The Progesterone Receptor Exon 4 Val660Leu G/T Polymorphism and Risk of Breast Cancer in Australian Women¹

Although endogenous and exogenous exposure to estrogen is associated with an increased risk of breast cancer, there is conflicting evidence regarding the role of progesterone exposure in mammary gland biology. Progesterone antagonizes estrogen action and has antiproliferative effects in reproductive tissues such as the endometrium (1); numerous in vivo and in vitro studies on normal and malignant human breast cells have shown that progesterone can either stimulate or inhibit cell proliferation (2, 3, 4), and higher saliva concentrations of progesterone in the mid-luteal phase have been associated with a greater incidence of breast cancer (5).

Linkage disequilibrium has been reported to exist between three polymorphisms of the PR³gene: an intronic Alu insertion, an exon 4 amino acid substitution codon 660 T variant, and an exon 5 synonymous codon 700 T variant (6, 7). Although there are no peer-reviewed published data indicating that this complex of polymorphisms has functional significance, data from transient cotransfection assays (published in abstract form) indicate that the insertion allele shows higher mRNA stability, increased amount of protein, and higher transcriptional activity than the wild-type allele, with highest transcriptional activity for the heterodimer receptor (6, 7). This intronic Alu insertion polymorphism was reported recently to be associated with a reduced risk of breast cancer in a German population-based study of 554 cases and 559 controls (8). Compared with the common homozygote genotype, the rare Alu insertion homozygote was associated with a 0.3-fold risk of breast cancer (95% CI, 0.1–0.7), and the heterozygote was associated with a not significant 0.8-fold risk (95% CI, 0.6–1.1). Two smaller independent studies of British women (292 cases and 220 controls; Ref. 9) and North American women (68 cases and 101 controls; Ref. 10) did not find any evidence for an association between breast cancer risk and the Alu insertion. The Alu insertion polymorphism has also been investigated recently as a candidate modifier in BRCA1 and BRCA2 mutation carriers (11). Although this study of 195 ovarian cancer cases, 392 breast cancer cases, and 294 controls without breast or ovarian cancer found no association with risk of breast or ovarian cancer status overall, the insertion variant was more common in ovarian cancer cases compared with the subset of just 125 pooled breast cancer cases and controls without a history of oral contraceptive use, leading the authors to suggest that the Alu insertion variant may be associated with increased risk of ovarian cancer in carriers without exposure to oral contraceptives (11).

We have undertaken a case-control comparison to assess whether the PR exon 4 G to T Val660Leu amino acid substitution is associated with risk of female breast cancer in Australia. We chose this particular variant because it is the only amino acid substitution polymorphism reported to be in linkage disequilibrium with the Alu insertion and may account directly for the functional differences reported to be associated with the complex of three polymorphisms in which it is included.

A population-based, case-control-family study of breast cancer in women <40 years of age was carried out in Melbourne and Sydney from 1992 to 1995 (12, 13, 14) and extended from 1996 to 2000 to also include women up to 59 years of age (13). Cases were women with a diagnosis of a first primary breast cancer identified although the Victorian and New South Wales cancer registries. Controls were women without breast cancer selected from the electoral roll (adult registration for voting is compulsory in Australia) using stratified random sampling, frequency matched for age. Cases and controls were administered a questionnaire to record family history of cancer and other known or potential risk factors for breast cancer. With the subjects’ permission, all living parents, aunts, grandparents, and adult siblings were asked to participate and were administered the same risk factor questionnaire (13, 14). Ancestry was assessed by an open-ended question and from the country of birth of the respondents, their parents, and grandparents. The great majority of subjects’ parents and grandparents were born in Australia, the British Isles, or Western Europe. In subanalyses restricted to Caucasian women (1302 cases and 665 controls), subjects with any Australian aboriginal, Torres Strait Islander, or Maori heritage or any country of birth in the South Pacific, Indian Ocean, Caribbean islands, or Asia were excluded.

Family history of cancers was systematically collected from each case and control and included cancer history of all of their first- and second-degree relatives. This history was subsequently checked with each living relative at the time of their interview. A family history of breast cancer was defined as having at least one first- or second-degree relative with breast cancer. Verification of all cancers reported by subjects and their relatives was sought through personal interview, cancer registries, pathology reports, hospital records, clinicians, and death certificates.

Interviews were conducted for 1579 cases and 1021 controls. PR exon 4 G/T genotyping was performed on 1453 cases (92% of participating) and 793 controls (78% of participating) on the basis of DNA availability. Results were obtained for 1452 cases and all 793 controls, constituting a PCR success rate of 99.95%. Genotyped cases ranged in age at diagnosis from 22 to 59 years (average, 41.7 years; SD, 8.7), and genotyped controls ranged in age at interview from 20 to 60 years (average, 40.9 years; SD, 9.0).

Mutation detection within the BRCA1 and BRCA2 genes for these samples is ongoing. To date, by protein-truncation testing in specific exons covering ∼70% of the coding regions, manual sequencing of the coding regions in a subset, and some additional testing, a total of 35 cases included in the PR exon 4 G/T analysis have been found to have a deleterious mutation in BRCA1 or BRCA2 (15, 16, 17).⁴

This study was approved by the human research ethics committees of the University of Melbourne, the New South Wales Cancer Council, the Anti-Cancer Council of Victoria, and the Queensland Institute of Medical Research.

Collection of peripheral blood and DNA extraction have been described previously (18). The PR exon 4 Val660Leu G/T polymorphism (GenBank accession number P06401) was detected using the PE ABI Prism 7700 Sequence Detection System for multicolor real-time or endpoint fluorogenic PCR detection (PE Applied Biosystems, Foster City, CA), as described previously (19). A subset of samples of known PR exon 4 genotype (n = 52), as determined by Sequence Detection System analysis, were screened for the PR Alu insertion polymorphism by PCR using forward and reverse primers 5′-ATACGGTATCCATGACATGAG-3′ and 5′-AAGTATTTTCTTGCTAAATGTCTG-3′ spanning the Alu insertion. The 10-μl reaction mix contained 15 ng of DNA, primers (8 pmol each), deoxynucleotide triphosphates (200 nm), 1× Perkin-Elmer Taq polymerase buffer, 1 unit of Taq polymerase, and 1.5 mm MgCl₂. Amplification conditions were 5 min at 94°C, 35 cycles of 94°C for 20 s, 51°C for 20 s, and 72°C for 60 s, followed by a 10-min extension at 72°C. Reaction products, 79 bp for wild type and 385 bp for Alu insertion sequence, were resolved on 4.5% Nusieve gels.

Allele frequencies were estimated and compared, assuming that alleles within an individual were independent binomial variables. The HWE assumption was assessed for defined groups using maximum likelihood methods by comparing the observed numbers of different genotypes with those expected under HWE in that group and also for cases by assuming that the control genotypes were under HWE (20). Logistic regression was used to analyze genotype as a binary outcome, defined according to recessive or dominant mode of inheritance, as a function of measured variables [see McCredie et al. (14) for details on the categorization of variables]. The association between the PR exon 4 G/T genotype and risk of breast cancer was assessed using unconditional multiple logistic regression, with and without adjustment for other measured risk factors. All statistical tests were two-tailed, and statistical significance was taken as P < 0.05. SPSS (version 9.0), Epistat, and Ottutil software were used.

Table 1 shows that genotyped women were slightly older than nongenotyped women (40.7 versus 38.3 years for cases, 40.9 versus 38.6 years for controls) and more likely to report a family history of breast cancer (95% versus 91% for cases, 88% versus 75% for controls), whereas genotyped cases (but not controls) were more likely to be postmenopausal (95% versus 91%). Nevertheless, there was no difference between genotyped cases and controls with respect to age (P = 0.5) or menopausal status (P = 0.4).

Table 2 shows the genotype distribution of the PR exon 4 G/T polymorphism in cases and controls. The T allele frequency was no different in cases than in controls (P = 0.9), being 0.166 (95% CI, 0.152–0.179) in cases and 0.164 (95% CI, 0.146–0.182) in controls. Similarly, there was no difference in genotype distribution between cases and controls (P = 0.5) and no deviation from HWE in cases (P = 0.2) or controls (P = 0.5). Genotype distribution was also independent of age at diagnosis, family history, and menopausal status (all P > 0.1).

Table 3 shows that, compared with the GG genotype, neither the GT genotype nor the TT genotype was associated with risk of breast cancer [OR, 0.95 (95% CI, 0.78–1.15)] and [OR, 1.34 (95% CI, 0.78–2.30)], P = 0.6 and 0.3, respectively. Findings were unchanged by adjustment for age alone (data not shown) or for age and other measured variables (as shown in Table 3). Furthermore, there was no difference in the risk associated with the TT genotype under a recessive model, being 1.36 (95% CI, 0.79–2.34) before and 1.53 (95% CI, 0.88–2.68) after adjustment.

Results were unchanged when analyses were carried out excluding the 35 cases known to carry a deleterious BRCA1 or BRCA2 mutation. Under the codominant model, the crude OR was 1.00 (95% CI, 0.83–1.21) for the GT genotype (P = 1.0) and 1.44 (95% CI, 0.84–2.48) for the TT genotype (P = 0.2). Similarly, for analyses restricted to subjects of Caucasian ancestry, the crude OR was 0.92 (95% CI, 0.75–1.14) for the GT genotype (P = 0.4) and 1.23 (95% CI, 0.70–2.15) for the TT genotype (P = 0.5).

Overall, our findings did not support either the GT or TT genotypes being associated with risk of breast cancer. The confidence intervals for all estimates of risk associated with either genotype included 1.0. However, because the point estimate of the OR for the TT genotype was greater than unity, the more so after adjustment, we investigated the recessive model in more detail. Within cases and within controls, there was no evidence that the proportion with the TT genotype differed according to age, family history, or any of the measured variables shown (14) or known to influence breast cancer risk (all P ≥ 0.1). Stratification by family history, age, and menopausal status revealed no differences in the effect of the PR polymorphism on breast cancer risk between strata (all P > 0.6). For example, the crude OR for the TT genotype under the recessive model was 1.48 (95% CI, 0.49–4.53) for subjects reporting a family history (P = 0.5) and 1.34 (95% CI, 0.72–2.49) for those without (P = 0.4). Similarly, there was no difference between estimates for women <40 years of age (OR, 1.35; 95% CI, 0.68–2.69) and those 40–59 years of age (OR, 1.41; 95% CI, 0.59–3.38) nor between premenopausal woman (OR, 1.41; 95% CI, 0.75–2.63) and postmenopausal women (OR, 0.95; 95% CI, 0.31–2.96).

To assess the report of linkage disequilibrium between the PR Alu insertion polymorphism and the exon 4 T allele, a subset of 52 subjects of known exon 4 genotype (as established by the Sequence Detection System assay) was screened for the insertion polymorphism. There was perfect concordance between genotypes for the 12 TT, 20 GG, and 20 GT individuals screened, suggesting that linkage disequilibrium between the T allele and Alu insertion is complete. This is supported by the observation that the Alu allele frequency in the German control sample (8) is the same as the T allele frequency in the Australian control sample (this study), 0.16.

Given the apparent linkage disequilibrium between the PR Alu insertion and exon 4 T alleles, our results contrast with the protective association between breast cancer risk and the Alu insertion allele reported by Wang-Gohrke et al. (8) but not the negative results reported in two smaller studies of British (9) and North American (10) subjects. Our Australian study of more than twice as many women as the German study, and larger than the combined three studies above, did not detect an association between the PR exon 4 T allele and risk of breast cancer, overall, or stratified by family history, age, or menopausal status. The risks associated with both the heterozygote and homozygote genotypes were not different from unity. There was no support for a decreased risk of breast cancer associated with the rare TT genotype, as reported previously for German women for the linked Alu insertion variant (8). Our estimate of risk associated with the TT genotype was significantly different from the 0.3-fold risk reported from the German study for the linked Alu insertion variant (P = 0.001), and the lower end of the 95% CI for the risk estimate was ∼0.8. We had 90% power to detect an OR of 0.3 or less for the rare homozygous TT genotype and 80% power to detect an OR of 0.8 or less for the heterozygote genotype. Selection bias was unlikely to have affected the findings of this study. There was no difference between genotyped cases and controls with respect to age and menopausal status, and although individuals reporting a family history were more likely to participate in genetic studies, this shift in proportion was the same for cases and controls, and we adjusted for family history in the analyses. Furthermore, there was no association between genotype and measured variables known to influence breast cancer risk, suggesting that confounding attributable to these factors was unlikely. Similar to our study, the German study was a population-based study of mostly premenopausal women, and risk estimates from the latter study were reported to be unchanged by adjustment for age, study region, reproductive factors (including menopausal status), and family history (8). Assuming complete linkage disequilibrium between the exon 4 T allele and the Alu insertion, there was no difference in genotype frequencies of control samples from the two populations (P = 0.6); therefore, differences in genotype frequencies between the case samples (P = 0.002) are unlikely to be attributable to methodological differences between the two studies.

There was also no support for a greatly increased risk of breast cancer associated with the T allele, given that we had 80% power to detect risks of 1.3 and 2.0 associated with the GT and TT genotypes, respectively. We therefore conclude that this polymorphism is not associated with a markedly reduced or increased risk of breast cancer in Australian women <60 years of age. However, despite its considerable size, our study cannot exclude a small reduced or increased risk associated with the T allele, especially the rare TT genotype.

We are indebted to Drs. Jenny Chang-Claude, Katie Healey, and Alison Dunning for discussion of their research results. We thank Sarah Steinborner for sample preparation and Gillian Dite for management and access to subject epidemiological data. We are grateful to physicians, surgeons, and oncologists in Victoria and New South Wales who endorsed this project, to the interviewing staff, and to the many women and their relatives who participated in this research.