The production of estrogen from androgen via the estrogen biosynthesis pathway is catalyzed by aromatase P450 (cyp19). We have assessed the frequency of allelic variants of the CYP19 intron 4 [TTTA]n repeat in 327 breast cancer cases and 253 controls from southern England. Previous studies have suggested that the [TTTA]10 repeat and [TTTA]12 repeat variants represent low penetrance breast cancer susceptibility alleles. Compared with controls our breast cancer cases had a statistically significant positive association with the [TTTA]10 allele (1.5 versus 0.2%, P = 0.028) and the [TTTA]8 allele (13.5 versus 8.7%, P = 0.012). The frequency of the [TTTA]12 allele was not significantly elevated in our study group compared with controls (2.3 versus 2.2%, P = 1.00). The CYP19 intron 4 [TTTA]n repeat is unlikely to have a functional effect on aromatase activity and it is more likely that the [TTTA]8 and [TTTA]10 variants are in linkage disequilibrium with other functional CYP19 variants.

Studies aimed at identifying common polymorphisms in genes that may represent low penetrance cancer susceptibility alleles are becoming increasingly common (1,2). Although the penetrance of such allelic variants may be low, the fact that they are very common means they may account for a large proportion of cancers. Candidate low penetrance cancer predisposition genes may include those involved in detoxification of chemicals or DNA repair. In the context of hormone-sensitive cancers such as breast cancer, genes involved in hormone biosynthesis and metabolism are very attractive candidates. In particular, exposure to endogenous estrogens are known to increase the risk of breast cancer (3). Consequently, genetic variants of genes involved in estrogen production are plausible candidates for breast cancer predisposition.

The P450 aromatase (cyp19) enzyme catalyzes the conversion of androgen to estrogen. Genetic variation at this locus may alter gene activity and thereby predispose carriers to increased breast cancer risk. The potential importance of this gene for breast cancer susceptibility has evoked several recent studies of genetic variation within the introns and exons of this gene. A [TTTA]n tetranucleotide repeat polymorphism within intron 4 of CYP19 has been analyzed for possible associations between the repeat number and breast cancer susceptibility (48). Seven alleles have been reported, ranging from 7 to 13 repeats (Figure 1). The [TTTA]7, [TTTA]10 and [TTTA]12 alleles have previously been implicated as possible breast cancer susceptibility alleles (4,6,8). Siegelmann-Danieli and Buetow (6) observed a higher [TTTA]7 allele frequency among 348 breast cancers compared with 148 controls (18.5 versus 13.4%). In particular, homozygotes for the [TTTA]7 allele were 5.4 times more likely to be in the case group than in the controls. However, this association was not observed in a subsequent study (7). Siegelmann-Danieli and Buetow (6) also observed a lower frequency of the [TTTA]12 allele among the case groups compared with the controls (1.6 versus 5.3%), suggesting that this may represent a low risk allele. This was not verified in another study (8) and indeed Kristensen et al. (4) observed a significant excess of the [TTTA]12 allele among a group of 367 sporadic and familial breast cancers from Scandinavia (P = 0.029). The most convincing association with breast cancer is with the [TTTA]10 allele. In a meta analysis of four studies of CYP19 [TTTA]n repeat alleles in breast cancer Healy et al. (7) only found a significant positive association with breast cancer with the [TTTA]10 allele (OR 2.31, 95% CI 1.35–4.10). Haiman et al. (8) observed a significant increase in the frequency of the [TTTA]10 allele among 462 breast cancer cases compared with 618 controls (OR 2.87, 95% CI 1.2–6.87). Nevertheless, not all studies have observed an excess of the [TTTA]10 allele in breast cancers. Probst-Hensch et al. (5) found that the [TTTA]10 allele frequency varied considerably among African-American, Japanese, Latino and non-Latino white breast cancer cases (0–3%) and in all these groups the [TTTA]10 allele frequency was higher among the controls than the cases.

In this study we sought to obtain independent verification of associations between the CYP19 intron 4 [TTTA]n repeat polymorphism and breast cancer risk among breast cancer cases from a British population. We genotyped lymphocyte DNA from 327 breast cancer patients as well as 253 non-cancer controls, all resident in and around Southampton, UK. The breast cancers were selected on the basis of an age at onset under 40 years, a family history of breast cancer (defined as two or more cases of breast cancer in a first or second degree female relative) or bilateral breast cancer irrespective of family history or age at onset. The age range of the breast cancer cases was 19–79 with a mean age of 38. The controls were all white female volunteers who were either staff at the Princess Anne Hospital or patients attending for non-neoplastic disease conditions. The age of the controls ranged from 18 to 84 with a mean age of 39 years. The majority of these cases and controls have been the subject of other allele association studies (9,10).

The [TTTA]n repeat was amplified using the primers and conditions described by Polymeropoulos et al. (11). The forward primer was labeled with a fluorescent dye. Bands were detected using a scanning laser fluorescence imager (Bio-Rad Molecular Imager FX) after electrophoresis in 6% denaturing polyacrylamide gels. We detected seven [TTTA]n tetranucleotide repeat alleles of CYP19, as shown in Figure 1. Representative alleles were sequenced to confirm their repeat number status (not shown). The smallest allele is not part of the [TTTA]n polymorphism and is due to a 3 base TCT insertion/deletion 50 bp upstream of the [TTTA]n tract (7). The insertion/deletion is in strong linkage disequilibrium with the [TTTA]7 allele and we did not observe it outside this context. The frequencies of these alleles among the breast cancer cases and controls are given in Table I. The [TTTA]7 allele frequencies are shown as separate [TTTA]7 –TCT and [TTTA]7 +TCT allele frequencies. The Hardy–Weinberg equilibrium assumption was assessed by comparing the observed numbers of individuals with different genotypes with those expected under Hardy–Weinberg equilibrium for the estimated allele frequency. We did not observe a departure from Hardy–Weinberg equilibrium in the breast cancer patients or the control groups.

We found no evidence of an association of the [TTTA]7, [TTTA]9, [TTTA]11, [TTTA]12 or [TTTA]13 alleles with breast cancer. Siegelmann-Danieli and Buetow (6) recorded an over-representation of the [TTTA]7 +TCT allele in breast cancer cases (OR = 1.47; 95% CI = 0.99 – 2.17). Strikingly, 13 of the 14 homozygotes detected in their study were from the breast cancer case group. In our breast cancer cases the frequency of the [TTTA]7 +TCT allele was not significantly different from the control frequency. Specifically, there were 6/253 [TTTA]7 +TCT homozygotes in the controls and 3/327 [TTTA]7 +TCT homozygotes in breast cancer cases. Our findings agree with those of Haiman et al. (8) in showing no association between breast cancer and the [TTTA]7 +TCT allele.

Haiman et al. (8) and Kristensen et al. (4) both reported a higher frequency of the [TTTA]12 allele in breast cancer cases than controls (3.1 versus 2.1%, P = 0.021 and 3.6 versus 1.6%, P = 0.029, respectively). Conversely, Siegelmann-Danieli and Buetow (6) have reported controls with the [TTTA]12 allele at five times the frequency of cases (5.63 versus 1.12%), suggesting that it may act as a potential low risk allele. Our studies have not shown any significant difference in the [TTTA]12 allele among the breast cancers compared with the controls (2.2 versus 2.3%, P = 1.00).

The [TTTA]8 allele was not associated with breast cancer in any of the previous studies. However, we observed a significant elevation of the [TTTA]8 allele frequency among the breast cancer cases compared with the controls (13.5 versus 8.7%, P = 0.012). The number of homozygotes for the [TTTA]8 allele was also higher among the breast cancers than controls (2.1 versus 1.6%), but this was not statistically significant (P = 0.763).

The only allele that has been associated with an increased risk of breast cancer in the majority of previous studies is the rare [TTTA]10 allele. In our study we also observed an increased frequency of the [TTTA]10 allele among breast cancer cases versus the controls (1.5 versus 0.2%, P = 0.028, OR 7.965, 95% CI 1.016–62.426). There were no homozygotes for the [TTTA]10 allele among the controls or breast cancer cases. Haiman et al. (8) observed a significant correlation between the [TTTA]10 allele and breast cancer, with 2.3% of 462 cases and 0.65% of 618 controls carrying the allele (P = 0.005). They also observed a higher frequency of the [TTTA]10 allele in more advanced cases. Healey et al. (7) reported an increased [TTTA]10 allele frequency in their UK based study (1.3% in cases versus 0.8% in controls) and although the increase was not statistically significant, their meta analysis of four previously published studies did reveal a significant positive association with breast cancer (OR 2.31, 95% CI 1.35–4.10). Not all studies have observed a positive association of the [TTTA]10 allele with breast cancer. Probst-Hensch et al. (5) reported a multi-racial study of the CYP19 [TTTA]n polymorphism within small cohorts of African-Americans, Japanese, Latino and non-Latino whites from Los Angeles and Hawaii, USA. Only three from a total of 191 cases (1.6%) were carriers of a [TTTA]10 allele (one Latino and two non-Latino whites) compared with 11 out of a total of 619 (1.8%) of the controls.

There are a number of possible causes for the lack of consistent findings between the different studies. Firstly, the selection criteria for the cases differed between studies, with some including unselected cases or cases under 55 years of age or cases with advanced stage tumors. Although our cases were selected on the basis of early age of onset or strong family history, the association with the [TTTA]10 allele has also been observed in unselected cases (8). It is also possible that differences in the statistical methods applied in each study may have led to spurious associations. In our study we performed multiple comparisons as if each of the seven alleles were independent, since there is no evidence that this polymorphism has any functional significance of itself and therefore there is no reason to believe that the number of repeats is related directly to breast cancer risk. Indeed, when we applied linear correlation (Pearson) to the average number of repeats, the smallest repeat or the largest repeat in each case, there was no statistically significant difference between the cases and controls (P = 0.23, P = 0.55 and P = 0.72, respectively).

As mentioned above, there is no definitive data concerning the functional significance of the [TTTA]n polymorphism on aromatase activity. Two studies have examined associations between CYP19 repeat genotypes and plasma hormone levels, but no significant associations were observed in either study (5,8). Because of the rarity of the [TTTA]10 allele neither study was able to examine a sufficient number of cases with this genotype to determine if there was any association with aromatase activity. While it is possible that the [TTTA]10 allele itself may be associated with an increased risk of breast cancer, it is more likely to be in linkage disequilibrium with another functional allele in the CYP19 gene. Interestingly, in a Norwegian population the [TTTA]12 allele has been shown to be in strong linkage disequilibrium with a putative high activity C→T substitution in exon 10 of CYP19 (12). Linkage disequilibrium of the [TTTA]n alleles to this and other as yet unidentified functional polymorphisms could explain the lack of consensus with respect to disease association of particular [TTTA]n alleles reported between the different studies.

Table I.

CYP19 intron 4 [TTTA]n repeat allele frequencies in breast cancer cases and controls

aFisher's exact test. 
Repeat number Product size (bp) Number of alleles (%) P valuea OR (95% CI) 
  Controls Cases   
[TTTA]7 – TCT 168 181 (35.8) 209 (32.0) 0.188 0.843 (0.660–1.078) 
[TTTA]7 + TCT 171  68 (13.4)  84 (12.8) 0 793 0.949 (0.674–1.338) 
[TTTA]8 175  44 (8.7)  88 (13.5) 0.012 1.633 (1.114–2.393) 
[TTTA]9 179  4 (0.8)  7 (1.1) 0.765 1.358 (0.395–4.665) 
[TTTA]10 183  1 (0.2)  10 (1.5) 0.028 7.965 (1.016–62.426) 
[TTTA]11 187 192 (37.9) 237 (36.2) 0.581 0.930 (0.731–1.182) 
[TTTA]12 191  11 (2.2)  15 (2.3) 1.000 1.056 (0.481–2.321) 
[TTTA]13 195  5 (1.0)  4 (0.6) 0.515 0.617 (0.165–2.309) 
aFisher's exact test. 
Repeat number Product size (bp) Number of alleles (%) P valuea OR (95% CI) 
  Controls Cases   
[TTTA]7 – TCT 168 181 (35.8) 209 (32.0) 0.188 0.843 (0.660–1.078) 
[TTTA]7 + TCT 171  68 (13.4)  84 (12.8) 0 793 0.949 (0.674–1.338) 
[TTTA]8 175  44 (8.7)  88 (13.5) 0.012 1.633 (1.114–2.393) 
[TTTA]9 179  4 (0.8)  7 (1.1) 0.765 1.358 (0.395–4.665) 
[TTTA]10 183  1 (0.2)  10 (1.5) 0.028 7.965 (1.016–62.426) 
[TTTA]11 187 192 (37.9) 237 (36.2) 0.581 0.930 (0.731–1.182) 
[TTTA]12 191  11 (2.2)  15 (2.3) 1.000 1.056 (0.481–2.321) 
[TTTA]13 195  5 (1.0)  4 (0.6) 0.515 0.617 (0.165–2.309) 
Fig. 1.

Relative mobilities of CYP19 [TTTA]n alleles in a 6% denaturing polyacrylamide gel. The [TTTA]n repeat number and product size are shown on the right of the figure. DNA was prepared from blood lymphocytes as described previously (13). The [TTTA]n repeat was amplified using the following primers: forward, 5′-gcaggtacttagttagctac-3′; reverse, 5′-ttacagtgagccaaggtcgt-3′ (11). For detection the forward primer was labeled with a 5′-fluorescent dye (FITC). PCR was carried out in a reaction volume of 10 μl containing 10–200 ng genomic DNA, primers (25 nM each), 1× reaction buffer (Applied Biotechnologies, UK), 200 nM dATP, dTTP, dGTP and dCTP (Promega, UK) and 0.2 U Taq DNA polymerase (Red Hot Taq; Applied Biotechnologies, UK). PCR consisted of an initial denaturation at 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 60 s followed by one cycle of 72°C for 10 min. PCR products were denatured by addition of 20 μl of 95% formamide and heating at 94°C for 5 min. The alleles were then separated by electrophoresis through 6% denaturing polyacrylamide gels. The fluorescently labeled alleles were detected using a Bio-Rad Molecular Imager FX scanning laser fluorescence imager with a 488 nm laser.

Fig. 1.

Relative mobilities of CYP19 [TTTA]n alleles in a 6% denaturing polyacrylamide gel. The [TTTA]n repeat number and product size are shown on the right of the figure. DNA was prepared from blood lymphocytes as described previously (13). The [TTTA]n repeat was amplified using the following primers: forward, 5′-gcaggtacttagttagctac-3′; reverse, 5′-ttacagtgagccaaggtcgt-3′ (11). For detection the forward primer was labeled with a 5′-fluorescent dye (FITC). PCR was carried out in a reaction volume of 10 μl containing 10–200 ng genomic DNA, primers (25 nM each), 1× reaction buffer (Applied Biotechnologies, UK), 200 nM dATP, dTTP, dGTP and dCTP (Promega, UK) and 0.2 U Taq DNA polymerase (Red Hot Taq; Applied Biotechnologies, UK). PCR consisted of an initial denaturation at 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 60 s followed by one cycle of 72°C for 10 min. PCR products were denatured by addition of 20 μl of 95% formamide and heating at 94°C for 5 min. The alleles were then separated by electrophoresis through 6% denaturing polyacrylamide gels. The fluorescently labeled alleles were detected using a Bio-Rad Molecular Imager FX scanning laser fluorescence imager with a 488 nm laser.

2
To whom correspondence should be addressed Email: i.campbell@pmci.unimelb.edu.au

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