ORIGINAL PAPER

The influence of genetic polymorphisms in MDR1 gene onbreast cancer risk factors in Chinese

Yunhe Jia • Wenjing Tian • Shuai Sun •

Peng Han • Weinan Xue • Mingqi Li •

Yanlong Liu • Shixiong Jiang • Binbin Cui

Received: 16 April 2013 / Accepted: 3 May 2013 / Published online: 21 May 2013

� Springer Science+Business Media New York 2013

Abstract Breast cancer (BC) is the most common cancer

among women in the world. The human multidrug resis-

tance 1 gene (MDR1) is potentially an important gene

influencing the susceptibility to breast cancer. This study

aimed to assess the association of MDR1 genetic poly-

morphisms with the susceptibility to BC. Overall, 353 BC

patients and 360 cancer-free controls were enrolled. The

clinical characteristics were summarized by questionnaires.

The c.1564A [ T genetic polymorphism was genotyped

using created restriction site–polymerase chain reaction

method. We found that no significant differences in the

genotypic and allelic frequencies between BC patients and

cancer-free controls. Furthermore, the distribution of BC

patients’ risk factors was not significantly different among

AA, AT, and TT genotypes. Our findings indicate that the

c.1564A [ T genetic polymorphism is not significantly

associated with the susceptibility to BC in Chinese Han

populations.

Keywords Breast cancer � MDR1 gene �Single-nucleotide polymorphisms � Risk factors �Cancer susceptibility

Introduction

Breast cancer (BC) remains the most common cancer and

the leading cause highly cancer-related deaths among

women in the world [1–5]. Over the recent decades, the

incidence and mortality of BC have arosed rapidly world-

wide [5, 6]. Nowadays, several substantial progress has

been made in the treatment and diagnosis of BC [7]. An

effort has been made to detect genetic factors and risk

factors that contribute to the risk of BC development [7–

10]. There were many studies indicated that the genetic

factors have significant function in the pathogenesis of BC

[4–14]. The human multidrug resistance 1 gene (MDR1) is

potentially an important gene influencing BC susceptibil-

ity, which encodes P-glycoprotein (Pgp), a transmembrane

efflux transporter conferring resistance to natural cytotoxic

drugs and potentially toxic xenobiotics [15–17]. It has been

indicated that the MDR1 genetic polymorphisms contribute

to affect the expression and function of Pgp, thus influ-

encing various diseases susceptibility such as BC [7, 11,

13, 14, 18, 19]. Up to date, many genetic polymorphisms of

MDR1 gene have been reported, and one of these genetic

polymorphisms, the C3435T genetic polymorphism, was

found to alter Pgp function and decrease tissue protein

expression and activity [20, 21]. The potential association

between the risk factors of BC and C3435T or other genetic

polymorphisms have been investigated [7, 11, 13, 14, 19,

22, 23]. However, there are no related studies which have

reported the association between BC risk factors and

c.1564A [ T genetic polymorphism. Therefore, our study

Yunhe Jia and Wenjing Tian contributed equally to this paper.

Y. Jia (&) � S. Sun � P. Han � W. Xue � M. Li � Y. Liu �S. Jiang � B. Cui (&)

The Colorectal Cancer Center, Tumor Hospital, Harbin Medical

University, No. 150 Haping Road, Nangang District, Harbin

150086, Heilongjiang Province, People’s Republic of China

e-mail: yunhe_jia@sina.cn

B. Cui

e-mail: binbin_cui@sina.cn

W. Tian

Department of Epidemiology, Public Health College, Harbin

Medical University, Harbin 150086, Heilongjiang Province,

People’s Republic of China

123

Med Oncol (2013) 30:601

DOI 10.1007/s12032-013-0601-0

focused on detecting the distribution of this genetic

polymorphism and determining the influence on BC

susceptibility.

Materials and methods

Subjects

In total, 713 subjects were enrolled in this study, consisting

of 353 patients with primary BC and 360 healthy age-

matched women who had no history of any breast diseases

as the control group. There was no significant difference

regarding age and gender between case and control groups

(P [ 0.05). All subjects were unrelated Chinese Han

population. According to the previous published studies [7,

13, 24–26], the patients’ general characteristics and related

risk factors, such as age, age of first pregnancy, age of

menarche, oral contraceptives consumption, number of

pregnancies, number of abortions, duration of education,

smoking status, body mass index (BMI), cancer stage,

tumor size, node involvement, number of involved nodes,

and family history of BC were collected from question-

naires. The ethics committee of Tumor Hospital of Harbin

Medical University approved this study, and the written

informed consent was obtained from each subject.

DNA extraction and genotyping

Genomic DNA was isolated form peripheral venous blood

samples using the standard extraction method and then

stored at -80 �C until analyzed [27]. The polymerase

chain reaction (PCR) primers were designed using Primer

Premier 5.0 software. Table 1 performed the information

of primers sequences, annealing temperature, fragment

region, and size. The PCR reactions were carried out in

20 lL solution containing 50 ng template DNA, 19buffer

(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 0.5 U Taq DNA polymerase (Promega, Mad-

ison, WI, USA). The PCR protocol was as followed: 94 �C

for 5 min, followed by 32 cycles of 94 �C for 35 s, 57.2 �C

for 35 s and 72 �C for 35 s, and a final extension at 72 �C

for 8 min. The c.1564A [ T genetic polymorphism was

genotyped using created restriction site-PCR (CRS-PCR)

method with one of the primers including a nucleotide

mismatch, which enables the use of selected restriction

enzymes for discriminating sequence variations [28–32].

Aliquots of 5 lL PCR products were digested for 10 h at

37 �C with 2 U MaeIII restriction enzyme (MBI Fermen-

tas, St. Leon-Rot, Germany). The digested products were

analyzed by electrophoresis on a 2 % agarose gel and

observed under ultraviolet light. To ensure concordance

with the CRS-PCR genotyping results, we selected about

10 % of random samples to verify using DNA sequencing

method (ABI3730xl DNA Analyzer, Applied Biosystems,

Foster City, CA).

Statistical analysis

The chi-squared (v2) test evaluated the Hardy–Weinberg

equilibrium (HWE) in allelic/genotypic distributions and

clinical characteristics. All statistical analyses were ana-

lyzed using the Statistical Package for Social Sciences

Table 1 The primer sequences, PCR, and CRS-PCR analysis for c.1564A [ T genetic polymorphism in MDR1 gene

Primer sequences Annealing

temperature (�C)

Amplification

fragment (bp)

Region Restriction

enzyme

Genotype (bp)

50-GGTTTTCTGTGGTAGAAATTTGTC-3 57.2 212 Exon15 MaeIII AA: 191, 21

50-GTTGGTTTGAACTAAGCCTCAC-30 AT: 212, 191, 21

TT: 212

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

Table 2 The genotypic and allelic frequencies of MDR1 c.1564A [ T genetic polymorphism in the studied subjects

Groups Genotypic frequencies (%) Allelic frequencies (%) v2 P

AA AT TT A T

Case group (n = 353) 170 (48.16) 140 (39.66) 43 (12.18) 480 (67.99) 226 (32.01) 2.7876 0.2481

Control group (n = 360) 196 (54.44) 131 (36.39) 33 (9.17) 523 (72.64) 197 (27.36) 2.5734 0.2762

Total (n = 713) 366 (51.33) 271 (38.01) 76 (10.66) 1,003 (70.34) 423 (29.66) 5.6666 0.0588

v2 = 3.3933, P = 0.1833 v2 = 3.694, P = 0.0546

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Table 3 The association between c.1564A [ T genetic polymorphism in MDR1 gene and risk factors for breast cancer in patients

Risk factors Genotype Frequency (%) Total P value v2

AA AT TT

Age (M = 0) \35 62 (47.33) 51 (38.93) 18 (13.74) 131 0.7892 0.4734

C35 108 (48.65) 89 (40.09) 25 (11.26) 222

Total 170 (48.16) 140 (39.66) 43 (12.18) 353

Stage (M = 27) Earlya 102 (50.75) 80 (39.80) 19 (9.45) 201 0.3874 1.8968

Advancedb 59 (47.20) 48 (38.40) 18 (14.40) 125

Total 161 (49.39) 128 (39.26) 37 (11.35) 326

Tumor size (M = 8) T0, T1, T2 110 (50.46) 89 (40.82) 19 (8.72) 218 0.3919 1.8737

T3, T4 61 (48.03) 49 (38.58) 17 (13.39) 127

Total 171 (49.57) 138 (40.00) 36 (10.43) 345

Node involvement (M = 11) Yes 101 (48.10) 85 (40.48) 24 (11.42) 210 0.7492 0.5776

No 68 (51.52) 48 (36.36) 16 (12.12) 132

Total 169 (49.42) 133 (38.88) 40 (11.70) 342

Number of involved nodes (M = 9) N0, N1 97 (50.00) 78 (40.21) 19 (9.79) 194 0.5104 1.3453

N2, N3 68 (45.34) 62 (41.33) 20 (13.33) 150

Total 165 (47.96) 140 (40.70) 39 (11.34) 344

Family history of breast cancer (M = 16) Yes 60 (56.08) 35 (32.71) 12 (11.21) 107 0.2351 2.8956

No 108 (46.96) 97 (42.17) 25 (10.87) 230

Total 168 (49.85) 132 (39.17) 37 (10.98) 337

Duration of education (M = 11) \9 years 58 (45.31) 57 (44.53) 13 (10.16) 128 0.5238 1.2934

C9 years 107 (50.00) 82 (38.32) 25 (11.68) 214

Total 165 (48.25) 139 (40.64) 38 (11.11) 342

Smoking (M = 0) Yes 55 (44.00) 52 (41.60) 18 (14.40) 125 0.4359 1.6607

No 115 (50.44) 88 (38.60) 25 (10.96) 228

Total 170 (48.16) 140 (39.66) 43 (12.18) 353

Age of menarche (M = 5) \14 81 (47.93) 69 (40.83) 19 (11.24) 169 0.9720 0.0568

C14 88 (49.16) 71 (39.66) 20 (11.18) 179

Total 169 (48.56) 140 (40.23) 39 (11.21) 348

Age of first pregnancy (M = 30) \25 90 (48.92) 76 (41.30) 18 (9.78) 184 0.6652 0.8153

C25 62 (44.60) 60 (43.17) 17 (12.23) 139

Total 152 (47.05) 136 (42.11) 35 (10.84) 323

Number of pregnancies (M = 24) B1 113 (50.22) 91 (40.44) 21 (9.34) 225 0.0610 5.5937

[1 50 (48.08) 35 (33.65) 19 (18.27) 104

Total 163 (49.54) 126 (38.30) 40 (12.16) 329

Number of abortions (M = 24) \1 121 (50.42) 98 (40.83) 21 (8.75) 240 0.0563 5.7527

C1 38 (42.70) 35 (39.33) 16 (17.97) 89

Total 159 (48.32) 133 (40.43) 37 (11.25) 329

Oral contraceptives consumption (M = 10) Yes 62 (53.45) 43 (37.07) 11 (9.48) 116 0.5411 1.2282

No 107 (47.14) 95 (41.85) 25 (11.01) 227

Total 169 (49.27) 138 (40.23) 36 (10.50) 343

Body mass index (M = 0) \22 kg/m2 91 (48.15) 78 (41.27) 20 (10.58) 189 0.5712 1.1200

C22 kg/m2 79 (48.17) 62 (37.80) 23 (14.03) 164

170 (48.16) 140 (39.66) 43 (12.18) 353

M missing dataa Early stages include I, IIA, and IIBb Advanced stages include IIIA, IIIB, IIIC, and IV

Med Oncol (2013) 30:601 Page 3 of 5

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software (SPSS, Windows version release 15.0; SPSS Inc.;

Chicago, IL, USA). A P value of 0.05 was considered as

statistically significant level.

Results

MDR1 genetic polymorphisms identification

In this study, we detected a genetic polymorphism

(c.1564A [ T) within exon15 of MDR1 gene through the

CRS-PCR method. The sequence analyses indicated that

this genetic polymorphism was a non-synonymous muta-

tion in exon15 of MDR1 gene, which caused by A ? T

mutation and resulted in threonine (Thr) to serine (Ser)

amino acid replacement (p.Thr522Ser). The PCR products

were digested with MaeIII restriction enzyme (MBI Fer-

mentas, St. Leon-Rot, Germany) and divided into three

genotypes: AA (191 and 21 bp), AT (212, 191, and 21 bp),

and TT (212 bp, Table 1).

Allelic and genotypic frequencies

The allelic and genotypic frequencies were showed in

Table 2. The allele-A and genotype AA frequencies were

maximums in the cases and controls. The allele-A fre-

quencies in BC subjects and healthy controls were 67.99

and 72.6 %, and for allele T, frequencies were 32.01 and

27.36 %. The frequencies of genotype AA, AT, and TT in

BC patients were 48.16, 39.66, and 12.18 %, while these

genotypes frequencies of healthy subjects were 54.44,

36.39, and 9.17 %, respectively. The results of chi-square

test (v2) indicated that the distributions of this genetic

polymorphism were accordance with HWE in the studied

populations (P [ 0.05, Table 2).

Association between the MDR1 genetic polymorphism

and breast cancer

The potential association between the c.1564A [ T genetic

polymorphism and risk factors of BC in patients were

performed on Table 3. Our data indicated no statistically

significant association between this genetic polymorphism

and risk factors of BC in the current study (P [ 0.05).

Discussion

BC is the most polygenic malignant solid cancers in

women and is increasing in both developed and developing

countries [1–5]. It is caused from complex interactions

between genetic factors and environmental factors, and

genotypic variation plays key functions in human

phenotypic variability of cancer susceptibility [14]. The

potential association have been analyzed with several

MDR1 genetic polymorphisms and risk factors of BC. Most

of these studies were focused on the C3435T genetic

polymorphism [7, 11, 14, 19, 22, 23]. The C3435T genetic

polymorphism is located on exon26 of MDR1 gene, and it

has been founded to alter Pgp function and decreased tissue

protein expression and activity [20, 21]. Tatari et al. [7]

observed that the C3435T genetic polymorphism was not

associated with the susceptibility to BC in Iranian popu-

lation. Turgut et al. [11] detected a 1.5-fold increased risk

for the development of BC in allele T carriers for C3435T

genetic polymorphism. In the present study, we examined

the relevance of MDR1 genetic polymorphisms in relation

to BC susceptibility, clinical, and pathological character-

istics of BC using association analysis. We firstly detected

the c.1564A [ T genetic polymorphism in exon15 of

MDR1 gene through CRS-PCR method and evaluated the

potential correlation with risk factors of BC. Results from

our study suggested that no statistically significant differ-

ences were found in the distribution of BC patients’ risk

factors among different genotypes (Table 3, P [ 0.05).

There were no significant differences in the genotypic and

allelic frequencies (v2 = 3.3933, P = 0.1833; v2 =

3.6940, P = 0.0546, Table 2) between BC patients and

cancer-free controls. It was indicated that the c.1564A [ T

genetic polymorphism was not statistical significantly

associated with BC susceptibility in the population studied.

Our data demonstrated that for the c.1564A [ T genetic

polymorphism in MDR1 gene, BC patients do not differ

from cancer-free subjects in Chinese women. Our findings

add to the growing literature that suggests that the corre-

lation between genetic polymorphisms in MDR1 gene is

complex and incompletely understood, and further larger

and more detailed studies on different populations are

essential to confirm these findings and to reach to more

reliable results.

Conflict of interest The authors declare that they have no conflicts

of interest.

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