Exposure to sex hormones is a major risk factor for breast cancer and current treatments include hormone modifying drugs, among them aromatase inhibitors. We studied the association of CYP19 (Val80 and [TTTA]n) polymorphisms, the gene translated to aromatase, and the risk of breast cancer in BRCA carriers and noncarriers. The study consisted of 958 cancer cases and 931 healthy controls, including 474 carriers and 1,415 noncarriers. Cases and controls came from a population-based study of breast cancer in Israel, enriched with BRCA carriers from a clinical familial cancer service. Val80 G/G genotype was associated with significantly increased risk of breast cancer compared with the Val80 A/A genotype in BRCA1 carriers ages <50 years (odds ratio, 2.81; 95% confidence interval, 1.09-7.22; P = 0.032) but not in BRCA2 carriers or noncarriers of any age. A similar magnitude suggestive association, although nonstatistically significant, was found between Val80 polymorphism and estrogen receptor-negative status of the breast tumors. A common haplotype composed of the Val80 G allele and three haplotype-tagging single nucleotide polymorphisms (rs727479, rs10046, and rs4646) in the CYP19 coding region showed a trend to association with breast cancer risk in BRCA1 carriers ages <50 years. Published expression data show higher estrogen levels with higher repeats in [TTTA]n found in linkage disequilibrium with Val80. The present study suggests that the CYP19 Val80 polymorphism and a haplotype that includes this polymorphism are associated with increased breast cancer risk in young women with BRCA1 mutations. (Cancer Epidemiol Biomarkers Prev 2009;18(5):1617–23)

Breast cancer is one of the leading causes of cancer morbidity and mortality worldwide (1). In the past decade, two major genes have been shown to be related to breast (and ovarian) cancer susceptibility, BRCA1 and BRCA2 (2). Three Ashkenazi Jewish founder mutations in these genes (BRCA1: 185delAG and 5382insC and BRCA2: 6174delT) have a combined frequency of 2.5% in the Ashkenazi population and appear in ∼10% (3-5) of breast cancer cases in Ashkenazi Jewish women. Estimates of penetrance vary greatly across different studies, ranging from 37% to 90% (6-12). This heterogeneity in risk among women who carry BRCA1/2 mutations suggests the existence of modifying genetic and/or environmental factors. Polymorphisms in several genes have been suggested to modify breast and ovarian cancer risk in BRCA1 and BRCA2 carriers, although most have not been replicated (13-21). The only confirmed BRCA2 breast cancer risk modifier is RAD51 135G>C (22). No studies have, as yet, evaluated the association between polymorphisms in the genes involved in estrogen metabolism and breast cancer risk in BRCA mutation carriers.

Because estrogens play an important role in carcinogenesis and progression of breast cancer (23, 24), genes encoding for enzymes involved in estrogen biosynthesis and metabolism are plausible candidates as breast cancer susceptibility genes. One such candidate is CYP19 (p450arom), which encodes for aromatase, an enzyme that converts androgens to estrogens. The association of polymorphisms in the CYP19 with breast cancer risk has previously been studied, with particular focus on the [TTTA]n repeats polymorphism (25-31). A single study observed a slightly higher [TTTA]7 allele frequency among breast cancer cases in the Caucasian population (31). In this study, the allele A of the silent variant at exon 3 [rs700518 (Val80Val)] is in complete linkage disequilibrium (LD) with [TTTA]7. Subsequent studies of [TTTA]n repeats showed inconsistent results. The following alleles have been implicated as possible breast cancer susceptibility alleles: [TTTA]7delTCT and [TTTA]>10 (32, 33), [TTTA]7 repeats (31), [TTTA]8 and [TTTA]10 (26, 27), [TTTA]11 (34), and [TTTA]12 (27, 29).

Several studies have observed an association between CYP19 polymorphisms and concentrations of serum circulating estrogen-related metabolites. The alleles [TTTA]7 and [TTTA]<9 were associated with lower estrogen levels; conversely, the alleles [TTTA]8, [TTTA]>9, rs727479(T), rs10046(T), and rs4646(G) were associated with higher levels of estrogens (27, 35-39). The Val80 (rs700518) G allele was found to be associated with elevated aromatase expression (40).

Estrogen receptor (ER) status is an important prognostic factor of breast tumors. A higher prevalence of ER-negative breast tumors in BRCA1 carriers was described (41). It has been shown that this association is neither a consequence of the young age at onset nor high grade but is an intrinsic property of BRCA1-related cancers (42). Recent studies found a significant association of the CYP19 [TTTA]7(delTCT) allele with ER-positive and CYP19 Trp39Arg (TC/CC) genotypes with ER-negative breast tumors (32, 43).

Previous genotyping efforts established the LD haplotype block structure of CYP19 (44). In block 4, which spans the entire coding region of CYP19, four common haplotypes cover 88% of haplotype diversity in Caucasians and can be distinguished by only three single nucleotide polymorphisms (SNP; rs727479, rs10046, and rs4646). None of these haplotypes was found to be significantly associated with breast cancer risk in a large multiethnic case-control study (44), but individual alleles rs727479(T), rs10046(T), and rs4646(G) were shown to be in association with elevated estrogen levels in postmenopausal women (39).

The present study investigates the causality of breast cancer in the unique population of Ashkenazi Jewish women, some of which carry the BRCA1 and BRCA2 founder mutations. Based on the previously published data concerning the role of CYP19 polymorphisms in breast cancer predisposition, it was hypothesized that the Val80 polymorphism, the [TTTA]n repeat polymorphism, or specific haplotypes in the CYP19 coding region can modify breast cancer risk. The aim of the study is to determine the clinical value of CYP19 polymorphisms in assessing breast cancer risk and their interaction with known genetic risk factors.

Study Population

The study population included 474 Ashkenazi Jewish women (223 breast cancer cases 251 healthy controls) carrying Jewish founder mutations in the BRCA1 and BRCA2 genes and 1,415 Ashkenazi Jewish noncarrier women (733 cases and 680 controls; Table 1). Carriers were determined to have one of the three Jewish founder mutations in BRCA1 (185delAG and 5382insC) or BRCA2 (6174delT) and were cared for by the Clalit Health Services National Familial Cancer Consultation Service at the time of study. The series of 1,405 noncarriers derived from an ongoing population-based case-control study of the molecular and environmental etiology of breast cancer in Israel. In this study, which was initiated in 2000, all incident breast cancer cases in a distinct geographic region in Northern Israel are invited to participate after signing an informed consent approved by the Carmel Medical Center Institutional Review Board Committee. Controls are randomly sampled from the list of all women enrolled in the healthcare program provided by Clalit Health Services, the largest health services provider in Israel covering the majority of the Israeli population, and matched on age, residence, and Jewish/Arab status. Participants are interviewed by trained nurses to evaluate risk factors, including a detailed three-generation family history of cancer. Blood is drawn from each subject for DNA extraction and molecular analysis, including BRCA1 and BRCA2 founder mutation genotyping. DNA extracted from the blood is studied for a variety of molecular events, among them the existence of one or more of the Jewish founder mutations in the BRCA1 and BRCA2 genes. Participants who are found to be BRCA carriers are referred to the Clalit Health Services National Familial Cancer Consultation Service. This Service is a referral center, which can be approached by the population at large or by health professionals for advice. Most women who are evaluated by this service with regards to the breast/ovary syndromes either have a significant family history or have a personal history of breast cancer appearing at an early age, bilateral breast cancer, or breast cancer appearing in conjunction with ovarian cancer. Medical records were extracted, when available, for all study participants with breast cancer. Clinical data extracted from these records includes the ER and progesterone receptor status of the primary tumor when available.

Table 1.

Study population characteristics

BRCA status*Age category (y)Breast cancer cases
Controls
Total
nMean (SD) agenMean (SD) age
BRCA1 carriers <50 83 39.7 (6.2) 97 32.9 (9.1) 180 
 >50 62 62.8 (8.7) 54 60.8 (10.4) 116 
 Total 145 48.9 (13.5) 151 42.9 (16.5) 296 
BRCA2 carriers <50 41 41.1 (6.1) 57 35.2 (9.5) 98 
 >50 37 61.1 (8.8) 43 61.8 (11.4) 80 
 Total 78 50.6 (12.5) 100 46.6 (16.8) 178 
Noncarriers <50 117 43.6 (5.0) 94 32.2 (5.0) 211 
 >50 618 66.5 (10.3) 586 66.6 (10.3) 1,204 
 Total 735 62.8 (12.8) 680 63.5 (12.5) 1,415 
BRCA status*Age category (y)Breast cancer cases
Controls
Total
nMean (SD) agenMean (SD) age
BRCA1 carriers <50 83 39.7 (6.2) 97 32.9 (9.1) 180 
 >50 62 62.8 (8.7) 54 60.8 (10.4) 116 
 Total 145 48.9 (13.5) 151 42.9 (16.5) 296 
BRCA2 carriers <50 41 41.1 (6.1) 57 35.2 (9.5) 98 
 >50 37 61.1 (8.8) 43 61.8 (11.4) 80 
 Total 78 50.6 (12.5) 100 46.6 (16.8) 178 
Noncarriers <50 117 43.6 (5.0) 94 32.2 (5.0) 211 
 >50 618 66.5 (10.3) 586 66.6 (10.3) 1,204 
 Total 735 62.8 (12.8) 680 63.5 (12.5) 1,415 
*

Ashkenazi founder mutations in BRCA1 (185delAG and 5382insC) and BRCA2 (6174delT).

Breast cancer combines breast cancer and breast/ovarian cancer cases.

The carriers group included 474 carriers of Jewish founder mutations: 296 of BRCA1 (218 of 185delAG and 78 of 5382insC) and 178 of BRCA2 (6174delT). Carriers with ovarian cancer only and two compound heterozygotes with mutations in both BRCA genes were not included into this study.

DNA Extraction and Genotyping

Genomic DNA was extracted from whole blood using a commercially available kit according to the manufacturer's protocol (Puregene DNA extraction kit; Gentra Systems). Genetic testing for BRCA1 (185delAG and 5382insC) and BRCA2 (6174delT) was done using the Pronto BRCA kit (Pronto). Genomic DNA from breast cancer patients known to carry one of the three mutations (185delAG, 5382insC, and 6174delT) served as positive controls for each assay. All positive samples were confirmed by restriction enzyme digestion as described previously (4).

The genotyping of CYP19 rs727479 (C_4749_10), Val80 (rs700518; C_8794675_10), rs10046 (C_8234731_1_), and rs4646 (C_8234730_1_) was done by allelic discrimination using the 5′-nuclease Assay-on-Demand on 7900HT sequence detection system (Applied Biosystems). The assay was done in a 15 μL reaction volume containing 1× TaqMan PCR core reagents (Applied Biosystems), 5 mmol/L MgCl2, 200 nmol/L each PCR primer, 100 nmol/L MGB probes (Applied Biosystems), 0.5 units AmpliTaq Gold, 0.2 units AmpErase UNG, and 40 ng genomic DNA. The Val80 5′-nuclease assay was validated by genotyping of 587 individuals using TaqMan and designed restriction fragment length polymorphism. No discrepancies were detected in the validation process.

The region of [TTTA]n repeats was amplified by PCR using the following primers: TTTA-F 5′-GCAGGTACTTAGTTAGCTAC-3′ and TTTA-R 5′-TTACAGTGAGCCAAGGTCGT-3′. The PCR was carried out in a total volume of 25 μL containing 3 μL DNA template (∼50 ng), 10 pmol of each primer, and 1 unit Taq polymerase (TaKaRa). The reaction was incubated at 95°C for 5 min before 30 cycles of denaturation of 30 s at 95°C, annealing of 1 min at 55°C, and extension of 30 s at 72°C followed by a final extension of 10 min at 72°C. The amplified products were run on 2% agarose gels, excised, and purified by Qiagen MinElute Gel Extraction Kit (Qiagen). The cleaned PCR products were sequenced at Weizmann Institute DNA Sequencing Service.

Statistical Methods

Hardy-Weinberg equilibrium was tested in controls using the χ2 goodness-of-fit test. CYP19 (Val80) genotype frequencies were compared between cases and controls using the Pearson χ2 test and Armitage's test for trend. Genotype odds ratios (OR) and their 95% confidence intervals (95% CI) were obtained using logistic regression, with Val80 A/A genotype as the reference category.

Multivariate logistic regression was used to adjust OR estimates for age and to test for gene-gene interaction. Genotype tests and trend tests were done using number of Val80 G alleles as categorical and continuous variable, respectively.

The carriers group included 474 women clustered in 341 families. To take into account the possible correlations within these families, we applied logistic regression models for correlated data (GEE model, SAS Genmod). As these additional analyses gave similar results to the ordinary logistic regression results, only the latter are presented.

The distribution of [TTTA] alleles was compared between cases and controls using Pearson χ2 test and Armitage's trend test. Exact test was employed when appropriate. Logistic regression based on allelic data was used to estimate ORs per 1 repeat increase. For this purpose, number of repeats was considered as a continuous variable and the associated P value was reported as Ptrend. When the global test for association was significant, allelic OR of a specific [TTTA]n repeat was estimated, where the group with [TTTA]7 and [TTTA]7delTCT alleles served as a reference category.

We used the haplo.stats package (45) for R for reconstruction of haplotypes and analysis of their potential association with breast cancer. Haplotype frequencies were estimated and compared using age-adjusted global and haplotype-specific score tests (“haplo.score” function). Rare haplotypes (combined frequency <10%) were pooled. In addition, based on inferred haplotype data, possible haplotype pairs and their posterior probability were calculated using the haplo.em function in R. A continuous variable counting the number of haplotype copies as well as indicator variables for one and two copies were created. Expectations of these variables were computed for each subject to account for the haplotype ambiguity. Using logistic regression, expected haplotype values were used to estimate age-adjusted ORs in both log-additive and dominant model. Pairwise LD (D′ and r2) between the haplotype-tagging SNPs was tested using the χ2 statistic for LD in the “genetic” package for R.

In all analyses, P values < 0.05 were considered statistically significant. Unless otherwise specified, all results are age-adjusted. Analyses were done in SPSS (version 14) and SAS (version 9.1).

The frequency of the CYP19 Val80 G allele among controls was 50% in BRCA1 carriers, 50% in BRCA2 carriers, and 49% in noncarriers. CYP19 genotypes did not deviate from the Hardy-Weinberg equilibrium in controls.

In all BRCA1 mutation carriers, CYP19 Val80 was not associated with breast cancer risk (OR per G allele increase, 1.17; 95% CI, 0.83-1.63; P = 0.370). However, it was found that, among the 180 BRCA1 carriers ages <50 years, the Val80 G allele was associated with increased breast cancer risk (Ptrend = 0.028; G/G versus A/A; OR, 2.81; 95% CI, 1.09-7.22; P = 0.032; Table 2). No significant association between CYP19 Val80 and breast cancer risk was found in either BRCA1 carriers of older age (age ≥50 years; Ptrend = 0.321) or BRCA2 carriers (Ptrend = 0.888 and 0.946 for ages <50 and ≥50 years, respectively; Table 2). There was no difference between cases and controls in Val80 genotype distribution among the noncarriers of BRCA1/2 mutations (Ptrend = 0.300, 0.628, and 0.346 for the overall and ages <50 and ≥50 years, respectively; Table 2).

Table 2.

CYP19 Val80 genotype and breast cancer risk in BRCA1/2 carriers and noncarriers

BRCA1/2 statusVal80 genotypeCases/controlsOR (95% CI)PPtrend
<50 y      
    BRCA1 carriers A/A 15/24 Reference  0.028 
 G/A 39/54 1.41 (0.61-3.26) 0.418  
 G/G 27/19 2.81 (1.09-7.22) 0.032  
 Total 81/97    
    BRCA2 carriers A/A 10/13 Reference  0.888 
 G/A 22/29 1.21 (0.42-3.47) 0.719  
 G/G 8/15 0.91 (0.26-3.20) 0.885  
 Total 40/57    
    Noncarriers A/A 28/26 Reference  0.628 
 G/A 67/51 1.22 (0.59-2.38) 0.549  
 G/G 22/17 1.20 (0.51-3.21) 0.664  
 Total 117/94    
≥50 y      
    BRCA1 carriers A/A 15/12 Reference  0.321 
 G/A 33/25 1.05 (0.42-2.63) 0.922  
 G/G 13/17 0.6 (0.21-1.71) 0.336  
 Total 61/54    
    BRCA2 carriers A/A 9/10 Reference  0.946 
 G/A 20/27 0.82 (0.28-2.39) 0.716  
 G/G 7/8 0.98 (0.25-3.82) 0.978  
 Total 36/45    
    Noncarriers A/A 153/146 Reference  0.346 
 G/A 299/303 0.94 (0.69-1.24) 0.677  
 G/G 166/137 1.16 (0.83-1.63) 0.366  
 Total 618/586    
BRCA1/2 statusVal80 genotypeCases/controlsOR (95% CI)PPtrend
<50 y      
    BRCA1 carriers A/A 15/24 Reference  0.028 
 G/A 39/54 1.41 (0.61-3.26) 0.418  
 G/G 27/19 2.81 (1.09-7.22) 0.032  
 Total 81/97    
    BRCA2 carriers A/A 10/13 Reference  0.888 
 G/A 22/29 1.21 (0.42-3.47) 0.719  
 G/G 8/15 0.91 (0.26-3.20) 0.885  
 Total 40/57    
    Noncarriers A/A 28/26 Reference  0.628 
 G/A 67/51 1.22 (0.59-2.38) 0.549  
 G/G 22/17 1.20 (0.51-3.21) 0.664  
 Total 117/94    
≥50 y      
    BRCA1 carriers A/A 15/12 Reference  0.321 
 G/A 33/25 1.05 (0.42-2.63) 0.922  
 G/G 13/17 0.6 (0.21-1.71) 0.336  
 Total 61/54    
    BRCA2 carriers A/A 9/10 Reference  0.946 
 G/A 20/27 0.82 (0.28-2.39) 0.716  
 G/G 7/8 0.98 (0.25-3.82) 0.978  
 Total 36/45    
    Noncarriers A/A 153/146 Reference  0.346 
 G/A 299/303 0.94 (0.69-1.24) 0.677  
 G/G 166/137 1.16 (0.83-1.63) 0.366  
 Total 618/586    

NOTE: Adjusted for age.

Of the 956 breast cancer cases in the study, ER status was available for 772 (81%) cases (108 BRCA1 carriers, 55 BRCA2 carriers, and 609 noncarriers). There were 18 (2.9%) tumors diagnosed with ductal carcinoma in situ. Tumors were ER-negative in 67% of the BRCA1 carriers (72 of 108), 29.1% of the BRCA2 carriers, and 20.4% of the noncarriers. A case-only analysis of BRCA1 carriers ages <50 years showed a trend toward association between Val80 G/G and ER-negative tumors although not statistically significant (G/G versus A/A; OR, 3.53; 95% CI, 0.58-21.51; P = 0.171; Ptrend = 0.173; Table 3).

Table 3.

CYP19 Val80 genotype and ER-negative status in BRCA1/2 carriers

BRCA1/2 statusVal80 genotypeER(−)ER(+)OR (95% CI)PPtrend
<50 y       
    BRCA1 carriers A/A 1.00  0.173 
 G/A 21 1.95 (0.40-9.44) 0.405  
 G/G 16 3.53 (0.58-21.51) 0.171  
 Total 43 14    
≥50 y       
    BRCA1 carriers A/A 1.00  0.747 
 G/A 14 11 0.75 (0.18-3.21) 0.697  
 G/G 0.76 (0.14-4.09) 0.746  
 Total 28 21    
BRCA1/2 statusVal80 genotypeER(−)ER(+)OR (95% CI)PPtrend
<50 y       
    BRCA1 carriers A/A 1.00  0.173 
 G/A 21 1.95 (0.40-9.44) 0.405  
 G/G 16 3.53 (0.58-21.51) 0.171  
 Total 43 14    
≥50 y       
    BRCA1 carriers A/A 1.00  0.747 
 G/A 14 11 0.75 (0.18-3.21) 0.697  
 G/G 0.76 (0.14-4.09) 0.746  
 Total 28 21    

NOTE: Adjusted for age.

We genotyped haplotype-tagging SNPs in haplotype block 4 covering the CYP19 coding region [rs727479, rs700518 (Val80), rs10046, and rs4646] and reconstructed haplotypes. Haplotype analysis of the younger BRCA1 carriers showed an increase trend in breast cancer risk associated with the common TGTG haplotype (frequency, 48.1%; OR per haplotype copy, 1.56; Ptrend = 0.066; OR for TGTG haplotype homozygotes versus no copies of TGTG, 2.42; 95% CI, 0.95-6.16; P = 0.063; Table 4). This is consistent with the suggestive association between the rs10046 T allele and risk of breast cancer among younger BRCA1 carriers (Ptrend = 0.062; OR for T/T versus C/C, 2.46; 95% CI, 0.96-6.28; P = 0.060) and the OR of rs4646 G/G versus T/T was 2.69 (95% CI, 0.72-10.05; P = 0.140; data not shown). The intron 4 [TTTA]n polymorphism was genotyped in a subset of 104 noncarriers and 284 BRCA1 carriers and was found to be in complete LD with CYP19 Val80. A higher number of repeats (>7) was linked with the Val80 G allele (D′ = 1; r2 = 1; data not shown). The rare [TTTA]13 allele was not found in our sample. Among the younger BRCA1 mutation carriers, increasing number of repeats tended to be associated with increased breast cancer risk (OR per 1 repeat increase, 1.18; P = 0.053; data not shown).

Table 4.

CYP19 coding region TGTG haplotype and breast cancer risk in BRCA1 carriers before and after age 50 y and BRCA2 carriers

HaplotypesFrequency
OR (95% CI)PPtrend
Case (%)Control (%)
BRCA1 <50 y      
    Other/other 21.5 30.7 Reference  0.063 
    TGTG/other 50.8 50.7 1.63 (0.72-3.66) 0.241  
    TGTG/TGTG 27.7 18.5 2.42 (0.95-6.16) 0.063  
BRCA1 >50 y      
    Other/other 28.6 25.4 Reference  0.454 
    TGTG/other 53.7 50.6 0.94 (0.38-2.30) 0.887  
    TGTG/TGTG 17.7 24.0 0.65 (0.22-1.93) 0.440  
BRCA2 <50 y      
    Other/other 32.2 24.9 Reference  0.803 
    TGTG/other 48.3 50.6 0.99 (0.36-2.75) 0.983  
    TGTG/TGTG 19.4 24.5 0.85 (0.25-2.93) 0.793  
BRCA2 >50 y      
    Other/other 25.7 25.5 Reference  0.977 
    TGTG/other 60.8 61.4 0.98 (0.34-2.83) 0.973  
    TGTG/TGTG 13.5 13.1 1.03 (0.23-4.58) 0.965  
HaplotypesFrequency
OR (95% CI)PPtrend
Case (%)Control (%)
BRCA1 <50 y      
    Other/other 21.5 30.7 Reference  0.063 
    TGTG/other 50.8 50.7 1.63 (0.72-3.66) 0.241  
    TGTG/TGTG 27.7 18.5 2.42 (0.95-6.16) 0.063  
BRCA1 >50 y      
    Other/other 28.6 25.4 Reference  0.454 
    TGTG/other 53.7 50.6 0.94 (0.38-2.30) 0.887  
    TGTG/TGTG 17.7 24.0 0.65 (0.22-1.93) 0.440  
BRCA2 <50 y      
    Other/other 32.2 24.9 Reference  0.803 
    TGTG/other 48.3 50.6 0.99 (0.36-2.75) 0.983  
    TGTG/TGTG 19.4 24.5 0.85 (0.25-2.93) 0.793  
BRCA2 >50 y      
    Other/other 25.7 25.5 Reference  0.977 
    TGTG/other 60.8 61.4 0.98 (0.34-2.83) 0.973  
    TGTG/TGTG 13.5 13.1 1.03 (0.23-4.58) 0.965  

NOTE: Adjusted for age.

The haplotypes consist of haplotype-tagging SNPs tagging haplotype block 4 of CYP19 gene that cover the entire coding region of the gene: rs727479, rs700518 (Val80), rs10046, and rs4646. Note that Val80 (rs700518) was not included by Haiman et al. (44) to haplotype-tagging SNPs.

In the present study, we found an association between SNPs and haplotypes of the CYP19 gene and breast cancer risk in BRCA1 mutation carriers ages <50 years. Although many studies have been published supporting the role of CYP19 polymorphisms in breast cancer (26, 27, 29, 31-33), the modifying effect of these polymorphisms on BRCA1-related breast cancer risk had not been previously studied. Our finding may shed light on possible mechanisms by which the penetrance of BRCA mutations is altered. Such partial penetrance reflects the possible existence of modifying genes or lifestyle factors that have been formerly suggested (46).

The previously reported prevalence of ER-negative tumors in premenopausal BRCA1 breast cancer (42) and relatively high frequency of breast cancer in the general population may bring to conclusion about higher frequency of phenocopies among women with postmenopausal breast cancer. From this point of view, the association shown in our study of CYP19 polymorphisms with breast cancer risk among premenopausal women is reasonable. In addition, a recent study found the association of CYP19 rs10046 T/T with elevated breast cancer risk in middle age group (ages 45-54 years; ref. 47).

The association of CYP19 with breast cancer risk was found in BRCA1 carriers ages <50 years but not in BRCA2 carriers. This agrees with known biological and clinical differences between breast tumors in BRCA1 and BRCA2 carriers (2). BRCA1-related tumors are more commonly ER-negative, differently related to reproductive risk factors, less often detected by mammography, and more commonly involve ovarian cancers than are BRCA2-related tumors. These differences may be attributed to differences in estrogen metabolism or availability.

The complete LD between [TTTA]n repeats and Val80 observed in our study is in line with former reports (26, 27, 29, 31-34). Several studies found an association between shorter [TTTA]n alleles ([TTTA]7 and [TTTA]<9) and lower blood estrogen levels, whereas longer alleles ([TTTA]8 and [TTTA]>9) were found to be associated with higher blood estrogen levels (27, 35, 38). These observations support our finding that the Val80 G allele (and therefore [TTTA]>7 alleles) increases the risk of breast cancer compared with shorter [TTTA]7 allele, which may be due to higher lifetime exposure to estrogens. Furthermore, our results show a suggestive positive association between the Val80 G/G genotype ([TTTA]>7 homozygotes) and ER-negative tumors in young BRCA1 carriers. Although this association did not reach statistical significance, this may be due to a lack of power from missing data on tumors collected at the beginning of the study. A potential association between Val80 G/G genotype and ER-negative tumors would have implications for our understanding of the factors that cause a breast cancer cell to develop into an ER-positive or ER-negative tumor. Higher circulating levels of estrogens associated with [TTTA]>7 homozygotes (or Val80 G/G) in ER-negative tumors can thus be a key element in the tumor microenvironment directing its receptor commitment.

Suitability for hormonal chemoprevention or treatment with selective ER modulators or aromatase inhibitors is another implication of the potential association between Val80 and ER status. Whereas most studies show that ER-negative tumors do not respond to hormonal interventions, one case-control study (48) in BRCA carriers suggested the opposite. The role of estrogen in BRCA1-related breast cancer pathogenesis (and not in BRCA2) may depend on the direct carcinogenic effect of estrogen metabolites and not on ER binding (49). The ER-negative breast stem cells are surrounded by ER-positive cells that can exert a paracrine effect on these stem cells (49, 50). BRCA1 is potentially involved in the maturation of ER-negative stem cells into mature ER-positive cells (42). In BRCA1 mutation carriers, the differentiation is impaired (51) and the enlarged pool of stem cells has greater chance of malignant transformation. The higher estrogen levels potentially associated with the Val80 G/G genotype can promote the synthesis of growth factors (such as epidermal growth factors and insulin-like growth factors) by ER-positive mature breast cells surrounding ER-negative stem cells (52). Growth factors could further enhance stem cells proliferation, which in turn could increase the rate of stem cell transformation.

In spite of the demonstrated modifying effect of Val80 and [TTTA]n polymorphisms, it is likely that the biologically functional change in the aromatase protein is due to an unknown causal allele in the coding region of CYP19, related to the risk haplotype TGTG. Breast cancer risk modification seems to be restricted to two SNPs of these haplotypes, Val80 (rs700518) and rs10046 (3′-untranslated region). These SNPs are in very strong LD (D′ = 0.93, r2 = 0.88) and cover almost the entire coding region of CYP19 from exons 3 to 10. Nevertheless, our findings provide what seems to be a breast cancer risk modifier for younger women carrying BRCA1 gene mutations.

No potential conflicts of interest were disclosed.

Grant support: Chief Scientist Office, Israeli Ministry of Health, and Israel Cancer Association. Partial results have been presented as posters in AACR Workshop on SNPs and Cancer, Key Biscayne, FL 2002, at the Clalit Health Services Cancer Prevention Conference, Dead Sea 2003, and in the American Society of Clinical Oncology Annual Meeting in Chicago, 2007.

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
Bray F, McCarron P, Parkin DM. The changing global patterns of female breast cancer incidence and mortality.
Breast Cancer Res
2004
;
6
:
229
–39.
2
Narod SA, Foulkes WD. BRCA1 and BRCA2: 1994 and beyond.
Nat Rev Cancer
2004
;
4
:
665
–76.
3
Tonin P, Weber B, Offit K, et al. Frequency of recurrent BRCA1 and BRCA2 mutations in Ashkenazi Jewish breast cancer families.
Nat Med
1996
;
2
:
1179
–83.
4
Abeliovich D, Kaduri L, Lerer I, et al. The founder mutations 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 appear in 60% of ovarian cancer and 30% of early-onset breast cancer patients among Ashkenazi women.
Am J Hum Genet
1997
;
60
:
505
–14.
5
Rennert G, Bisland-Naggan S, Barnett-Griness O, et al. Clinical outcomes of breast cancer in carriers of BRCA1 and BRCA2 mutations.
N Engl J Med
2007
;
357
:
115
–23.
6
Easton DF, Ford D, Bishop DT. Breast and ovarian cancer incidence in BRCA1-mutation carriers. Breast Cancer Linkage Consortium.
Am J Hum Genet
1995
;
56
:
265
–71.
7
Struewing JP, Hartge P, Wacholder S, et al. The risk of cancer associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews.
N Engl J Med
1997
;
336
:
1401
–8.
8
Hopper JL, Southey MC, Dite GS, et al. Population-based estimate of the average age-specific cumulative risk of breast cancer for a defined set of protein-truncating mutations in BRCA1 and BRCA2. Australian Breast Cancer Family Study.
Cancer Epidemiol Biomarkers Prev
1999
;
8
:
741
–7.
9
Thorlacius S, Struewing JP, Hartge P, et al. Population-based study of risk of breast cancer in carriers of BRCA2 mutation.
Lancet
1998
;
352
:
1337
–9.
10
Ballard-Barbash R, Klabunde C, Paci E, et al. Breast cancer screening in 21 countries: delivery of services, notification of results and outcomes ascertainment.
Eur J Cancer Prev
1999
;
8
:
417
–26.
11
Offit K. BRCA mutation frequency and penetrance: new data, old debate.
J Natl Cancer Inst
2006
;
98
:
1675
–7.
12
Risch HA, McLaughlin JR, Cole DE, et al. Population BRCA1 and BRCA2 mutation frequencies and cancer penetrances: a kin-cohort study in Ontario, Canada.
J Natl Cancer Inst
2006
;
98
:
1694
–706.
13
Breast cancer and breastfeeding: collaborative reanalysis of individual data from 47 epidemiological studies in 30 countries, including 50302 women with breast cancer and 96973 women without the disease.
Lancet
2002
;
360
:
187
–95.
14
Jakubowska A, Narod SA, Goldgar DE, et al. Breast cancer risk reduction associated with the RAD51 polymorphism among carriers of the BRCA1 5382insC mutation in Poland.
Cancer Epidemiol Biomarkers Prev
2003
;
12
:
457
–9.
15
Kadouri L, Easton DF, Edwards S, et al. CAG and GGC repeat polymorphisms in the androgen receptor gene and breast cancer susceptibility in BRCA1/2 carriers and non-carriers.
Br J Cancer
2001
;
85
:
36
–40.
16
Kadouri L, Kote-Jarai Z, Easton DF, et al. Polyglutamine repeat length in the AIB1 gene modifies breast cancer susceptibility in BRCA1 carriers.
Int J Cancer
2004
;
108
:
399
–403.
17
Levy-Lahad E, Lahad A, Eisenberg S, et al. A single nucleotide polymorphism in the RAD51 gene modifies cancer risk in BRCA2 but not BRCA1 carriers.
Proc Natl Acad Sci U S A
2001
;
98
:
3232
–6.
18
Rebbeck TR, Kantoff PW, Krithivas K, et al. Modification of BRCA1-associated breast cancer risk by the polymorphic androgen-receptor CAG repeat.
Am J Hum Genet
1999
;
64
:
1371
–7.
19
Rebbeck TR, Wang Y, Kantoff PW, et al. Modification of BRCA1- and BRCA2-associated breast cancer risk by AIB1 genotype and reproductive history.
Cancer Res
2001
;
61
:
5420
–4.
20
Redston M, Nathanson KL, Yuan ZQ, et al. The APCI1307K allele and breast cancer risk.
Nat Genet
1998
;
20
:
13
–4.
21
Wang WW, Spurdle AB, Kolachana P, et al. A single nucleotide polymorphism in the 5′ untranslated region of RAD51 and risk of cancer among BRCA1/2 mutation carriers.
Cancer Epidemiol Biomarkers Prev
2001
;
10
:
955
–60.
22
Antoniou AC, Sinilnikova OM, Simard J, et al. RAD51 135G->C modifies breast cancer risk among BRCA2 mutation carriers: results from a combined analysis of 19 studies.
Am J Hum Genet
2007
;
81
:
1186
–200.
23
Clemons M, Goss P. Estrogen and the risk of breast cancer.
N Engl J Med
2001
;
344
:
276
–85.
24
Hsieh CC, Trichopoulos D, Katsouyanni K, Yuasa S. Age at menarche, age at menopause, height and obesity as risk factors for breast cancer: associations and interactions in an international case-control study.
Int J Cancer
1990
;
46
:
796
–800.
25
Polymeropoulos MH, Xiao H, Rath DS, Merril CR. Tetranucleotide repeat polymorphism at the human aromatase cytochrome P-450 gene (CYP19).
Nucleic Acids Res
1991
;
19
:
195
.
26
Baxter SW, Choong DY, Eccles DM, Campbell IG. Polymorphic variation in CYP19 and the risk of breast cancer.
Carcinogenesis
2001
;
22
:
347
–9.
27
Haiman CA, Hankinson SE, Spiegelman D, et al. A tetranucleotide repeat polymorphism in CYP19 and breast cancer risk.
Int J Cancer
2000
;
87
:
204
–10.
28
Healey CS, Dunning AM, Durocher F, et al. Polymorphisms in the human aromatase cytochrome P450 gene (CYP19) and breast cancer risk.
Carcinogenesis
2000
;
21
:
189
–93.
29
Kristensen VN, Andersen TI, Lindblom A, Erikstein B, Magnus P, Borresen-Dale AL. A rare CYP19 (aromatase) variant may increase the risk of breast cancer.
Pharmacogenetics
1998
;
8
:
43
–8.
30
Probst-Hensch NM, Ingles SA, Diep AT, et al. Aromatase and breast cancer susceptibility.
Endocr Relat Cancer
1999
;
6
:
165
–73.
31
Siegelmann-Danieli N, Buetow KH. Constitutional genetic variation at the human aromatase gene (Cyp19) and breast cancer risk.
Br J Cancer
1999
;
79
:
456
–63.
32
Miyoshi Y, Ando A, Hasegawa S, et al. Association of genetic polymorphisms in CYP19 and CYP1A1 with the oestrogen receptor-positive breast cancer risk.
Eur J Cancer
2003
;
39
:
2531
–7.
33
Miyoshi Y, Iwao K, Ikeda N, Egawa C, Noguchi S. Breast cancer risk associated with polymorphism in CYP19 in Japanese women.
Int J Cancer
2000
;
89
:
325
–8.
34
Ahsan H, Whittemore AS, Chen Y, et al. Variants in estrogen-biosynthesis genes CYP17 and CYP19 and breast cancer risk: a family-based genetic association study.
Breast Cancer Res
2005
;
7
:
R71
–81.
35
Tworoger SS, Chubak J, Aiello EJ, et al. Association of CYP17, CYP19, CYP1B1, and COMT polymorphisms with serum and urinary sex hormone concentrations in postmenopausal women.
Cancer Epidemiol Biomarkers Prev
2004
;
13
:
94
–101.
36
Dunning AM, Dowsett M, Healey CS, et al. Polymorphisms associated with circulating sex hormone levels in postmenopausal women.
J Natl Cancer Inst
2004
;
96
:
936
–45.
37
Paynter RA, Hankinson SE, Colditz GA, Kraft P, Hunter DJ, De Vivo I. CYP19 (aromatase) haplotypes and endometrial cancer risk.
Int J Cancer
2005
;
116
:
267
–74.
38
Gennari L, Masi L, Merlotti D, et al. A polymorphic CYP19 TTTA repeat influences aromatase activity and estrogen levels in elderly men: effects on bone metabolism.
J Clin Endocrinol Metab
2004
;
89
:
2803
–10.
39
Haiman CA, Dossus L, Setiawan VW, et al. Genetic variation at the CYP19A1 locus predicts circulating estrogen levels but not breast cancer risk in postmenopausal women.
Cancer Res
2007
;
67
:
1893
–7.
40
Riancho JA, Valero C, Naranjo A, Morales DJ, Sanudo C, Zarrabeitia MT. Identification of an aromatase haplotype that is associated with gene expression and postmenopausal osteoporosis.
J Clin Endocrinol Metab
2007
;
92
:
660
–5.
41
Lakhani SR, Van De Vijver MJ, Jacquemier J, et al. The pathology of familial breast cancer: predictive value of immunohistochemical markers estrogen receptor, progesterone receptor, HER-2, and p53 in patients with mutations in BRCA1 and BRCA2.
J Clin Oncol
2002
;
20
:
2310
–8.
42
Foulkes WD, Metcalfe K, Sun P, et al. Estrogen receptor status in BRCA1- and BRCA2-related breast cancer: the influence of age, grade, and histological type.
Clin Cancer Res
2004
;
10
:
2029
–34.
43
Hirose K, Matsuo K, Toyama T, Iwata H, Hamajima N, Tajima K. The CYP19 gene codon 39 Trp/Arg polymorphism increases breast cancer risk in subsets of premenopausal Japanese.
Cancer Epidemiol Biomarkers Prev
2004
;
13
:
1407
–11.
44
Haiman CA, Stram DO, Pike MC, et al. A comprehensive haplotype analysis of CYP19 and breast cancer risk: the Multiethnic Cohort.
Hum Mol Genet
2003
;
12
:
2679
–92.
45
Schaid DJ, Rowland CM, Tines DE, Jacobson RM, Poland GA. Score tests for association between traits and haplotypes when linkage phase is ambiguous.
Am J Hum Genet
2002
;
70
:
425
–34.
46
Thompson D, Easton D. The genetic epidemiology of breast cancer genes.
J Mammary Gland Biol Neoplasia
2004
;
9
:
221
–36.
47
Ralph DA, Zhao LP, Aston CE, et al. Age-specific association of steroid hormone pathway gene polymorphisms with breast cancer risk.
Cancer
2007
;
109
:
1940
–8.
48
Narod SA, Brunet JS, Ghadirian P, et al. Tamoxifen and risk of contralateral breast cancer in BRCA1 and BRCA2 mutation carriers: a case-control study. Hereditary Breast Cancer Clinical Study Group.
Lancet
2000
;
356
:
1876
–81.
49
Clarke RB, Howell A, Anderson E. Estrogen sensitivity of normal human breast tissue in vivo and implanted into athymic nude mice: analysis of the relationship between estrogen-induced proliferation and progesterone receptor expression.
Breast Cancer Res Treat
1997
;
45
:
121
–33.
50
Zeps N, Bentel JM, Papadimitriou JM, D'Antuono MF, Dawkins HJ. Estrogen receptor-negative epithelial cells in mouse mammary gland development and growth.
Differentiation
1998
;
62
:
221
–6.
51
Furuta S, Jiang X, Gu B, Cheng E, Chen PL, Lee WH. Depletion of BRCA1 impairs differentiation but enhances proliferation of mammary epithelial cells.
Proc Natl Acad Sci U S A
2005
;
102
:
9176
–81.
52
Rudland PS, Fernig DG, Smith JA. Growth factors and their receptors in neoplastic mammary glands.
Biomed Pharmacother
1995
;
49
:
389
–99.