyses indicate that
the c.159G [ T SNP is a synonymous mutation and causes
by G to T mutations (p.valine (Val) 53Val). As for the
c.1564A [ T SNP, it is a nonsynonymous mutation and
causes by A to T mutations, which resulted in threonine
(Thr) to serine (Ser) amino acid replacement (p.Thr522Ser,
reference sequences: GenBank IDs: NG_011513.1,
NM_000927.4, and NP_000918.2).
Allelic and genotypic frequencies
The PCR amplified products of c.159G [ T SNP were
digested with TaqI restriction enzyme and divided into
three genotypes: GG (208 bp), GT (208, 189, and 19 bp),
and TT (189 and 19 bp). As for c.1564A [ T SNP, the
PCR amplified products were digested with MaeIII
restriction enzyme and divided into three genotypes: AA
(192 and 23 bp), AT (215, 192, and 23 bp), and TT
(215 bp). The allele and genotype frequencies are given in
Table 3. The frequencies of allele G in c.159G [ T and
allele A in c.1564A [ T SNPs were maximums in the
gastric cancer patients and cancer-free controls. As for
c.159G [ T, the allele frequencies of gastric cancer
patients (G 68.49 %, T 31.51 %) were statistically signif-
icantly different from cancer-free controls (G 73.43 %,
T 26.57 %; v2 = 4.3354, P = 0.0373). The genotype dis-
tributions in gastric cancer patients were statistically sig-
nificantly different from cancer-free controls (v2 = 8.9400,
P = 0.0114). As for c.1564A [ T, the allele frequencies of
gastric cancer patients (A 66.71 %, T 33.29 %) were sta-
tistically significantly different from cancer-free controls
(A 72.21 %, T 27.79 %, v2 = 5.2099, P = 0.0225). The
genotype frequencies in gastric cancer patients were not
consistent with cancer-free controls, the differences being
statistically significant (v2 = 8.9671, P = 0.0113). The
genotype distributions of these two SNPs in the studied
populations did not significantly deviate from Hardy–
Weinberg equilibrium (all P values [0.05).
Association between the MDR1 SNPs and gastric cancer
Table 4 shows the potential association between the
c.159G [ T and c.1564A [ T SNPs of MDR1 gene and
gastric cancer risk. As for c.159G [ T, we found statisti-
cally significantly increased risk of gastric cancer in
homozygote comparison (TT versus (vs.) GG: OR 2.34,
95 % CI 1.31–4.19, v2 = 8.55, P = 0.003), recessive
model (TT vs. GT/GG: OR 2.32, 95 % CI 1.32–4.08,
v2 = 8.91, P = 0.003), and allele contrast (T vs. G: OR
1.27, 95 % CI 1.01–1.59, v2 = 4.33, P = 0.037). Simi-
larly, as for c.1564A [ T, we also detected statistically
significant increased risk of gastric cancer in homozygote
comparison (TT vs. AA: OR 2.27, 95 % CI 1.31–3.93,
Table 2 The CRS-PCR analysis for MDR1 genetic polymorphisms
SNPs Primer sequences Annealing
temperature
(�C)
Amplification
fragment
(bp)
Region Restriction
enzyme
Genotype (bp)
c.159G [ T 50-CAAAGGTAGAGGGTGTCTTGGACT-30 62.9 208 Exon5 TaqI GG: 208
50-GATGATGGCAGCCAAAGTTCG-30 GT: 208, 189, 19
TT: 189, 19
c.1564A [ T 50-TGGGTTTTCTGTGGTAGAAATTTGTC-30 63.6 215 Exon15 MaeIII AA: 192, 23
50-GGTTGGTTTGAACTAAGCCTCACTG-30 AT: 215, 192, 23
TT: 215
SNPs single nucleotide polymorphisms, PCR polymerase chain reaction, CRS-PCR created restriction site-polymerase chain reaction. Underlined
nucleotides mark nucleotide mismatches enabling the use of the selected restriction enzymes for discriminating sequence variations
Med Oncol (2013) 30:643 Page 3 of 6
123
v2 = 8.74, P = 0.003), recessive model (TT vs. AT/AA:
OR 2.21, 95 % CI 1.30–3.75, v2 = 8.82, P = 0.003), and
allele contrast (T vs. A: OR 1.30, 95 % CI 1.04–1.62,
v2 = 5.21, P = 0.023).
Discussion
Gastric cancer is a common and polygenic malignant solid
cancer. It causes from complex interactions between genetic
and environmental factors. The genetic variants of candidate
genes which influence the development of gastric cancer play
key roles in the pathogenesis of gastric cancer. In this case–
control study, the influence of genetic variants in MDR1 gene
on gastric cancer risk in Chinese Han population was eval-
uated by association analysis. The different genotypes of
c.159G [ T and c.1564A [ T SNPs were detected by CRS-
PCR and DNA sequencing methods. Our data indicated that
the distribution of allele and genotype frequencies in gastric
cancer patients was significantly different from cancer-free
controls (P \ 0.05, Table 3). As for c.159G [ T, the geno-
type-TT was statistically associated with increased gastric
cancer risk compared with genotype-GG and GT/GG-carri-
ers (P \ 0.05, Table 4). As for c.1564A [ T, the genotype-
TT was statistically associated with increased gastric cancer
risk compared with genotype-AA and AT/AA-carriers
(P \ 0.05, Table 4). The allele-T and genotype-TT for both
these two SNPs could contribute to increase gastric cancer
risk. Results from this study demonstrated that these two
SNPs in MDR1 gene were statistically associated with gastric
cancer risk in Chinese Han population and could be used as
molecular markers for detecting gastric cancer susceptibility.
Recently, there are several similar studies have evaluated the
potential association of other genetic variants of MDR1 gene
with the risk of gastric cancer [9–13, 15, 18]. Sugimoto et al.
[9] reported that the association with MDR1 C3435T poly-
morphism and risk for developing Helicobacter pylori-rela-
ted gastric cancer in Japanese was low. Sabahi et al. [10]
suggested that C3435T polymorphism may be associated
with gastric cancer in an ethnic Iranian population; the
polymorphic homozygote (T/T) genotype showed significant
association with the incidence of gastric cancer compared
with controls (P \ 0.05). Tahara et al. [11] found that the
C3435T polymorphism of MDR1 influenced H. pylori-rela-
ted inflammatory conditions in the stomach, especially in
older subjects. In another study, Tahara et al., reported that
the MDR1 3435 TT genotype showed a significantly higher
frequency in controls than in gastric cancer patients (OR
0.43; 95 % CI 0.23–0.79). Tahara et al. [15] suggested that
3435T/T polymorphism of MDR1 is associated with a
reduced risk of gastric cancer in the Japanese population.
Oliveira et al. [18] indicated that no correlation was observed
between the C3435T polymorphism of MDR1 gene and Ta
ble
3T
he
gen
oty
pe
and
alle
lefr
equ
enci
eso
fM
DR
1g
enet
icp
oly
mo
rph
ism
sin
case
san
dco
ntr
ols
Gro
ups
c.159G
[T
c.1564A
[T
Gen
oty
pe
freq
uen
cies
(%)
All
ele
freq
uen
cies
(%)
Gen
oty
pe
freq
uen
cies
(%)
All
ele
freq
uen
cies
(%)
GG
GT
TT
GT
v2P
AA
AT
GG
TC
v2P
Cas
es(n
=365)
176
(48.2
2)
148
(40.5
5)
41
(11.2
3)
500
(68.4
9)
230
(31.5
1)
1.3
370
0.5
125
167
(45.7
5)
153
(41.9
2)
45
(12.3
3)
487
(66.7
1)
243
(33.2
9)
1.1
529
0.5
619
Contr
ols
(n=
367)
191
(52.0
4)
157
(42.7
8)
19
(5.1
8)
539
(73.4
3)
195
(26.5
7)
3.4
111
0.1
817
185
(50.4
1)
160
(43.6
0)
22
(5.9
9)
530
(72.2
1)
204
(27.7
9)
2.7
270
0.2
558
Tota
l(n
=732)
367
(50.1
3)
305
(41.6
7)
60
(8.2
0)
1039
(70.9
7)
425
(29.0
3)
0.0
918
0.9
551
352
(48.0
9)
313
(42.7
6)
67
(9.1
5)
1017
(69.4
7)
447
(30.5
3)
0.0
468
0.9
769
v2=
8.9
400,
P=
0.0
114
v2=
4.3
354,
P=
0.0
373
v2=
8.9
671,
P=
0.0
113
v2=
5.2
099,
P=
0.0
225
Page 4 of 6 Med Oncol (2013) 30:643
123
gastric cancer risk or prognosis. These observations sug-
gested that MDR1 genetic variants may contribute to the
influences on gastric cancer risk. Results from this study
could provide more evidence to further analyze about the role
of MDR1 gene for the susceptibility to gastric cancer. Further
functional studies are needed to confirm these findings in
larger different populations and to elucidate the underlying
molecular mechanisms.
Conflict of interest The authors have no conflict of interests.
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SNPs single nucleotide
polymorphisms, OR odds ratio,
CI confidence interval, vs.
versus
SNPs Comparisons Test of association
OR (95 % CI) v2 value P value
c.159G [ T Homozygote comparison
(TT vs. GG)
2.34 (1.31–4.19) 8.55 0.003
Heterozygote comparison
(GT vs. GG)
1.02 (0.76–1.39) 0.02 0.883
Dominant model
(TT/GT vs. GG)
1.17 (0.87–1.56) 1.07 0.301
Recessive model
(TT vs. GT/GG)
2.32 (1.32–4.08) 8.91 0.003
Allele contrast
(T vs. G)
1.27 (1.01–1.59) 4.33 0.037
c.1564A [ T Homozygote comparison
(TT vs. AA)
2.27 (1.31–3.93) 8.74 0.003
Heterozygote comparison
(AT vs. AA)
1.06 (0.78–1.44) 0.14 0.711
Dominant model
(TT/AT vs. AA)
1.21 (0.90–1.61) 1.59 0.208
Recessive model
(TT vs. AT/AA)
2.21 (1.30–3.75) 8.82 0.003
Allele contrast
(T vs. A)
1.30 (1.04–1.62) 5.21 0.023
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