C3435T polymorphism in the MDR1 gene and breast cancer risk in northeastern Mexico

Abstract

The multidrug resistance gene 1 (MDR1) encodes a membrane-bound phosphoglycoprotein (P-gp). It functions as a transmembrane efflux pump for various structurally unrelated carcinogens and toxins. Polymorphism C3435T of MDR1 has been investigated for its association with breast cancer in different populations. However, the results are inconsistent and inconclusive. The objective of this study was to determine whether an association exists between the MDR1 C3435T polymorphism and the risk of breast cancer in a population from northeastern Mexico, which displays ethnic characteristics that differentiate it from other populations of the country. Genotypes were determined for 243 women with histologically confirmed breast cancer and 118 control subjects. Polymorphism of MDR1 C3435T was analyzed by DNA microarray. We found an increased breast cancer risk associated with CT and CC genotypes (OR = 1.88, 95% CI: 1.04-3.39, P = 0.033 for CT vs. TT; OR = 2.91, 95% CI: 1.48-5.74, P = 0.001 for CC vs. TT). Furthermore, there was significantly increased risk of breast cancer associated with the C allele (OR = 1.59, 95% CI: 1.16-2.18, P = 0.003). In conclusion, we found an association between the MDR1 C3435T polymorphism and risk of breast cancer in subjects from northeastern Mexico. Identification of inter-individual variability in this polymorphism may be useful for individualizing breast cancer genetic screening and therapeutic intervention.

Introduction

Breast cancer is the most common type of cancer and the most common cause of cancer death among women worldwide [1]. The disease has important genetic and environmental factors, most of them still unknown. It is possible that the mechanism of breast carcinogenesis may representa complex interplay between exposure to potential toxins and carcinogens and the genes involved in the detoxification pathways [2].

The multidrug resistance gene 1 (MDR1) encodes a membrane-bound phosphoglycoprotein (P-gp). It functions as a transmembrane efflux pump for various structurally unrelated carcinogens and toxins such as organic cations, amino acids, polysaccharides, proteins, and antibiotics [3,4].

MDR1 is highly polymorphic with more than 50 single-nucleotide polymorphisms (SNPs) identified [5]. One of the most important SNPs is C3435T in exon 26, which alters gene expression, protein activity, and substrate specificity. It may limit the local detoxification activity in breast tissue and be a risk factor for cancer development and behavior [6-8]. Several studies have investigated the relationship between the C3435T polymorphism and the risk of breast cancer in different populations. However, the findings of these studies are still inconclusive [8-19, see also meta-analyses 20-25].

In Mexico, two related studies have been carried out, in which only subjects from the central-western area of the country were analyzed [26,27]. We previously investigated the relationship between several genetic polymorphisms and breast cancer in northeastern Mexico and found significant differences with other populations of the country [28,29]. The purpose of this study was to determine whether an association exists between the MDR1 C3435T polymorphism and the risk of breast cancer in subjects from northeastern Mexico.

Material and methods

Study population

The subjects and methods have been described in detail in previous reports [28-30]. The case subjects were 243 women with histologically confirmed breast cancer treated at the University Cancer Center of the University Hospital “Dr José E González” of the Autonomous University of Nuevo Leon and the Hospital of Specialties number 25 of the Mexican Institute of Social Security, both located in Monterrey, Nuevo Leon, Mexico. Both are reference centers for breast cancer patients from throughout the northeastern area of Mexico, which includes the states of Coahuila, Nuevo Leon, San Luis Potosi, Tamaulipas, and Zacatecas. One hundred eighteen controls with no previous history of any type of cancer or other vital disease were also studied. The study was performed in accordance with the tenets of the Declaration of Helsinki, and was approved by the local Ethics Committee (registration HU BI10-002), and all participants provided written informed consent.

Genotyping

For genotype analysis, genomic DNA was obtained from peripheral blood samples either using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol or using TSNT lysis buffer (1% Triton, 1% sodium dodecyl sulfate, 100 mM NaCl, 10 mM Tris-HCl [pH 8.0], and 1 mM EDTA) followed by phenol-chloroform extraction and ethanol precipitation. The extracted DNA was stored at -20°C until use. Analysis of MDR1 C3435T polymorphism was performed using the PHARMAchip^® DNA microarray following the manufacturer’s protocols (Progenika Biopharma SA, Derio, Spain).

Statistical analysis

The difference in genotype or allele frequencies between breast cancer patients and control subjects was analyzed by a Crosstab and Pearson’s chi-square test. The data were input to the Statistical Package for Social Sciences software (SPSS, Windows version release 22.0; SPSS Inc.; Chicago, IL) for handling and further statistical analyses. Hardy-Weinberg equilibrium was examined by the Chi-square test using the public software developed by Tim M. Strom and Thomas F. Wienker (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association between the polymorphism and cancer risk (Epi Info program version 7.1.3, CDC, Atlanta, GA, USA). Statistical significance was set at P<0.05 for all tests, and all test were two-sided.

Results

The distribution of MDR1 C3435T genotype in patients (P = 0.048) but not for controls (P = 0.350) showed significant deviation from a Hardy-Weinberg equilibrium.

The CC genotype was found in 32.8%, CT genotype was found in 54.2%, and TT genotype was found in only 13.0% in the breast cancer patients. In the control group, frequencies of genotypes were 21.2% for CC, 54.2% for CT and 24.6% for TT. This difference was found to be statistically significant (P = 0.007). Furthermore, in the stratified analysis, there were significantly increased risks of breast cancer associated with CT and CC genotypes (OR = 1.88, 95% CI: 1.04-3.39, P = 0.033 for CT vs. TT; OR = 2.91, 95% CI: 1.48-5.74, P = 0.001 for CC vs. TT).

In the patient group, the C allele frequency was 60.0% and the T allele frequency was 40.0%, and in the control group the frequencies were 48.0% for C allele and 52.0% for T allele. There was significantly increased risk of breast cancer associated with the C allele (OR = 1.59, 95% CI: 1.16-2.18, P = 0.003). Results are summarized in Table 1.

Table 1.

Genotype and allele frequencies of the C3435T polymorphism of the multidrug resistance gene 1 (MDR1) in breast cancer patients and healthy controls from northeastern Mexico

Variable	Cases* (%)	Controls (%)	OR (95% CI)	P
Genotypes
TT	31 (13.0)	29 (24.6)	Reference
CT	129 (54.2)	64 (54.2)	1.88 (1.04-3.39)	0.033
CC	78 (32.8)	25 (21.2)	2.91 (1.48-5.74)	0.001
Alleles
T	95 (40.0)	61 (52.0)	Reference
C	143 (60.0)	57 (48.0)	1.59 (1.16-2.18)	0.003

^*Numbers may not sum to total due to missing data.

OR-odds ratio; CI-confidence interval.

Discussion

Several studies have investigated the relationship between the C3435T polymorphism of the MDR1 gene and the risk of breast cancer in different populations. The purpose of this study was to determine whether an association exists between this polymorphism and the risk of developing breast cancer in subjects from northeastern Mexico.

Some studies have reported a link between the C3435T polymorphism and the risk of developing breast cancer [8,9,11,14,17,18,21-25,27], while others have failed to observe an association [10,12,13,15,16,19,20,26]. Our results indicate that the frequencies of the CC and CT genotypes and of the C allele were significantly higher in breast cancer patients compared to controls (Table 1).

P-gp, encoded by MDR1, can prevent intracellular accumulation of potentially toxic substances and metabolites [31]. Its dysfunction would reduce the protection for cells and would contribute to development of breast cancer [9,23]. Functional analyses have shown that the C3435T polymorphism significantly decreases both MDR1 expression and P-gp activity [32,33]. Most of the studies that have found a relationship between the C3435T polymorphism and breast cancer have also reported the association of TT genotype and T allele with high risk of developing the disease [9,11,14,17,21-25,27].

However, other studies did not establish an association between the C3435T polymorphism and MDR1 expression or P-gp activity [34,35]. Furthermore, results obtained by Zubor et al. [8] and Abuhaliema et al. [18] revealed that subjects carrying CC genotype of the C3435T polymorphism had increased risk of breast cancer. Similarly, Ghafouri et al. [19] reported that the frequencies of CC genotype and C allele were higher in patients than in controls, although the differences were not statistically significant. It has been hypothesized that in patients with the CC genotype there might be linkage disequilibrium with other polymorphisms in the MDR1 gene as well as errors in the post-translational modification in the protein structure of P-gp, which might make it defective [18,36].

The discrepancies among studies may be due to several factors, including differential exposure to specific environmental agents, variation in methodologies employed, and ethnic differences among analyzed populations. Lu et al. [20], Wang et al. [21], and Sheng et al. [22] found association between the C3435T polymorphism and the risk of breast cancer in Caucasians but not in Asians. More recently, Tazzite et al. [24] found that patients with TT genotype had a significantly increased risk of breast cancer in Asians, Caucasians, and North African but not among mixed populations. Thus, ethnicity might have a great effect on the relationship between the C3435T polymorphism and breast cancer risk.

In Mexico, two related studies have been carried out, in which only subjects from the central-western area of the country were analyzed.Macías-Gómez et al. [26] showed that there was no significant difference between breast cancer patients and control groups for the C3435T polymorphism. Gutierrez-Rubio et al. [27] did not find differences in the presence of this polymorphism between total breast cancer cases and the reference group. However, pre-menopausal T allele carriers showed a 2-fold increased risk of breast cancer with respect to the reference and post-menopausal groups. The differences between these studies and our results are most probably related to ethnicity.

The Mexican population has a high degree of genetic variability. Mestizos constitute the main population in Mexico resulting from admixture between Spaniards, Amerindians, and Africans, principally [37]. Analyses with autosomal short tandem repeats (STRs) have demonstrated significant differences in ancestral components of Mestizo populations throughout the Mexican territory, displaying an increasing North-to-South gradient of Amerindian ancestry, and vice versa regarding the European component [38]. Moreno-Estrada et al. examined local patterns of variation from nearly 1 million genome-wide autosomal SNPs for individuals from several populations, covering most geographic regions of Mexico. They demonstrated a high degree of fine-scale genomic structure across the country, shaped by pre-Columbian population dynamics and affecting the present-day genomes of Mexican mestizos, which is of both anthropological and biomedical relevance. The study unveiled genetic differences among Mexicans as extensive as the variations between some Europeans and Asians [39]. The genetic variability among Mexicans from different regions of the country has been corroborated with analyses of other genetic systems, such as mitochondrial DNA (mtDNA) [40], and Y-chromosome markers, including Y-STRs [41] and Y-SNPs [42].

Our study examined subjects from northeastern Mexico, whereas in the previous studies [26,27] subjects from the country’s central-western zone were analyzed. Thus, ethnic diversity may have contributed to the discrepancies observed between these studies. In fact, we previously investigated the relationship between several genetic polymorphisms and breast cancer in northeastern Mexico and found significant differences with populations from central [28] and central-western [29] areas of the country.

In conclusion, we analyzed the association between the MDR1 C3435T polymorphism and the risk of developing breast cancer in subjects from northeastern Mexico. A significant increase in the risk of developing breast cancer was associated with the presence of the CC and CT genotypes and of the C allele. Identification of inter-individual variability in MDR1 C3435T polymorphism may be useful for individualizing breast cancer genetic screening and therapeutic intervention. Further studies estimating the effect of gene-gene and gene-environment interactions may provide a better understanding of the role of MDR1 C3435T polymorphism in breast cancer predisposition.

Disclosure of conflict of interest

None.