Abstract: Male breast cancer is an uncommon disease that has been the focus of limited research. In the present study, we specifically investigated whether combination of common genetic variants in the X-ray repair cross-complementation group 1 gene (XRCC1; exon 10, codon 399 Arg/Gln and exon 6, codon 194 Arg/Trp) and loss-of-function deletion polymorphisms in glutathione S-transferases T1 (GSTT1) and M1 (GSTM1), were associated with an altered risk of male breast cancer. Blood samples from 10 males with breast cancer and 15 age- and sex-matched healthy persons were collected. The GSTM1, GSTT1 and XRCC1 genotypes were determined using PCR-based method. The null genotypes of GSTM1 and GSTT1 and 399Gln and 194Trp alleles of XRCC1 assumed as high-risk genotypes and alleles. Statistical analysis showed that the χ2 for linear trend for 0, 1, 2 and 3 putative high-risk genotypes is equal to 5.673 (p = 0.017).
Introduction
Male Breast Cancer (MBC) is a rare disease that has been the focus of limited research. It accounts for less than 1% of all cases of cancer in men and about 0.7% of all diagnosed breast cancer cases (Giordano, 2005). Very little is known about the genetic background of MBC. Polymorphisms in a few genes (such as androgen receptor, BRCA1, BRCA2) have been associated with predisposition to the development of male breast cancer (Basham et al., 2002; Csokay et al., 1999; MacLean et al., 2004). The strongest known risk factors for MBC are mutations in BRCA1 and particularly BRCA2 gene (Basham et al., 2002; Csokay et al., 1999; MacLean et al., 2004).
Enzymes involved in the detoxification of carcinogenic compounds as well as DNA repair system may play an important role(s) in the susceptibility to develop some types of cancers. GSTs constitute a super family of ubiquitous, multi functional enzymes, which play a key role in detoxification. The GSTs catalyze the conjugation of the tripeptide glutathione (GSH) to a wide variety of exogenous and endogenous chemicals with electrophilic functional group. Both GSTM1 (a member of GSTμ) and GSTT1 (a member of GSTθ) are known to be polymorphic in human and both of them have null alleles resulting in gene deletion and complete absence of enzyme activity. A number of studies have considered GSTM1 and GSTT1 polymorphism in relation to female breast cancer risk, with conflicting results (Egan et al., 2004; Saadat and Saadat, 2004; Vogl et al., 2004).
The X-ray repair cross-complementation group 1 (XRCC1) protein plays an important role in base excision repair (Shu et al., 2003). It is reported that the Arg194Trp and Arg399Gln polymorphisms of XRCC1 may modulate repair function and are involved in cancer susceptibility (Divine et al., 2001; Duell et al., 2001; Smith et al., 2003; Stren et al., 2001; Sturgis et al., 1999). The 399 Gln and 194Arg alleles have been linked to an increased risk of several types of cancers (Divine et al., 2001; Stren et al., 2001; Sturgis et al., 1999). In additional, several studies have reported a positive association between polymorphisms of XRCC1 at codon 399 and/or codon 194 and female breast cancer but the results are inconsistent (Duell et al., 2001; Smith et al., 2003).
Because there is no published data on the GSTM1, GSTT1 and polymorphism of XRCC1 at codons 399 and 194 and susceptibility to male breast cancer, the present study was done.
Materials and Methods
Subjects
This case-control study consisted of 10 males with breast cancer that were
recruited from chemotherapy department of Nemazee hospital in Shiraz, Iran,
during February 2005 to February 2006. A total of 15 healthy male matched with
patients according to age (±5 years), as a control group. The mean age
of the patients and the controls was 63.5±11.1 years and 58.13±8.4
years, respectively. Subjects were unrelated Iranian Muslims. Informed consent
was obtained from each subject before the study.
DNA Extraction and Genotyping Analysis
Blood sample were collected from the subject. Genomic DNA was extracted
from whole blood samples. The PCR condition for determining GSTM1 and
GSTT1 genotypes were the same as that reported previously (Saadat and
Saadat, 2001). To test for contamination, negative controls (tubes containing
the PCR mixture without the DNA template) were included in every run. A 1030
bp fragment was amplified by PCR with the GSTM1 primers and a 480 bp
fragment was amplified by PCR with the GSTT1 primers. The absence of
amplified product was consistent with the null genotypes. Successful amplification
by albumin specific primers confirmed the proper function of the PCR.
XRCC1 genotypes were examined using PCR-RFLP assays. Genotypic analysis for the XRCC1 gene was determined in DNA by PCR assay, using the PCR primers described by Lunn et al. (1999) for the codons 194 and 399. These primers generate 491 and 615 bp products containing the polymorphic site at codons 194 and 399, respectively. The XRCC1 Arg399Gln polymorphism was genotyped by restriction enzyme digestion of the PCR product with the MspI enzyme. The MspI restricted products, Arg/Arg, Arg/Gln and Gln/Gln genotypes had band sizes of 374/221, 615/374/221 and 615 bp, respectively (Abdel-Rahman and El-Zein, 2000). The XRCC1 Arg/Trp polymorphism at codon 194 was genotype by digestion of the PCR fragment with the PvuII enzyme. The PvuII restricted products, Arg/Arg, Arg/Trp and Trp/Trp genotypes had band sizes of 490, 490/294/196 and 294/196 bp (Hu et al., 2001).
Statistical Analysis
Based on the published data the null genotypes of GSTM1 and GSTT1
and 399Gln and 194Trp alleles of XRCC1 assumed as high-risk
genotypes and alleles, respectively (Divine et al., 2001; Duell et
al., 2001; Egan et al., 2004; Saadat and Saadat, 2001; Smith et
al., 2003; Stren et al., 2001; Sturgis et al., 1999). In order
to count the high-risk genotypes we counted the genotypes as follows:
| • | Positive genotype of GSTM1 = 0, Null genotype of GSTM1 = 1 |
| • | Positive genotype of GSTT1= 0, Null genotype of GSTT1 = 1 |
| • | Genotypes of Arg/Arg, Arg/Gln and Gln/Gln at codon 399 XRCC1 counted as 0, 1 and 2, respectively. |
| • | Genotypes of Arg/Arg and Arg/Trp at codon 194 XRCC1 counted as 0 and 1, respectively. |
The relative associations between the combination of genotypes and breast cancer risk were assessed by calculating Odds Ratios (OR) and 95% Confidence Intervals (CIs). Also the χ2 for linear trend was calculated. Data analysis was performed using SPSS software version 11.5. A probability of p<0.05 considered statistically significant difference.
Results and Discussion
Table 1 shows the genotypes of 25 males comprising of 10 breast cancer cases and 15 healthy subjects as control group. Table 2 shows the number of putative high risk genotypes in patients and control subjects. As shown in Table 2, there is no patient with 0 high risk genotypes, whereas about 13% of control subjects belong to this combination of genotypes. However, 50% of cases and about 6% of controls had 3 putative high risk genotypes. The presence of 3 putative high-risk genotypes results in an almost 20-fold risk of the diseases in comparison with 0 and/or 1 putative high-risk genotypes (OR = 19.99, 95% CI: 1.42-282.4, p = 0.027). Statistical analysis showed that χ2 for linear trend for 0, 1, 2 and 3 putative high risk genotypes is equal to 5.673 (p = 0.017). This means that the male breast cancer risk increased as a function of the number of high-risk genotypes of GSTM1, GSTT1 and 399Gln and 194Trp of XRCC1. There are several potential explanations for our findings. First, the high-risk genotypes have additive effect. Same effects were reported previously for developing some types of multifactorial disease (Saadat and Saadat, 2001; Saadat et al., 2004). It may be suggested that inability to detoxify many xenobiotics, including an array of environmental carcinogens and endogenously derived reactive oxygen species because of null genotypes of GSTM1 and/or GSTT1 (Hayes and Strong 1995; Saadat et al., 2004) and decreased DNA repair capacity or fidelity because of the presence 399Gln and/or 194Trp alleles of XRCC1 (Abdel-Rahman and El-Zein, 2000), perpetuate the DNA damage and consequently increase male breast cancer risk. Second, we can not rule out chance or random association. Less is known about the genetic influences in male breast cancer development.
| Table 1: | Genotypes of GSTM1, GSTT1 and XRCC1 (at codons 194 and 399) in studied subjects |
| Table 2: | Association between the combination of GSTM1, GSTT1 and XRCC1 (at codons 194 and 399) genotypes and male breast cancer |
| χ2 for linear trend for 0, 1, 2 and 3 putative high-risk genotypes is equal to 5.673 (p = 0.017) | |
This is due to the fact that this disease is rare and large-scale genetic epidemiologic studies have been difficult to carry out. With limited sample size, present results allow for preliminary conclusions and future large studies are warranted to further test DNA repair genetic variants and polymorphisms of genes involved in detoxification in male breast cancer susceptibility.
Acknowledgments
The authors are indebted to the participants for their close cooperation. The authors are indebted to Dr. Maryam Ansari-Lari for critical reading of the manuscript and for her contribution in discussion. This study was supported by Shiraz University.