SHORT COMMUNICATION
Breast cancer risk not only was not associated with CYP17/A2 allele but also was related to A1 allele

Introduction

Breast cancer is the second most common cancer in the world and the most common cancer in Iranian women in terms of rate [1-3].
A large population study in Iranian patients shows that breast cancer is the most common, with an incidence of about 22 per 100,000 [4].
Polymorphisms of the CYP 17 genes were involved in oestrogens biosynthesis in modulating the susceptibility to breast cancer [5-8].
A single nucleotide polymorphism (SNP) creates a restriction recognition site, resulting in two alleles designated A1 and A2 [T (A1) to C (A2)] [6]. Ethnic differences of the variant allele frequencies were found by other groups between affected women [1, 2, 9-12].
The aim of our study was to show the allelic variants in the CYP17 gene in Iranian sporadic breast cancer patients.

Material and methods



Patient data

Analyses were conducted for 53 patients and 53 controls genotyped for CYP17, including 14 patients and 21 controls of premenopausal women, and 39 patients and 32 controls of postmenopausal women, and ages were 35-55 years.
This study was ethically approved by the local Ethical Committee of Islamic Azad University from the point of view of patients’ and also controls’ rights.
All patients participated in the Special Medical Centre, part of chemotherapy, Tehran, Iran. A questionnaire including questions on breast cancer risk factors were completed and each patient completed a consent form. The blood samples were collected from patients and controls prior to the start of treatment. Subjects were genotyped for CYP17 SNP using genomic DNA extracted from peripheral blood lymphocytes. DNA was isolated from peripheral blood using the FelxiGene DNA extraction kit (Qiagen Germany).

Genotyping

Bio-Rad PCR System was used to amplify 414 bp fragments, which include the polymorphic site in the promoter region of CYP 17. The cycling condition for CYP17 A1/A2 gene polymorphism was set according to a previously published method [6], with some changes, such as: one cycle at 94°C for 5 min, 35 cycles at 94°C for 40 s, 55°C for 45 s, 72°C for 90 s, and one final extension cycle at 72°C for 7 min.
The genotypes and allelic frequencies of CYP17 A1/A2 polymorphisms in patient and control groups were analysed using c2 and Fisher’s exact tests.
Genotyping of the MspA1 polymorphism in the 5’- untranslated region of the CYP17 gene was determined by PCR restriction fragment length polymorphism, which includes the polymorphic site in the promoter region of CYP17, according to a previously published method [2], with slight modifications.
The PCR products were digested with restriction enzyme MspA1I (Biolab, USA), separated by 3% agarose gel electrophoresis and identified with ethidium bromide staining. This method is able to detect all three possible genotypes for the polymorphism: A1A1 (homozygous wild type), A1A2 (heterozygous variant type) and A2A2 (homozygous variant type) (Figure 1). The primers used for PCR were designed as follows:
Forward 5’-CATTCGCACTCTGGAGTC-3’
Reverse 5’-AGGCTCTTGGGGTACTTG-3’

Results

Analyses of affected and controls show that A1/A2 genotype has the highest frequency in both groups (54.7 in patients and 62.3 in control group).
The A1 homozygote genotype has an increase in the patient group (34.0) compared with controls (13.2) and A2 homozygote genotype has a decrease in the patient group (11.3) compared to the control group (24.5) (Table I).
Comparison between genotypes, odds ratio and p value showed that p value of A1/A1 / A2/A2 was p = 0.008. Moreover, p value for A1/A1 / A1/A2 was p = 0.033 and for A2/A2 / A1/A2 was 0.242.
Odds ratio for A1/A1 / A2/A2 was 5.571 (95% confidence interval, 1.514-20.506), was 2.926 for A1/A1 / A1/A2 (95% confidence interval, 1.071-7.998) and was 1.904 for A2/A2 / A1/A2 (95% confidence interval, 641-5.654).

Discussion

The impact of CYP17 genetic polymorphism on the risk of breast cancer received a lot of interest after Feigelson and colleagues first reported an increase in risk of advanced breast cancer for carriers of the A2 allele [13].
Some population studies including young Indian, Korean, and Russian women suggest that CYP17 homozygote A2 allele gene polymorphism might play a significant role in breast cancer development [14-16]. The published data in American Caucasian women showed that women with homozygote A2 alleles were more likely to share characteristics associated with greater breast cancer susceptibility [2], although a few exceptions, in the study of Bergman et al. and Spurdle et al., were found [17, 18]. However, most subsequent studies did not find an overall increase in risk with the A2/A2 genotype [2, 19-23]. On the other hand, interestingly, some population-based studies showed no overall association between CYP17 gene polymorphism and breast cancer risk [5, 24, 25].
In agreement with the majority of the previous studies on CYP17 polymorphism and breast cancer risk, this study did not reveal any significant association between the CYP17 A2 allele and overall risk of breast cancer in Iranian women.
In the present study, we found that the A2 allele did not increase breast cancer risk. The difference between patient and control groups, regarding the genotype ratio of A1/A2 heterozygote, was non-significant. On the other hand, comparison of A1/A1 genotype and A2/A2 genotype ratio in the two groups was significant.
However, no association was found between the presence of A2 allele and breast cancer susceptibility. Nevertheless, there is a significant association between presence of A1 allele and breast cancer occurrence.
Our results showed that A1/A1 was significantly increased in the patient group and A2/A2 polymorphism was decreased in the control group.
In conclusion, that not only was A2/A2 in our patients not associated with breast cancer risk but also there is a relation between presence of A1/A1 and increase of breast cancer risk.

Acknowledgments

We would like to thank all the patients for their kind collaboration in our projects, Dr. M.S. Fallah Mahboobpasand for his kind consultation in statistical analysis, and the Islamic Azad University for supporting this research.
Finally, we would like to thank the head and physicians of the Special Medical Centre, Tehran, Iran, who helped us during this project.

References

1. Picado-Leonard J, Miller WL. Cloning and sequence of the human gene for P450c17 (steroid 17 alpha-hydroxylase/17, 20 lyase): similarity with the gene for P450c21. DNA 1987; 6: 439-48.
2. Ambrosone CB, Moysich KB, Furberg H, et al. CYP17 genetic polymorphism, breast cancer, and breast cancer risk factors. Breast Cancer Res 2003; 5: R45-51.
3. Mousavi SM, Montazeri A, Mohagheghi MA, et al. Breast cancer in Iran: an epidemiological review. Breast J 2007; 13: 383-91.
4. Harirchi I, Karbakhsh M, Kashefi A, Momtahen AJ. Breast cancer in Iran: Results of a multi-center study. Asian Pac J Cancer Prev 2004; 5: 24-7.
5. Verla-Tebit E, Wang-Gohrke S, Chang-Claude J. CYP17 5’-UTR MspA1 polymorphism and the risk of premenopausal breast cancer in a German population-based patient – control study. Breast Cancer Res 2005; 7: R455-64.
6. Carey AH, Waterworth D, Patel K, et al. Polycystic ovaries and premature male pattern baldness are associated with one allele of the steroid metabolism gene CYP17. Hum Mol Genet 1994; 3: 1873-6.
7. Silva SN, Cabral MN, Bezerra de Castro G, et al. Breast cancer risk and polymorphisms in genes involved in metabolism of estrogens (CYP17, HSD17beta1, COMT and MnSOD): possible protective role of MnSOD gene polymorphism Val/Ala and Ala/Ala in women that never breast fed. Oncol Rep 2006; 16: 781-8.
8. Miyoshi Y, Iwao K, Ikeda N, Egawa C, Noguchi S. Genetic polymorphism in CYP17 and breast cancer risk in Japanese women. Eur J Cancer 2000; 36: 2375-9.
9. Warren R, Skinner J, Sala E, et al. Associations among mammographic density, circulating sex hormones, and polymorphisms in sex hormone metabolism genes in postmenopausal women. Cancer Epidemiol Biomarkers Prev 2006; 15: 1502-8.
10. Yanase T. 17 alpha-hydroxylase/17, 20-lyase defects. J Steroid Biochem Mol Biol 1995; 53: 153-7.
11. Franks S, White D, Gilling-Smith C, Carey A, Waterworth D, Williamson R. Hypersecretion of androgens by polycystic ovaries: the role of genetic factors in the regulation of cytochrome P450c17 alpha. Baillieres Clin Endocrinol Metab 1996; 10: 193-203.
12. Crocitto LE, Feigelson HS, Yu MC, et al. Short report on DNA marker at candidate locus: a polymorphism in intron 6 of the CYP17 gene. Clin Genet 1997; 52: 68-9.
13. Feigelson HS, Coetzee GA, Kolonel LN, Ross RK, Henderson BE. A polymorphism in the CYP17 gene increases the risk of breast cancer. Cancer Res 1997; 57: 1063-5.
14. Artamonov VV, Liubchenko LN, Shabanov MA, Babenko OV, Nemtsova MV, Zaletaev DV. Association of polymorphism of genetic markers of CYP19 and CYP17 with sporadic breast cancer [Russian]. Mol Biol 2003; 37: 975-82.
15. Bergman-Jungeström M, Gentile M, Lundin AC, Wingren S. Association between CYP17 gene polymorphism and risk of breast cancer in young women. Int J Cancer 1999; 84: 350-3.
16. Spurdle AB, Hopper JL, Dite GS, et al. CYP17 promoter polymorphism and breast cancer in Australian women under age forty years. J Natl Cancer Inst 2000; 92: 1674-81.
17. Hamajima N, Iwata H, Obata Y, et al. No association of the 5’ promoter region polymorphism of CYP17 with breast cancer risk in Japan. Jpn J Cancer Res 2000; 91: 880-5.
18. Mitrunen K, Jourenkova N, Kataja V, et al. Steroid metabolism gene CYP17 polymorphism and the development of breast cancer. Cancer Epidemiol Biomarkers Prev 2000; 9: 1343-8.
19. Wu AH, Seow A, Arakawa K, Van Den Berg D, Lee HP, Yu MC. HSD17B1 and CYP17 polymorphism and breast cancer risk among Chinese women in Singapore. Int J Cancer 2003; 104: 450-7.
20. Spurdle AB, Hopper JL, Dite GS, et al. CYP17 promoter polymorphism and breast cancer in Australian women under age forty years. J Natl Cancer Inst 2000; 92: 1674-81.
21. Dunning AM, Healey CS, Pharoah PD, et al. No association between a polymorphism in the steroid metabolism gene CYP17 and the risk of breast cancer. Br J Cancer 1998; 77: 2045-7.
22. Einarsdóttir K, Rylander-Rudqvist T, Humphreys K, et al. CYP17 gene polymorphism in relation to breast cancer risk: a Patient-control study. Breast Cancer Res 2005; 7: 890-6.
23. Hsieh YY, Tsai FJ, Chang CC, et al. Cytochrome P450c17alpha (CYP17) gene polymorphism is not associated with leiomyoma susceptibility. Genet Mol Biol 2002; 25: 361-4.
24. Han DF, Zhou X, Hu MB, et al. Polymorphisms of estrogen-metabolizing genes and breast cancer risk: a multigenic study. Chin Med J 2005; 118: 1507-16.
25. Chang JH, Gertig DM, Chen X, et al. CYP17 genetic polymorphism, breast cancer, and breast cancer risk factors: Australian Breast Cancer Family Study. Breast Cancer Res 2005; 7: R513-21.