Association Between the c.3073A>C Genetic Polymorphism of the MDR1 Gene and Susceptibility to Gastric Cancer in the Chinese Han Population

Abstract

Objective: Gastric cancer is one of the most common malignancies in the world. The multidrug resistance 1 gene (MDR1) is an important candidate gene for influencing the susceptibility to gastric cancer. The objective of this study was to find out the association of MDR1 genetic variants with gastric cancer susceptibility in the Chinese Han population. Methods: In total, 375 gastric cancer patients and 378 cancer-free controls were included. The c.3073A>C genetic polymorphism of the MDR1 gene was genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. Results: We found that the genotypes/alleles from c.3073A>C genetic polymorphisms were statistically associated with gastric cancer risk. The risk of gastric cancer was significantly higher for the CC genotype as compared to the AA wild genotype (odds ratio [OR]=1.85, 95% confidence interval [CI] 1.15-2.97, p=0.010). The allele C may contribute to the susceptibility to gastric cancer (OR=1.37, 95% CI 1.10-1.70, p=0.005). Conclusion: These preliminary results indicate that the c.3073A>C genetic polymorphism of the MDR1 gene is potentially related to the susceptibility to gastric cancer in the Chinese Han population.

Introduction

Gastric cancer is one of the most common malignancies worldwide (Parkin et al., 2005; Ferlay et al., 2010; Jemal et al., 2011; Ferlay et al., 2013). Although the incidence and mortality rate of gastric cancer have been decreasing over the last few decades, gastric cancer remains as one of the leading causes of cancer death in China (Stadtlander and Waterbor, 1999; Shen et al., 2000; Tahara et al., 2011; Chen et al., 2012). It is widely accepted that genetic factors play key roles in the pathogenesis of gastric cancer. The multidrug resistance 1 (MDR1) gene is considered to be one of the most important candidate genes for influencing the susceptibility to gastric cancer (Tahara et al., 2007; Lee et al., 2008; Sugimoto et al., 2008; Sabahi et al., 2010; Tahara et al., 2010; Li et al., 2011; Tahara et al., 2011; Sheng et al., 2012; Wang et al., 2012). The potential associations of genetic variants of the MDR1 gene with the risk of gastric cancer have been assessed, such as the C3435T genetic variant (Tahara et al., 2007; Sugimoto et al., 2008; Sabahi et al., 2010; Tahara et al., 2011; Sheng et al., 2012; Wang et al., 2012). However, up to date, no similar studies have reported the potential association of the c.3073A>C genetic variant of the MDR1 gene with the risk factors of gastric cancer. Therefore, the objective of this study aimed to find out the association of MDR1 c.3073A>C genetic variant with the susceptibility to gastric cancer.

Materials and Methods

Subjects

In this case-control study, 375 gastric cancer patients with a pathology-confirmed diagnosis and 378 healthy age-matched subjects who had no history of any gastric diseases as controls were enrolled from the First Affiliated Hospital of Soochow University. Demographic clinical characteristics of subjects enrolled in this study are summarized in Table 1. The Ethics Committee of the First Affiliated Hospital of Soochow University approved the protocol of this study, and the written informed consent form was obtained from each subject.

Table 1.

The Clinical Characteristics of Gastric Cancer Cases and Cancer-Free Controls

Characteristics	Cases (n)		Controls (n)		χ²-value	p-Value
Number	375	49.80	378	50.20
Age (years)					0.21704	0.64131
Mean±SD	61.66±12.75		63.15±13.45
<60	201	53.60	209	55.29
≥60	174	46.40	169	44.71
Gender (n)					0.84188	0.35886
Male	172	45.87	186	49.21
Female	203	54.13	192	50.79
Alcohol drinking					1.31401	0.25167
Yes	214	57.07	200	52.91
No	161	42.93	178	47.09
Tobacco smoking					2.80011	0.09426
Yes	226	60.27	205	54.23
No	149	39.73	173	45.77
Family history of gastric cancer (n)					1.65896	0.19774
Yes	211	56.27	195	51.59
No	164	43.73	183	48.41
H. pylori infection (n)					1.26522	0.26067
Yes	172	45.87	158	41.80
No	203	54.13	220	58.20

Genotyping

Genomic DNA was isolated from peripheral venous blood from each subject, using the standard extraction method and then stored at −80°C (Daly et al., 1996). The polymerase chain reaction (PCR) primers were designed by Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, CA). The primers sequences, annealing temperature, fragment region, and size are shown in Table 2. For PCR, 50 ng template DNA was amplified in 20 μL of reaction mixture containing 1× buffer (Tris-HCl 100 mM, pH 8.3; KCl 500 mM), 0.25 μM primers, 2.0 mM MgCl₂, 0.25 mM dNTPs, and 0.5U Taq DNA polymerase (Promega, Madison, WI). The PCR amplified PCR protocol was carried out at 94°C for 5 min, followed by 32 cycles of 94°C for 32 s, 56.0°C for 32 s, and 72°C for 32 s, and a final extension at 72°C for 8 min. The c.3073A>C genetic polymorphism of the MDR1 gene was genotyped by the PCR-restriction fragment length polymorphism (PCR-RFLP) method. The PCR products (5 μL) were digested with the 2U MaeIII restriction enzyme (MBI Fermentas, St. Leon-Rot, Germany) at 37°C for 10 h. The digested products were separated by electrophoresis on an agarose gel, and then observed under UV light. To make sure of concordance with the genotyping results of PCR-RFLP, about 10% of random samples were verified by DNA sequencing (ABI3730×l DNA Analyzer, Applied Biosystems, Foster City, CA).

Table 2.

The PCR-RFLP Analysis for the c.3073A>C Genetic Polymorphism in the MDR1 Gene

Primer sequences	Annealing temperature (°C)	Amplification fragment (bp)	Region	Restriction enzyme	Genotype (bp)
5′-GAAGCATGAGTTGTGAAGATAATAT-3′	56.0	234	Exon22	MaeIII	AA:154,80
5′-CTTTCAGTGCTTGTCCAGACA-3′					AC:234,154,80
					CC:234

PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism.

Statistical analysis

All statistical analyses were performed by SPSS software (Windows version release 15.0; SPSS, Inc., Chicago, IL). The Hardy-Weinberg equilibrium in genotypic distributions, and clinical characteristics were evaluated by the chi-squared (χ²) test. A p value less than 0.05 was considered as statistically significant.

Results

Subjects characteristics

In total, 753 subjects were recruited in the present study. As shown in Table 1, no statistically significant differences between gastric cancer patients and healthy controls were found with regard to age, gender, alcohol drinking, tobacco smoking, H.pyori infection, and family history of gastric cancer (All p values>0.05).

MDR1 genetic polymorphism identification

We assessed the c.3073A>C genetic polymorphism within human MDR1 gene exon22 through the PCR-RFLP and DNA sequencing methods. The sequence analyses suggest that this genetic polymorphism is a nonsynonymous mutation. It is caused by an A to C mutation and resulted in leucine (Leu) to phenylalanine (Phe) amino acid replacement (p.Leu860Phe, reference sequences: GenBank IDs: NG_011513.1, NM_000927.4, and NP_000918.2). The amplified PCR products were digested with the MaeIII restriction enzyme (MBI Fermentas, St. Leon-Rot, Germany) and divided into three genotypes: AA (154 and 80 bp), AC (234,154, and 80 bp), and CC (234 bp, Table 2). The allelic and genotypic frequencies are shown in Table 3. The allele frequencies of gastric cancer patients (A, 64.80%; C, 35.20%) were statistically significantly different from cancer-free controls (A, 71.56%; C, 28.44%; χ²=7.9342, p=0.0049). The genotype frequencies of gastric cancer patients (AA, 44.00%; AT, 41.60%; TT, 14.40%) were not consistent with cancer-free controls (AA, 52.38%; AC, 38.36%; CC 9.26%), the differences being statistically significant (χ²=7.4463, p=0.0242). The chi-square test (χ²) indicated that genetic polymorphisms corresponded with the Hardy-Weinberg equilibrium (p>0.05, Table 3).

Table 3.

The Genotype and Allele Frequencies of the MDR1 c.3073A>C Genetic Polymorphism in Cases and Controls

	Genotypic frequencies (%)			Allelic frequencies (%)
Groups	AA	AC	CC	A	C	χ²	p
Case group (n=375)	165 (44.00)	156(41.60)	54 (14.40)	486 (64.80)	264 (35.20)	2.9108	0.2333
Control group (n=378)	198 (52.38)	145 (38.36)	35 (9.26)	541 (71.56)	215 (28.44)	1.2523	0.5346
Total (n=753)	363 (48.21)	301 (39.97)	89 (11.82)	1027 (68.19)	479 (31.81)	4.6426	0.0981
	χ²=7.4463, p=0.0242			χ²=7.9342, p=0.0049

Association between MDR1 genetic polymorphism and gastric cancer

The association between MDR1 genetic polymorphism and the risk of gastric cancer is summarized in Table 4. The statistically significantly increased risk of gastric cancer was found in homozygote comparison (CC vs. AA: OR=1.85, 95% CI 1.15-2.97, χ²=6.61, p=0.010), dominant model (CC/AC vs. AA: OR=1.40, 95% CI 1.05-1.87, χ²=5.29, p=0.021), recessive model (CC vs. AC/AA: OR=1.65, 95% CI 1.05-2.59, χ²=4.77, p=0.029), and allele contrast (C vs. A: OR=1.37, 95% CI 1.10-1.70, χ²=7.93, p=0.005, Table 4). We failed to detect a statistically significantly increased gastric cancer risk in the heterozygote comparison (AC vs. AA: OR=1.29, 95% CI 0.95-1.75, χ²=2.67, p=0.102).

Table 4.

Association Between MDR1 c.3073A>C Gene Polymorphism and Gastric Cancer Risk

	Test of association
Comparisons	OR (95% CI)	χ²-value	p-Value
Homozygote comparison	1.85 (1.15-2.97)	6.61	0.010
(CC vs. AA)
Heterozygote comparison	1.29 (0.95-1.75)	2.67	0.102
(AC vs. AA)
Dominant model	1.40 (1.05-1.87)	5.29	0.021
(CC/AC vs. AA)
Recessive model	1.65 (1.05-2.59)	4.77	0.029
(CC vs. AC/AA)
Allele contrast	1.37 (1.10-1.70)	7.93	0.005
(C vs. A)

OR, odds ratio; CI, confidence interval.

Discussion

Gastric cancer is a polygenic malignant solid cancer arising from complex interactions between the environmental and genetic factors. It is well known that genetic variants of candidate genes for influencing the development of gastric cancer play key roles in the pathogenesis of cancer. In the current study, we investigated the influencing of genetic variants of the MDR1 gene on gastric cancer risk in the Chinese Han population by association analysis. The different genotypes of c.3073A>C genetic variant in exon22 of the MDR1 gene were detected by PCR-RFLP and DNA sequencing methods. The distribution of allele and genotype frequencies in gastric cancer patients was significantly different from cancer-free controls (p<0.05, Table 3). The genotype CC was statistically associated with increased gastric cancer risk compared with genotype AA and AC/AA carriers (p<0.05, Table 4). The allele C and genotype CC of this genetic variant could contribute to increase gastric cancer risk in the Chinese Han population. Our data demonstrated that the c.3073A>C genetic variant of the MDR1 gene was statistically associated with gastric cancer risk in the Chinese Han population, and could be used as a molecular marker for detecting the susceptibility to gastric cancer. In recent years, several similar studies have reported the potential association of other genetic variants of the MDR1 gene with susceptibility risk (Tahara et al., 2007; Sugimoto et al., 2008; Sabahi et al., 2010; Tahara et al., 2011; Sheng et al., 2012; Wang et al., 2012). These observations are consistent with our results that MDR1 genetic variants may contribute to susceptibility risk. Our findings could provide more evidence for further analysis about the role of the MDR1 gene for gastric cancer susceptibility. Further functional studies are needed to confirm these findings in larger populations, and to elucidate the molecular mechanisms of genetic variants in influencing susceptibility risk.

Footnotes

Author Disclosure Statement

The authors have no conflicts of interest.

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