Hereditary breast and ovarian cancer (HBOC) is an autosomal dominant syndrome with incomplete penetrance. The two most commonly mutated genes in HBOC are BRCA1 and BRCA2, which are essential components of the double-strand break repair system (1). Almost 3,500 cancer-associated mutations, scattered throughout the two genes, have so far been reported in the Breast Cancer Information Core (BIC) database (http://research.nhgri.nih.gov/bic/). The present study reports a new germline nucleotide 3020insCT/c.2901insCT mutation detected in the BRCA1 gene. In general, germline mutations in known breast cancer risk genes account for ~20% of breast cancers associated with a family history. It is therefore crucial to identify these individuals to offer appropriate cancer management and understand the contribution of BRCA1 and BRCA2 mutation-associated risks.

A 55-year-old non-Ashkenazi Spanish female diagnosed with breast cancer (at 51 years old) and ovarian cancer (at 55 years old) and treated at the University Clinic of Navarra (CUN; Pamplona, Navarra, Spain), was transferred to the genetic counseling unit. The clinical history of the patient lead us to consider the possibility of HBOC syndrome. Following verbal and written informed consent, genomic DNA was extracted from a peripheral blood sample and the BRCA1 and BRCA2 genes were sequenced on an automated analyzer (ABI PRISM^® 3130XL; Applied Biosystems, Foster City, CA, USA). The results were compared to the consensus wild-type sequences (Genbank NM_007294.2 for BRCA1 and Genbank NM_000059.1 for BRCA2). A 3020insCT/c.2901insCT frameshift mutation was identified in exon 11 of BRCA1 (Fig. 1). The insertion was confirmed by repeated analyses including reverse-primer sequencing. A BRCA1-Multiplex Ligation-dependent Probe Amplification (MLPA) analysis was performed, in order to investigate whether the mutation was able to lead to an exon rearrangement. The results indicated that none of the BRCA1 alleles showed deletion and/or duplication (results not shown).

Genetic analysis was recommended to the only other individual at risk in patient’s family, namely the twin sister, and the analysis showed that she did not carry the mutation.

A 3020insCT/c.2901insCT frameshift mutation in exon 11 of the BRCA1, which has yet to be reported in the BIC database, was detected in a 55-year-old non-Ashkenazi Spanish female diagnosed with breast and ovarian cancer. Since other family members were not available for genetic analysis, the segregation of the mutation could not be established. From the literature available, it may be deduced that the mutation leads to the deletion of the coiled-coil domain and BRCA1 C terminus (BRCT) domains of the BRCA1 protein. The coiled-coil region is critical for transcriptional activation through its interaction with the basic leucine zipper (bZIP) domain of the JunB protein. In vitro and in vivo experiments suggest that this BRCA1-JunB interaction is particularly important for the suppression of ovarian cancer (2). The lack of the coiled-coil domain in the present patient may have been closely correlated with the development of the ovarian cancer. However, BRCA1 has a pivotal function within the BRCA1-associated genome surveillance complex through the coordination of the actions of damage-sensing and executive repair proteins. Solyom et al(3) showed that the Abraxas protein serves as a central organizer of a large BRCA1 holoenzyme complex. Abraxas directly binds, via its phosphorylated C terminus, to the BRCA1 BRCT motifs, linking BRCA1 to a core protein complex dedicated to ubiquitin chain recognition and hydrolysis at DNA double-strand breaks (3,4). Moreover, BRCT domains in BRCA1 are able to bind DNA strand breaks and ends in vitro, which is enhanced by the formation of the BRCA1-BARD1 heterodimer (5). The structural studies of Kobayashi et al showed that the BRCT domain partially inserts into the major groove and makes extensive contacts with the DNA backbone (6), suggesting the possibility that proteins with BRCT domains may act as DNA sensors and transducers of DNA damage response signaling. The mutation identified in the present study would markedly compromise these functions, with profound biological consequences. The premature stop codon at amino acid 1000 leads to a truncated protein that has 70% of its normal length. The advantage of having mutant BRCA1 human breast cancer cell lines is that the impact of pathogenic human mutations may be evaluated in the context of a human genetic background. A previous study of 41 human breast cancer cell lines identified a BRCA1 mutant cell line, SUM149PT, with a nucleotide deletion at position 2288 (7). The resulting truncated BRCA1 protein lacked the C-terminal BRCT and coiled-coil domains similar to the present patient. Nuclear BRCA1 protein expression was not detectable in the cell line, therefore corroborating the tumor suppressor function of BRCA1 and the pathogenicity of the mutation.

The next step was to search for bibliographic evidence of the present mutation in BRCA1-knockout animal models. The homozygous loss of BRCA1 generally leads to early embryonic lethality, although it is possible to extend the viability though the removal of p53 function. Among the range of models available, McCarthy et al designed truncated human BRCA1f22-24/p53^+/− mice (harboring the second BRCT domain), that develop estrogen receptor-negative (ER⁻) and progesterone receptor-negative (PR⁻) tumors lacking HER2 protein overexpression and gene amplification (8). This phenotype is similar to 64–90% of human BRCA1-mutation breast cancers, so called ‘triple negative’ breast cancers. The immunophenotypic features of the present patient’s tumor indicated a noticeably different pattern, being ER⁺, PR⁺ and ErbB2-negative. It has been reported that 10–36% of BRCA1 mutation-related invasive breast cancers are, in fact, ER⁺. Furthermore, BRCA1 mutation carriers who are older or post-menopausal at the time of the diagnosis of breast cancer are more likely to have an ER⁺ breast cancer (9,10). With regard to the origin of these ER⁺BRCA1-related breast cancers, Lim et al observed the expansion of a committed luminal progenitor population, containing ER⁺ and ER⁻ cells, in preneoplastic tissues of BRCA1 mutation carriers and proposed the luminal progenitor cells as the cell of origin for BRCA1-associated cancers (11). In mouse models with the deletion of BRCA1, the expression of ER in the resulting tumors appears to depend on whether BRCA1 is deleted at an earlier or later stage of cell differentiation (12–14). These studies suggest that BRCA1-deficient ER⁺ tumors may derive from BRCA1 loss in an ER⁺ luminal progenitor cell.

Another key point is the therapeutic approach for ER⁺BRCA1-associated breast cancers. Given the availability of effective therapies that exploit defects in homologous recombination, such as PARP-1 inhibitors and cisplatin, it is increasingly important to determine whether these therapies are likely to be effective in ER⁺BRCA1-mutant cancers. A recent study by Kaplan et al indicated that ER⁺BRCA1-related breast cancers are indistinguishable from ER⁻BRCA1-related cancers in their nuclear expression of PARP-1, suggesting that ER⁺BRCA1-related breast cancers may respond well to drugs that exploit BRCA1 deficiency (15). ER⁺BRCA1-related breast cancers appear to be a unique group and efforts should be made to identify the individuals for whom estrogen-modifying agents are likely to be particularly effective.