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Cytochrome P450c17 Gene (CYP17) Polymorphism Predicts Use of Hormone Replacement Therapy¹

Departments of Preventive Medicine [H. S. F., R. M-C., M. C. P., B. E. H.] and Urology [G. A. C.], University of Southern California School of Medicine, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, California 90033-0800, and Cancer Etiology Program, Cancer Research Center of Hawaii, University of Hawaii Honolulu, Honolulu, Hawaii 96813 [L. N. K., A. M. Y. N., L. L.]

	ABSTRACT

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We investigated whether a polymorphism in the cytochrome P450c17 gene (CYP17), which is associated with higher endogenous hormone levels, influences the use of hormone replacement therapy (HRT). The study included 749 postmenopausal women ages 44–75 years at baseline randomly selected from a larger multiethnic cohort. African-American, Japanese, Latina, and white women were included in the study. Women who carry the CYP17 A2/A2 genotype were about half as likely as women with the A1/A1 genotype to be current HRT users (odds ratio = 0.52; 95% confidence interval, 0.31–0.86). This association was present in all four racial/ethnic groups and for women above and below the median weight of 150 pounds. These findings suggest that the actual risk of breast cancer associated with HRT use may be higher than previously reported.

	Introduction

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A recent meta-analysis of the many studies that have examined the association between HRT³ and breast cancer found that the relative risk for breast cancer increases with increasing duration of HRT use (1) . An important issue that has not been addressed in any of these studies is underlying genetic susceptibility to breast cancer. If such genetic susceptibility also is associated with HRT use, then this may be an important source of confounding.

The cytochrome P450c17 gene (CYP17) codes for the cytochrome P450C17 enzyme, which mediates both steroid 17-hydroxlyase and 17,20-lyase activities, and functions at key branch points in human steroidogenesis (2) . The 5'-untranslated region of CYP17 contains a single-bp polymorphism 34 bp upstream from the initiation of translation and 27 bp downstream from the transcription start site (3) . This bp change creates a recognition site for the MspAI restriction enzyme and has been used to designate two alleles, A1 (the published sequence) and A2.

We and others have recently shown that endogenous hormone levels are associated with a polymorphism in CYP17 (4 , 5) . Among young nulliparous women, we found that serum E₂ measured around day 11 of the menstrual cycle was 11% and 57% higher (P = 0.04), respectively, among women hetero- and homozygous for the CYP17 A2 allele compared to A1/A1 women (4) . Similarly, around cycle day 22, E₂ was 7% and 28% higher (P = 0.06), and progesterone was 24% and 30% higher (P = 0.04). In a group of 297 postmenopausal women, Haiman et al. (5) found that compared to women with the A1/A1 genotype, women with the A2/A2 genotype had statistically significantly elevated levels of estrone (+14.2%; P = 0.01) and dehydroepiandrosterone (+14.4%; P = 0.02) and similar but statistically nonsignificant elevations in E₂ (+18.8%; P = 0.08) and androstenedione (+17.1%; P = 0.06). There were smaller elevations of testosterone (+8.6%; P = 0.34) and dehydroepiandrosterones (+7.2%; P = 0.26).

Although the peripheral production of estrogen in adipose tissue of postmenopausal women differs from the ovarian production in premenopausal women, the role of CYP17 in the estrogen precursor metabolism pathway (in both the ovary and adrenal gland) is the same (3) . HRT use decreases with increasing weight, and this has been presumed to be due to the increase in endogenous estrogen. We therefore argue that endogenous estrogen levels controlled by CYP17 may influence HRT use.

	Materials and Methods

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Study Population.
This study is part of a larger ongoing multiethnic cohort study with an emphasis on studying diet and other life-style characteristics in the etiology of cancer. Details of this study are provided elsewhere (6) . Briefly, the cohort was accessed beginning in 1993 from driver’s license files in Hawaii and Los Angeles. The age range at baseline was restricted to 45–75 years to maximize both quality of data and cancer incidence. The cohort totals 215,251 men and women and includes African-Americans, Japanese, Hawaiians, Latinos, non-Latino whites, and small numbers of other racial/ethnic groups.

Baseline data were collected on all cohort members via a mailed questionnaire that contained five sections: (a) background, including medical history and family cancer history; (b) diet history; (c) medication use; (d) physical activity; and (e) female reproductive history, including the use of hormones. The questionnaire was extensively pretested and translated into Spanish for use with Los Angeles Latinos.

A 3% random sample of men and women from the cohort was generated to provide potential controls for nested case-control studies. These randomly selected cohort members are contacted by phone and asked to provide a blood specimen. At the time of the blood draw, informed consent forms are completed. Our participation rate in the blood collection phase of this study has been over 70% for all cohort controls in each of the four racial/ethnic groups.

For this analysis, we included women (n = 749) who had no current or prior history of cancer of the breast, ovaries, uterus, or cervix. Women who self-reported natural menopause or surgical menopause due to bilateral oophorectomy were included in the study. Women who self-reported surgical menopause due to hysterectomy (single or no oophorectomy) were included if they were 55 years at the time of blood draw. Analysis was restricted to four racial/ethnic groups (African-American, Japanese, Latina, and non-Latina white) because the remaining groups had too few members to be considered independently.

Statistical Analysis.
The odds of using HRT were calculated for female members of the cohort genotyped for CYP17. Unconditional logistic regression was used to calculate the ORs and corresponding 95% CIs. ORs reported in the text were adjusted for the following variables: (a) age at blood draw; (b) racial/ethnic group; and (c) weight (as categorical variables). Stratum-specific analyses were compared to evaluate potential effect modification for each of these variables (age in quartiles and weight < 150 pounds or 150 pounds). Women were characterized as current, past, or never users of HRT and by the duration of HRT use. To be certain that the inclusion of women with oophorectomies did not bias the findings, the analysis was repeated with these subjects excluded, and an adjustment was made in the analysis for menopausal type (although this adjustment was not kept in the final model because it did not change the results). Other potential confounders that were considered but not included in the final models because they had no effect on the results included family history of breast cancer, age at menarche, and age at menopause. Tests for trends were calculated by including genotype score (A1/A1 = 0, A1/A2 = 1, and A2/A2 = 2) as a continuous variable in the model.

Validation of HRT Use.
We have validated the data regarding use of HRT in our self-administered questionnaire using data from other ongoing or recently completed studies in both Hawaii and Los Angeles. We identified cohort members who were also participating in case-control studies of breast or ovarian cancer that have collected detailed information on the use of HRT by in-person interviews using a calendar approach with color photographs of various hormone preparations. We used these data as the gold standard to judge the validity of our questionnaire in 75 overlapping subjects. We found that 67 women (86.3%) gave the same response on ever use of HRT in the two studies. All of the eight discrepant results took estrogens for relatively short periods of time. Of the 31 HRT users in both studies, 19 had exact agreement on duration of use, and an additional 10 differed by only one category (often by just 1 year). The remaining two women differed by two categories. Thus, the agreement was very close in 94% of women (29 of 31 HRT users). Based on these findings, it appears that the cohort data on this important variable has a high degree of validity.

CYP17 Assay.
DNA was purified from buffy coats of peripheral blood samples for subjects using a rapid DNA preparation method (7) . The CYP17 assay has been described previously (3) . Briefly, a PCR fragment containing the bp change was generated using the following primers: (a) CYP-1, 5'-CATTCGCACTCTGGAGTC-3'; and (b) CYP-2, 5'-AGGCTCTTGGGGTACTTG-3'. PCR reactions were carried out in 25-µl aliquots containing about 50 ng of genomic DNA, 50 pmol of each primer, 1x reaction buffer, 100 µM deoxynucleotide triphosphates, and 1 unit of Taq polymerase (Pharmacia). The amplification was for 30 cycles with denaturation at 94°C for 1 min, annealing at 57°C for 1 min, and extension at 72°C for 1 min. An initial denaturation step of 5 min at 94°C and a final extension at 72°C for 5 min were used. The PCR products were digested for 3 h at 37°C using MspAI, separated by agarose gel electrophoresis, and stained with ethidium bromide to identify the bp change.

	Results

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The study included 749 women between the ages of 45 and 75 years at baseline. Four racial/ethnic groups were represented in the study: (a) African-American (34%); (b) Japanese (18%); (c) Latina (30%); and non-Latina white (18%). Characteristics of these women are shown in Table 1 . African-American and Latina women were more likely to be never users of HRT than either Japanese or non-Latina white women. Not only were Japanese and non-Latina white subjects more likely to be users of HRT, but they were more likely to be current users (51% and 43%, respectively) than past users (20% for each racial/ethnic group). The majority (79%) of current users took HRT for >3 years, whereas past users (65%) tended to be short-term users (<3 years).

	Discussion

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This is the first study to suggest that a specific genotype may predict patterns of exogenous hormone use. We found that women who were homozygous for the A2 allele were only about half as likely to be current users of HRT as A1 homozygotes. This association was present across all four racial/ethnic groups, who have differing cultural practices and access to medical care. This effect was most pronounced among women weighing 150 pounds or less, but it was still present among women over 150 pounds. Our previous work (8) , suggested that A2 women have an increased risk of breast cancer. These results, considered together, imply that the breast cancer risk from HRT has been underestimated, because the group at highest risk (A2) is underrepresented among HRT users.

In our study, past HRT use is independent of genotype. Past users of HRT typically used HRT only for a short time (<3 years). Whereas women begin HRT for a variety of both medical and cultural reasons, endogenous hormone levels, mediated in part by CYP17 and possibly by other estrogen metabolism genes, appear to influence whether a woman is a current user of HRT. Although current users tended to take HRT for longer periods than past users, we did not have data on whether the total duration of HRT use was continuous or intermittent; therefore, we could not adequately explore the relationship between CYP17 and the duration of HRT use. Details of how CYP17 affects HRT use are not clear. Additional data on why women start and continue using HRT (e.g., menopausal symptoms, disease prevention or treatment, or family history of different diseases) may explain this association, and we are currently collecting this information.

This CYP17 polymorphism is either in linkage disequilibrium with other, as yet unidentified, genetic variations located near this site or has a direct quantitative effect on CYP17 gene expression. Further laboratory investigation is needed to determine what specific variation at or near the CYP17 gene is responsible for these observed associations.

These findings must be considered preliminary, and additional studies are needed to clarify these relationships. This is an evolving field, with some studies finding associations between CYP17 and breast cancer, whereas others have not (5 , 9, 10, 11, 12) . The CYP17 genotype may provide an important piece of information for women seeking advice on HRT use and the associated risks and benefits. CYP17 is not the sole determinant of either endogenous hormone levels or exogenous hormone use, but these findings illustrate the importance of considering underlying genetic susceptibility in future studies on hormone use and perhaps when designing and evaluating clinical studies using exogenous hormone preparations.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge the contributions of Zhang Wu, Hank Huang, Kristine R. Monroe, and Faye Nagamine to this work.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by funds from the California Breast Cancer Research Program of the University of California (Grants 2FB-0212 and 4KB-0147) and by National Cancer Institute Grants CA54281 and CA63464.

² To whom requests for reprints should be addressed, at Department of Preventive Medicine, University of Southern California School of Medicine, University of Southern California/Norris Comprehensive Cancer Center 1441 Eastlake Avenue MS # 44, Los Angeles, CA 90033-0800. Phone: (323) 865-0419; Fax: (323) 865-0127.

³ The abbreviations used are: HRT, hormone replacement therapy; E₂, estradiol; OR, odds ratio; CI, confidence interval.

Received 4/27/99. Accepted 7/ 1/99.

	REFERENCES

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