Abstract
Aromatase, encoded by the CYP19A1 gene, is a key enzyme in the biosynthesis of estrogen. In an effort to screen for CYP19A1 single-nucleotide polymorphisms (SNPs) in Koreans, the CYP19A1 gene was directly sequenced in 50 normal subjects. A total of 19 variations were identified: four in exons, ten in introns, six in the 5′-untranslated region (UTR) and one in 3′-UTR. The distribution of CYP19A1 (TTTA)n polymorphisms was such that the most frequent allele was (TTTA)7 (66%), followed by (TTTA)11 (30%), (TTTA)12 (3%) and (TTTA)13 (1%). The order of the frequency distribution of CYP19A1 variations, other than that of the (TTTA)n variant, was IVS6-106T>G and IVS7-79A>G (57%); 1531C>T (56%); IVS5-16T>G and IVS6+36A>T (54%); −196A>C and −77G>A (49%); IVS2-59A>G and 240A>G (48%); −278C>T (31%); IVS4+27TCTI>D (29%); −144C>T and −588G>A (19%); 790C>T (16%); and other minor alleles with less than 5% frequency. Nineteen variations were used to characterize linkage disequilibrium (LD) structures at the CYP19A1 locus, which resulted in three LD blocks. Eight tagging SNPs in CYP19A1 were determined. Identification of CYP19A1 SNPs with LD blocks and tagging SNPs creates an important resource for genotype–phenotype association studies for estrogen-related phenotypes.
Similar content being viewed by others
Introduction
The cytochrome P450 19A1 (CYP19A1) gene encodes an aromatase that is responsible for the conversion of androgens to estrogens. CYP19A1 is located on chromosome 15q21 and spans 123 kb, including nine protein coding exons and a large 5′-untranslated region of 93 kb with alternative tissue-specific promoters.1 Several untranslated first exons are expressed under the control of a tissue-specific promoter and joined to the second exon through a common splice acceptor site.2 The expression of aromatase in humans has been demonstrated in gonads, adipose, placenta, skin, brain and bone. Given that estrogens have an important role in cell proliferation,3 many studies have suggested that variation in estrogen levels contributes to the risk of estrogen-dependent diseases, such as breast cancer,4 endometrial cancer5 and prostate cancer.6 In addition to cancer association studies, CYP19A1 has been implicated in the risk of obesity in Chinese women,7 in the risk of essential hypertension in males,8 as well as in bone mass and fracture risk.9 One of the important features of aromatase is evidenced by the fact that selective aromatase inhibitors are increasingly used to treat postmenopausal women with estrogen-responsive breast cancer.10 Therefore, CYP19A1 coding for aromatase has been an attractive target for identification of common genetic polymorphisms that may account for different levels of estrogen in humans. However, no efforts to resequence the CYP19A1 gene for a Korean population have been made. To determine the distribution of CYP19A1 alleles and haplotypes, we conducted direct DNA sequencing of the CYP19A1 gene in a Korean population for the first time. Analysis of the CYP19A1 allele distribution and haplotype structure with linkage disequilibrium (LD) was performed.
Materials and methods
Study subjects
DNA samples from 50 unrelated, healthy Koreans were obtained from the DNA repository bank at INJE Pharmacogenomics Research Center for direct DNA sequencing (Inje University College of Medicine, Busan, Korea).11 The Institutional Review Board (IRB) of Busan Paik Hospital (Busan, Korea) approved the research protocol for the use of human DNA from blood samples.
Sequencing and identification of SNPs
Genomic DNA was prepared from peripheral whole blood using the QiAamp blood kit (Valencia, CA, USA). Primers for PCR amplification were identical to those used previously.12 All exons, exon–intron boundaries and relevant promoter regions were PCR amplified. PCR contains primers at 0.2 μmol l−1 each, 2 U of r-Taq polymerase (Takara Bio, Shiga, Japan), and each dNTP at 0.2 mmol l−1 per reaction. PCR cycle parameters include initial denaturation (94 °C, 5 min, 1 cycle), 35 cycles of a denaturation/annealing/extension (94 °C, 30s/60 °C, 30s/72 °C, 30 s) and a final extension at 72 °C for 10 min. PCR was performed on a GeneAmp PCR 9700 (Applied Biosystems, Foster City,CA, USA). Amplified products were purified using a PCR purification kit (NucleoGen, Ansan, Korea), and sequencing was performed using an ABI Prism 3700XL Genetic Analyzer (Applied Biosystems). A software package, PC Gene (Oxford Molecular, Campbell, CA, USA), was used to identify variants with single-nucleotide substitutions in heterozygous or homozygous mutations.
Linkage disequilibrium and haplotype analysis
Hardy–Weinberg equilibrium and LD were analyzed by SNPAlyze software (version 4.1; Dynacom, Yokohama, Japan). ∣D′∣ and rho square (r2) values were used to access pairwise LD between single-nucleotide polymorphisms (SNPs), as described previously.11, 13
Tag SNP selection
Nineteen variations identified by direct DNA sequencing were applied to select tagging SNPs. The program Tagger was used to select tagging SNPs, which combines the simplicity of pairwise methods with the potential efficiency of multimarker approaches (http://www.broad.mit.edu/mpg/tagger/). The efficiency of tagged SNPs depends on the LD between itself and the tag SNP, as measured by the pairwise correlation coefficient (rp2). We selected a set of tag SNPs in which all known common variants (minor allele frequency >0.05) had an estimated rp2>0.8 with at least one tag SNP. Some SNPs are weakly correlated with other single SNPs but may be efficiently tagged by a haplotype defined by multiple SNPs, reducing the total number of tag SNPs needed. Therefore, we attempted to define the correlation coefficient between each SNP and a haplotype of tagging SNPs as rs2>0.8.
Result and Discussion
Genetic polymorphisms in CYP19A1 may change the function of the aromatase enzyme, resulting in altered levels of estrogen biosynthesis. Thus, its polymorphism may be implicated in the risk of estrogen-related diseases. Although CYP19A1 is a key enzyme in the process of estrogen synthesis, no resequencing efforts have yet been made in people of Korean descent. In this study, direct DNA sequencing analysis of the CYP19A1 gene in 50 Koreans revealed a total of 19 variations. A summary of their frequencies identified is provided in Table 1. The locations of SNPs in relation to the genomic structure of CYP19A1 are shown in Figure 1. χ2-tests were used to compare observed variants with expected variants in the study population. No deviation from Hardy–Weinberg equilibrium was observed for the SNPs identified (P>0.05). Frequencies of functionally important and relatively well-studied CYP19A1 variants in different ethnic groups were summarized in Table 2. Several studies have been conducted to address the association between CYP19A1 (TTTA)n polymorphisms and breast cancer risk as a genetic marker.14, 15 We observed that the most frequent CYP19A1 (TTTA) allele was (TTTA)7 (66%), followed by (TTTA)11 (30%), (TTTA)12 (3%) and (TTTA)13 (1%) (Table 3). Kim et al.16 reported that the frequency of the low type, which means that the TTTA copy number was under 10, was 55.5% and that of the high type (TTTA copy number with over 10) was 44.5% in 102 Korean control subjects. The similar report in a Korean population17 is described in Table 3. Although (TTTA)8 was not detected, this allele was the third most frequent allele in Caucasian and African-American populations, occurring at ∼12.5 and 2.5%, respectively.12 In the Korean population, the frequency of (TTTA)12 was 3%; however, this allele was detected in 10% of Japanese subjects,14 indicating that the allele frequency in one population cannot be assumed to be equally applicable in a similarly defined population. The promoter SNP −278C>T, known as a very rare allele in Caucasian and African-American populations, had an occurrence rate of 31% in our study, which is similar to that observed in other Asian subjects.12 The −278C>T allele may represent one of the Asian dominant alleles in racial comparisons. A 790C>T (Arg264Cys), reported at a lower frequency in Caucasians, was found at 16% frequency in our study. The 790C>T variant (Arg264Cys) has been reported to have a positive association with increased breast cancer risk in Asians.18 However, the results of another study did not support this hypothesis, and instead suggested that this allele is associated with a lowered risk of breast cancer.4 The 1531C>T (rs10046) allele has been studied extensively, which includes its potential gender-specific association with essential hypertension,8 increased level of estrogen,19 breast cancer risk in Japanese populations20 and endometrial cancer risk in Chinese women.21 This variant, observed at 56% frequency in this study and located in haplotype block 3, exhibited a haplotype structure with IVS7-79A>G, IVS6-106 T>G, IVS6+36A>T and IVS5-16T>G (D′=1, r2=0.92–0.96) in block 3 (Figures 2a and b). A set of tag SNPs, which will be useful for association mapping studies, were determined (Figure 2b). It is suggested that eight tag SNPs would be needed to track all important haplotype blocks in the CYP19A1 gene in Koreans. Nineteen variations were used to characterize LD structures at the CYP19A1 locus, resulting in three LD blocks (Figure 2a). The LD blocks identified were compared with the same SNPs reported in the HapMap database. Two SNPs (−77G>A and −196A>C), located in the 1.6 promoter, are in strong LD in Chinese and Japanese populations, as well as in the Korean group examined here (data not shown). These two SNPs were not linked with 1531C>T (rs10046) in Koreans (D′=0.48, 0.48; r2=0.17, 0.17). However, a relatively strong LD was observed in Japanese (D′=1, 0.94; r2=0.65, 0.64) and Chinese (D′=0.87, 0.87; r2=0.5, 0.5) populations between these two loci, as well as in 1531C>T (rs10046). It may be of interest to determine whether these mutations in the 1.6 promoter can cause population or environmental differences in the expression of the aromatase protein in the given populations.
In summary, we resequenced the CYP19A1 gene for the first time in a Korean population and identified 19 variations. Frequencies, haplotypes, LD structures and tagging SNPs were determined. Genetic information with respect to the degree of LD in Koreans might be useful for future genotype–phenotype association studies, especially in terms of breast cancer and hormone-related diseases.
Accession codes
References
Bulun, S. E., Sebastian, S., Takayama, K., Suzuki, T., Sasano, H. & Shozu, M. The human CYP19 (aromatase P450) gene: update on physiologic roles and genomic organization of promoters. J. Steroid Biochem. Mol. Biol. 86, 219–224 (2003).
Means, G. D., Kilgore, M. W., Mahendroo, M. S., Mendelson, C. R. & Simpson, E. R. Tissue-specific promoters regulate aromatase cytochrome P450 gene expression in human ovary and fetal tissues. Mol. Endocrinol. 5, 2005–2013 (1991).
Henderson, B. E., Ross, R. K., Pike, M. C. & Casagrande, J. T. Endogenous hormones as a major factor in human cancer. Cancer Res. 42, 3232–3239 (1982).
Haiman, C. A., Stram, D. O., Pike, M. C., Kolonel, L. N., Burtt, N. P., Altshuler, D. et al. A comprehensive haplotype analysis of CYP19 and breast cancer risk: the multiethnic cohort. Hum. Mol. Genet. 12, 2679–2692 (2003).
Setiawan, V. W., Doherty, J. A., Shu, X. O., Akbari, M. R., Chen, C., De Vivo, I. et al. Two estrogen-related variants in CYP19A1 and endometrial cancer risk: a pooled analysis in the Epidemiology of Endometrial Cancer Consortium. Cancer Epidemiol. Biomarkers Prev. 18, 242–247 (2009).
Modugno, F., Weissfeld, J. L., Trump, D. L., Zmuda, J. M., Shea, P., Cauley, J. A. et al. Allelic variants of aromatase and the androgen and estrogen receptors: toward a multigenic model of prostate cancer risk. Clin. Cancer Res. 7, 3092–3096 (2001).
Long, J. R., Shu, X. O., Cai, Q., Wen, W., Kataoka, N., Gao, Y. T. et al. CYP19A1 genetic polymorphisms may be associated with obesity-related phenotypes in Chinese women. Int. J. Obes (Lond). 31, 418–423 (2007).
Shimodaira, M., Nakayama, T., Sato, N., Saito, K., Morita, A., Sato, I. et al. Association study of aromatase gene (CYP19A1) in essential hypertension. Int. J. Med. Sci. 5, 29–35 (2008).
Masi, L., Becherini, L., Gennari, L., Amedei, A., Colli, E., Falchetti, A. et al. Polymorphism of the aromatase gene in postmenopausal Italian women: distribution and correlation with bone mass and fracture risk. J. Clin. Endocrinol. Metab. 86, 2263–2269 (2001).
Santen, R. J., Brodie, H., Simpson, E. R., Siiteri, P. K., Brodie, A. History of aromatase: saga of an important biological mediator and therapeutic target. Endocr. Rev. 30, 343–375 (2009).
Lee, S. S., Jeong, H. E., Yi, J. M., Jung, H. J., Jang, J. E., Kim, E. Y. et al. Identification and functional assessment of BCRP polymorphisms in a Korean population. Drug Metab. Dispos. 35, 623–632 (2007).
Ma, C. X., Adjei, A. A., Salavaggione, O. E., Coronel, J., Pelleymounter, L., Wang, L. et al. Human aromatase: gene resequencing and functional genomics. Cancer Res. 65, 11071–11082 (2005).
Gabriel, S. B., Schaffner, S. F., Nguyen, H., Moore, J. M., Roy, J., Blumenstiel, B. et al. The structure of haplotype blocks in the human genome. Science. 296, 2225–2229 (2002).
Miyoshi, Y. & Noguchi, S. Polymorphisms of estrogen synthesizing and metabolizing genes and breast cancer risk in Japanese women. Biomed. Pharmacother. 57, 471–481 (2003).
Mitrunen, K. & Hirvonen, A. Molecular epidemiology of sporadic breast cancer. The role of polymorphic genes involved in oestrogen biosynthesis and metabolism. Mutat. Res. 544, 9–41 (2003).
Kim, J. Y., Lee, C. S., Kim, H. O., Jo, Y. H., Lee, J., Jung, M. H. et al. The association between genetic polymorphisms in CYP19 and breast cancer risk in Korean women. Oncol. Rep. 22, 487–492 (2009).
Hur, S. E., Lee, S., Lee, J. Y., Moon, H. S., Kim, H. L. & Chung, H. W. Polymorphisms and haplotypes of the gene encoding the estrogen-metabolizing CYP19 gene in Korean women: no association with advanced-stage endometriosis. J. Hum. Genet. 52, 703–711 (2007).
Lee, K. M., Abel, J., Ko, Y., Harth, V., Park, W. Y., Seo, J. S. et al. Genetic polymorphisms of cytochrome P450 19 and 1B1, alcohol use, and breast cancer risk in Korean women. Br. J. Cancer. 88, 675–678 (2003).
Haiman, C. A., Dossus, L., Setiawan, V. W., Stram, D. O., Dunning, A. M., Thomas, G. et al. Genetic variation at the CYP19A1 locus predicts circulating estrogen levels but not breast cancer risk in postmenopausal women. Cancer Res. 67, 1893–1897 (2007).
Zhang, L., Gu, L., Qian, B., Hao, X., Zhang, W., Wei, Q. et al. Association of genetic polymorphisms of ER-alpha and the estradiol-synthesizing enzyme genes CYP17 and CYP19 with breast cancer risk in Chinese women. Breast Cancer Res. Treat. 114, 327–338 (2009).
Tao, M. H., Cai, Q., Zhang, Z. F., Xu, W. H., Kataoka, N., Wen, W. et al. Polymorphisms in the CYP19A1 (aromatase) gene and endometrial cancer risk in Chinese women. Cancer Epidemiol. Biomarkers Prev. 16, 943–949 (2007).
Nativelle-Serpentini, C., Lambard, S., Seralini, G. E. & Sourdaine, P. Aromatase and breast cancer: W39R, an inactive protein. Eur. J. Endocrinol. 146, 583–589 (2002).
Miyoshi, Y., Iwao, K., Ikeda, N., Egawa, C. & Noguchi, S. Breast cancer risk associated with polymorphism in CYP19 in Japanese women. Int. J. Cancer. 89, 325–328 (2000).
Olson, J. E., Ingle, J. N., Ma, C. X., Pelleymounter, L. L., Schaid, D. J., Pankratz, V. S., et al. A comprehensive examination of CYP19 variation and risk of breast cancer using two haplotype-tagging approaches. Breast Cancer Res. Treat. 102, 237–247 (2007).
Cai, H., Shu, X. O., Egan, K. M., Cai, Q., Long, J. R., Gao, Y. T. et al. Association of genetic polymorphisms in CYP19A1 and blood levels of sex hormones among postmenopausal Chinese women. Pharmacogenet. Genomics. 18, 657–664 (2008).
Song, C. G., Hu, Z., Yuan, W. T., Di, G. H., Shen, Z. Z., Huang, W. et al. Effect of R264C polymorphism in CYP19A1 gene on BRCA1/2-negative hereditary breast cancer from Shanghai population of China. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 23, 181–183 (2006).
Matsumoto, Y., Suzuki, A., Shibuya, N., Sadahiro, R., Kamata, M., Goto, K. et al. Effect of the cytochrome P450 19 (aromatase) gene polymorphism on personality traits in healthy subjects. Behav. Brain Res. 205, 234–237 (2009).
Probst-Hensch, N. M., Ingles, S. A., Diep, A. T., Haile, R. W., Stanczyk, F. Z., Kolonel, L. N. et al. Aromatase and breast cancer susceptibility. Endocr. Relat. Cancer. 6, 165–173 (1999).
Siegelmann-Danieli, N. & Buetow, K. H. Constitutional genetic variation at the human aromatase gene (Cyp19) and breast cancer risk. Br. J. Cancer. 79, 456–463 (1999).
Acknowledgements
This study was supported by a Korea Science and Engineering Foundation (KOSEF) grant funded by the Ministry of Education, Science and Engineering (MOEST; no. R13-2007-023-00000-0) and by a grant from the Korea Health 21 R&D Project, Ministry for Health, Welfare and Family Affairs (A030001), Republic of Korea. This work was also supported by a grant from Inje University, 2006.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lee, SJ., Kim, WY., Choi, JY. et al. Identification of CYP19A1 single-nucleotide polymorphisms and their haplotype distributions in a Korean population. J Hum Genet 55, 189–193 (2010). https://doi.org/10.1038/jhg.2010.6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/jhg.2010.6
Keywords
This article is cited by
-
Understanding polycystic ovary syndrome in light of associated key genes
Egyptian Journal of Medical Human Genetics (2023)
-
Association of CYP19A1 gene variations with adjuvant letrozole-induced adverse events in South Indian postmenopausal breast cancer cohort expressing hormone-receptor positivity
Breast Cancer Research and Treatment (2020)
-
Haplotype structures and functional polymorphic variants of the drug target enzyme aromatase (CYP19A1) in South Indian population
Medical Oncology (2013)
-
Aromatase (CYP19) gene variants influence ovarian response to standard gonadotrophin stimulation
Journal of Assisted Reproduction and Genetics (2012)
-
The association of aromatase (CYP19) gene variants with sperm concentration and motility
Asian Journal of Andrology (2011)