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Editor—About 3-10% of breast cancer cases have been estimated to result from a hereditary susceptibility to the disease.1 A significant proportion of these are the result of mutations in the BRCA1 gene.2 While several hundred BRCA1 gene mutations have been identified, the disease causing effect of some BRCA1 variants is a matter of current debate. In a recent issue of this journal, a 12 bp duplication in intron 20 of the BRCA1 gene has been proposed to be a regulatory mutation.3 This intronic variant had initially been described in a North American patient and was tentatively classified as a splicing mutation.2 4 It has also been detected in a population based sample of young breast cancer patients and was suggested to affect mRNA processing.5 In another study of Polish patients, a splicing effect was excluded but mRNA reduction has been suggested as a possible consequence of the variant.6 Robledoet al 3 provided experimental evidence to suggest that the intronic 12 bp duplication could act as a regulatory mutation resulting in the allele specific reduction of BRCA1 expression,3 although the underlying mechanism for such an effect remained unclear. Here we have investigated the expression of BRCA1 mRNA transcribed from the allele carrying the 12 bp duplication and provide evidence against a regulatory effect of this variant.
We have screened genomic PCR products from lymphocytes of breast cancer patients for mutations in exon 20 and flanking intronic sequences of the BRCA1 gene. In a series of 700 unselected breast cancer patients who had given their written informed consent to be tested, two German patients (0.3%) were identified as carriers of the 12 bp duplication in intron 20, starting at position +48 (fig 1). The presence of this variant was uncovered by the length difference of the respective PCR products on agarose gel electrophoresis and was confirmed by direct sequencing. One of the two patients was 69 years old at diagnosis and did not report any family history of breast cancer. The other patient was diagnosed by the age of 60 years and reported one grandmother with breast cancer. Her mother had eventually died from ovarian cancer by the age of 78 years but no material was available for confirmation. In order to investigate whether the 12 bp duplication could be a functionally significant BRCA1 mutation in our two patients, additional peripheral blood samples were obtained from these two index cases and total RNA was extracted from their lymphocytes. After reverse transcription with random hexamer primers, selected regions of the BRCA1 cDNA were amplified using primers located in different exons. When we amplified a cDNA region spanning BRCA1 exons 19-21, only product of wild type length was observed and no evidence for any aberrant splicing could be obtained in either case, which is in agreement with the results of others.3 6 We next investigated whether allele specific expression might be changed as a consequence of the intronic duplication. Because one of the two index patients (the one reporting a possible family history of breast cancer) was heterozygous for a neutral polymorphism, Ser1613Gly in exon 16 of the BRCA1 gene,7 we investigated whether both alleles were equally present in the cDNA from this patient. We thus amplified a cDNA region spanning exons 15 to 17 using the two PCR primers 5′-ACTACCCATCTCAAGAGGAGC-3′ and 5′-CTGGCAAACTTGTACACGAGC-3′ under standard PCR conditions. The RT-PCR products were subsequently digested with the restriction enzyme ScrFI, because one new recognition site for this enzyme is created by the presence of the Gly1613 allele. In two different cDNA preparations from the index patient whose DNA was heterozygous for the allele carrying the Gly1613 substitution and the 12 bp duplication, a clearly heterozygous pattern was also observed in the RT-PCR product (fig 2). Similar heterozygous patterns were obtained when using the enzymesAvaII or NlaIV to distinguish between both alleles (data not shown). These findings indicate that both BRCA1 alleles are equally expressed in the lymphocyte mRNA from this carrier of the 12 bp duplication. Any allele specific alteration in the BRCA1 mRNA levels in this sample would have to have been very subtle to remain undetected by our RT-PCR based assay, and therefore would be unlikely to result in a loss of BRCA1 function.
Thus, our observations are in agreement with the previously reported lack of aberrant splicing resulting from the intronic 12 bp duplication, but they differ from the results reported by Robledoet al 3 with respect to the relative expression of this variant allele. The reasons for these differences are unknown at present. It is possible, for example, that the presence of an undetected BRCA1 mutation could have resulted in the transcript instability reported in the previous study.3One caveat of both studies is that expression patterns in lymphocytes may not necessarily reflect expression patterns in other tissues such as breast epithelium. In addition, previous investigations have shown that BRCA1 mRNA expression in normal mammary epithelial cells in culture is highly sensitive to growth conditions and cell cycle status.8 Given these complexities of BRCA1 regulation, it is hard finally to rule out the possibility that the intronic 12 bp duplication could have a regulatory effect in any particular cell population under certain physiological circumstances. Although the intronic 12 bp duplication was seen only twice in our patient cohort, its relatively high incidence in some previously reported series indicates that a closer examination of this allele is important with respect to genetic diagnosis and counselling of breast cancer families. Because our present study, within its above mentioned limitations, failed to confirm the proposal that the presence of the intronic 12 bp insertion alone would be sufficient to have a real effect on BRCA1 expression in breast cancer, we suggest that this variant should not be regarded as a disease causing mutation unless definite proof can be obtained to show any impairment of BRCA1 level or function in carriers of the duplication.