Whilst studies have reported promising associations of cancer risk with 5' flanking region and promoter polymorphisms of several important genes such as estrogen receptor-alpha [24
] and MMP-2
], so far there has been no comprehensive investigation of BRCA1
promoter polymorphisms with breast cancer risk. Changes in transcriptional regulation of BRCA1
are likely to play an important role in the initiation or progression of sporadic breast cancer. Consistent decrease in BRCA1 expression has been observed in tumor samples, and epigenetic effects such as aberrant cytosine methylation [6
], histone hypoacetylation and chromatin condensation [26
] as possible mechanisms down-regulating BRCA1 expression have been demonstrated.
We report for the first time a comprehensive study of BRCA1
promoter polymorphisms and show that the common genetic variants c.-2804T>C, c.-2265C>T, c.-2004A>G and c.-1896(ACA)1
, could affect the binding affinity of nuclear protein and alter promoter activity. As supported by in-silico
prediction of putative transcription factor binding sites, the contribution of c.-2265C>T was most significantly demonstrated by EMSA assay which showed stronger binding for the T allele, whilst promoter activity assay showed that the most common mutant haplotype which contained the c.-2265T allele increased promoter activity by 70%. Genetic association study supported this finding demonstrating that carriers of the T allele were associated with a reduced risk of breast cancer. This association was stronger among older women, particularly those without a family history of breast cancer. Such a pattern of association is expected since germ-line mutations in high penetrant breast cancer susceptibility genes are less likely to be found in late-onset cases and cases without a family history of breast cancer [27
]. Moreover, this finding in Hong Kong Chinese was also replicated in an independently conducted population-based case-control study of Shanghai Chinese, which minimizes the possibility of type I error. Meta-analysis of both datasets by Mantel-Hanzel test showed no significant difference between reduced odds ratios between the two populations, allowing for combined odds ratio analysis, thus totaling 1484 cases and 1574 controls.
Previous studies had largely focused on association of BRCA1
coding SNPs. Dunning et al found homozygotes of Arg356 to be inversely associated with breast cancer risk [28
] which however could not be replicated by Cox et al [12
] or Freedman et al [13
]. Cox et al, identified one haplotype associated with a slight increased risk [12
] but the functional variant(s) responsible for this association remains unknown, and promoter SNPs were not included for analysis. Freedman et al's multiethnic cohort haplotype analysis found no significant association between the common variants of BRCA1
and breast cancer risk. Heterogeneity of BRCA1
haplotypes was noted among the ethnic groups tested, with the haplotypes of Japanese and Native Hawaiian populations showing relatively lower diversity and being different from those of African American, Latino, and Caucasian populations[13
]. Indeed one of Freedman et al's promoter SNPs, namely the c.-2613G>C variant was monoalleleic in the African-Americans, which we also found to be monoallelic in 90 healthy Hong Kong Chinese women (unpublished data). Two other SNPs (rs1546585 and rs2175957) included by Freedman are located much further upstream at the 5' of BRCA1
(18609bp and 8805bp respectively) and unlikely play an important role in promoter activity, and were hence not included in our study. As Thakur and Croce [29
] had demonstrated that substantial high level of promoter activity was maintained up to −1582bp, our study had included up to 1.6 kb of the promoter sequence for analysis.
It is noted that another gene, “NBR2”,
is present adjacent to BRCA1
in a head-to-head position [30
]. Indeed the 3'end of the BRCA1
promoter overlaps with 188 bp of the NBR2 promoter, with the remaining 5' portion of the BRCA1
promoter corresponding to complementary sequence of exon 1 to partial intron 1 of NBR2
. The c.-2265C>T SNP corresponds to 589 nucleotides downstream intron 1 of NBR2
and thus is less likely to give rise to a functional effect on NBR2 expression/translation.
In-silico TF-SEARCH had identifed OCT-1, GATA-1, GATA-2, GATA-3, USF and Elk-1 as potential transcription factors whose binding sites spanned over BRCA1 promoter polymorphisms and therefore could alter their binding affinity. Co-transfection of vectors expressing these putative factors with pGL3-basic/BRCA1-2265T or pGL3-basic/BRCA1-2265C was however unable to show significant change in the promoter activity assay (data not shown) and the identity of the transcriptional factors remains to be investigated.
Although it cannot entirely be ruled out that the observed functional effect could be attributed to other variants of BRCA1 not analyzed here, given our promoter assay results and the strong LD of the polymorphisms in this study, c.-2265C>T is a most justifiable tagging SNP.
The risk-reduction of 20–25% is consistent with that of low-penetrant gene effect. As this SNP (with minor allele T frequency over 35%) is relatively common in the Chinese population, its use as a predictive marker for reduced cancer risk is limited. However, as the most common mutant haplotype (which contains the c.-2265T allele) increased promoter activity by 70%, the functional consequences of such higher gene expression in BRCA1, a tumor-suppressor gene, may indeed confer an important protective role.
In summary, we are the first to show that the common genetic variants c.-2804T>C, c.-2265C>T, c.-2004A>G and c.-1896(ACA)1/(ACA)2, can affect the binding affinity of nuclear proteins and alter promoter activity, with the effect of c.-2265C>T being most significant. Our genetic association analysis of two independent Chinese cohorts totaling over 3,000 subjects support these findings by demonstrating that this BRCA1 promoter SNP was significantly associated with reduced breast cancer risk. Together with gene expression regulation by epigenetic mechanisms, promoter polymorphisms may indeed make an important contribution towards breast cancer development.