Biallelic Deleterious BRCA1 Mutations in a Woman with Early-Onset Ovarian Cancer

INTRODUCTION

Hereditary breast and ovarian cancer syndrome is predominantly caused by heterozygous, germline mutation in the BRCA1 or BRCA2 genes (1). Several forms of Fanconi Anemia, characterized by bone marrow failure and malignancy, can be a consequence of biallelic mutations in BRCA2 (2) or biallelic mutations in genes encoding BRCA2 and BRCA1 associated proteins PaLB2 and BRIP1 (3–7). Despite a frequency of approximately 1.5% in the Ashkenazi Jewish population for the BRCA1 mutations, 185delAG [Human Genome Variation Society (HGVS) c.68_69delAG] and 5382insC (HGVS c.5266dupC), no homozygous or compound heterozygote carriers of these mutations have been reported. Although Brca1 nullizygosity results in embryonic lethality in mice (8), genetically engineered mice harboring biallelic mutations that correspond to human cancer-associated missense mutations within the BRCA1 BRCT domain are viable through adulthood and display highly penetrant cancer susceptibility (9). These findings raise the possibility that a partially functional BRCA1 allele in trans with a deleterious truncating mutation (or with a similarly partially functioning mutation) in BRCA1 could be present within the same individual, and could contribute to familial cancer susceptibility in humans. Herein we document the presence of a functionally deleterious BRCA1 BRCT domain missense alteration in trans with a pathogenic BRCA1 alteration in a woman with dysmorphic features and early onset ovarian carcinoma.

RESULTS

The proband (Fig. 1A, ego 28, arrow) presented at age 28 with stage IV papillary serous ovarian carcinoma. Medical records revealed a history of microcephaly, short stature (adult height of 150 cm) and developmental delay with limited speech at age 4 years. Review of pictures provided by the family demonstrates coarse features with low anterior hairline, macrognathia, a prominent nasal bridge and small alae nasi. She did not have obvious abnormalities of her thumbs and had a normal complete blood count at the time of her cancer diagnosis. Neither ataxia nor telangiectasias were documented. This individual was found to have a known deleterious mutation in BRCA1 reported as 2576delC (HGVS c.2457delC; p.Asp821Ilefs*25) and a variant of unknown significance (VUS) in BRCA1 (HGVS c.5209T>C) p.Val1736Ala, as well as a VUS in BRCA2 (HGVS, c.971G>C; p.Arg324Thr). Treatment with carboplatin (target area under the concentration versus time curve in mg/mL•min (AUC) of 5) and paclitaxel (175 mg/m2) resulted in significant toxicity requiring hospitalization due to fever and grade 4 neutropenia (absolute neutrophil count (ANC) nadir of 160 per cubic millimeter), as well as grade 3 anemia (nadir hemoglobin 7.8g/dl) and grade 4 thrombocytopenia (nadir 3000 per cubic millimeter), for which she received red blood cell and platelet transfusions. She also developed grade ≥3 nausea, diarrhea and mucositis. As a result of the excess toxicity, carboplatin and paclitaxel were discontinued after two cycles. She received no further therapy and died six months following her diagnosis. Extreme sensitivity to the interstrand crosslinking agent carboplatin is not typically observed in heterozygous BRCA1 mutation carriers (10–12), but is seen in biallelic BRCA1 mutant cells and mice, suggesting that both BRCA1 alleles were compromised for DNA repair function.

The mother of ego 28 was diagnosed with ovarian cancer at age 53 and died at 55. A maternal great aunt (Fig. 1A, Family A, Ego 1) was diagnosed with breast and ovarian cancers at ages 59 and 69, respectively, and a contralateral breast cancer at age 76. A second maternal great-aunt (Ego 9, sister of Ego 1) was diagnosed with primary peritoneal cancer at age 67 and died at 68. Notably, both carried the BRCA1 p.Val1736Ala variant of uncertain significance (VUS) but not the known pathogenic mutation BRCA1 2576delC (HGVS c.2457delC). Additional genetic testing in the family revealed that the brother of the proband (ego 27) carries the BRCA1 c.2457delC mutation and the paternal lineage also had multiple cases of early onset breast cancer. To investigate this variant further, we were able to obtain pedigrees on 11 additional families with the BRCA1 p.Val1736Ala VUS. A total of 9 of these pedigrees which had additional genotyping of family members were used to assess co-segregation using methods described in Thompson et al (13) (a representative pedigree is shown in Fig. 1B and characteristics of the families are detailed in Supplementary table 1). The combined odds ratio in favor of p.Val1736Ala being pathogenic was 234:1 assuming the age-specific penetrance estimated in Antoniou et al. (14) Loss of heterozygosity (LOH) analysis was performed on genomic DNA extracted from BRCA1 p.Val1736Ala mutation positive tumors using a custom designed Taqman assay (Table 1 and Supplementary Fig. S1). Ovarian/primary peritoneal cancer tumor blocks from Family A egos 1 and 9 demonstrated LOH at the wild-type BRCA1 allele with retention of the p.Val1736Ala allele. Conversely, in ego 28, who carried germ-line BRCA1 c.2457delC and p.Val1736Ala alterations in trans, the ovarian tumor did not display LOH at either allele suggesting that p.Val1736Ala expression is not selected against in tumors.

The BRCA1 BRCT residue p.Val1736 is conserved across 18 different vertebrate species (Fig. 2A). In contrast with other BRCT residues that exhibit cancer-associated point mutations, structural models predict that p.Val1736 does not make direct contact with phospho-peptide ligands (Fig. 2B). Rather, Val1736 resides in a hydrophobic pocket, which may affect the stability of residues Pro1749 and Cys1697, both of which are required for BRCT function in DNA repair and tumor suppression. Transfection of a DNA double strand break (DSB) reporter cell line (Supplementary Fig. S2) (15) with an epitope tagged carboxy-terminal region of BRCA1 revealed that a wild-type (WT) BRCT fragment was observed at greater than 80% of DSBs, while the fragment containing p.Val1736Ala was reduced to below 40% (P = 0.0029), intermediate to the WT protein and known BRCT mutant p.Pro1749Arg, which was present at less than 20% of DSBs (P = 0.0017) (Fig. 2C, D). Similarly, co-immunoprecipitation experiments with the same epitope tagged BRCA1 fragments demonstrated significantly diminished interaction between p.Val1736Ala and RAP80, a BRCA1 BRCT interacting protein, in comparison to WT BRCT containing fragments (Fig. 2E). Consistent with these results, overexpression of the WT BRCT fragment acted as a dominant negative allele by reducing IR induced Rad51 foci formation and homology directed DSB repair by a significantly greater extent than overexpression of BRCA1 fragments containing either the BRCT mutations p.Pro1749Arg or p.Val1736Ala (Supplementary Fig. S3).

We are aware of only one previous report of biallelic deleterious mutations in BRCA1 in humans. In this report, a Scottish woman was found to be homozygous for BRCA12800delAA(HGVS c. 2681_2682delAA, p.Lys894Thrfs*8) (16). This individual was diagnosed with breast cancer at age 32, and subsequently developed a contralateral breast cancer. Homozygosity for this mutation was plausible particularly because it is a founder mutation in the studied population (17). Nevertheless, this report has long been questioned because potential primer bias in PCR-based genotyping could have led to preferential amplification of the putative mutant allele and hence masking of true heterozygosity (18). Because of the importance of this single report for the interpretation of our own results, we re-sequenced peripheral blood lymphocyte DNA from the reported biallelic carrier and found that only one BRCA1 allele harbored the designated mutation c. 2681_2682delAA, while the other allele was found to be wild-type at this position (Fig. 3A, B). Therefore, the purported homozygous carrier was in actuality heterozygous for a BRCA1 mutation.

DISCUSSION

Here we report the first individual with validated biallelic mutations in BRCA1. Compelling evidence is presented that BRCA1 p.Val1736Ala is both pathogenic and can support viability through adulthood in trans to a deleterious mutation in exon 11 of BRCA1 (BRCA1 2576delC). BRCA1 p.Val1736Ala diminishes protein-protein interaction with RAP80, localization to DSBs, and imparts cancer susceptibility independent of other BRCA1 or BRCA2 alterations. LOH analysis was also consistent with pathogenicity. Loss of the wild type allele occurred in both tumors that carried the p.Val1736Ala VUS in trans to wild type BRCA1, however LOH did not occur in the ovarian cancer of the proband (ego 28), which was compound heterozygous for p.Val1736Ala and 2576delC, indicative of a scenario in which selective pressure did not exist to delete either pathogenic allele.

Several features of the index patient were uncharacteristic for monoallelic BRCA1 mutation carriers. In addition to the aforementioned developmental delay, microcephaly and short stature, ovarian cancer was diagnosed below the age of 30, which is unusual for BRCA1 mutation carriers (19). She also had extreme sensitivity to the interstrand crosslinking agent carboplatin, a characteristic not typically displayed in heterozygous BRCA1 mutation carriers in vivo (10–12, 20).

While complete BRCA1 deficiency results in early embryonic lethality in mice, it should be considered that certain biallelic BRCA1 mutations that mimic human cancer associated mutations can support viability through adulthood in mice (9). Genetically engineered mice harboring biallelic BRCT mutations (p.Ser1598Phe) that corresponds to a known cancer-causing allele in humans (p.Ser1655Phe) were viable through adulthood and displayed similar cancer susceptibility to mice completely lacking BRCA1 gene function in the mammary gland (9). Moreover, complete deletion of exon 11 or introduction of a mutation that produces a stop codon in the BRCA1 exon 11 region, as predicted in the 2576delC allele, disrupts full length BRCA1 protein leaving intact an evolutionarily conserved exon 10 to exon 12 splice variant. The BRCA1 delta 11 splice product contains the BRCA1 RING domain and BRCT repeats, localizes to DNA damage sites, and can support viability in certain mouse backgrounds, yet still confers cancer susceptibility (21–23). The BRCA1 delta 11 splice isoform is expressed at both the RNA and protein levels in human cells (24), however, it is not known if this is the case in the context of the 2576delC mutation. It is therefore likely that partial DNA repair function of p.Val1736Ala (c.5209T>C), and possibly 2576delC (c.2457delC), or both of these mutant alleles is permissive for viability in humans.

Structural modeling suggests p.Val1736Ala is unique when compared to other BRCT missense mutations, in that it lies distal to the phosphopeptide-binding pocket. Prior studies have shown that p.Val1736Ala exhibits thermal instability and partial loss of function in transcriptional reporter assays (25, 26). Our results are also consistent that p.Val1736Ala is a hypomorphic alteration with respect to biochemical and cellular function. While BRCA1 p.Val1736Ala is predicted to be hypomorphic in terms of DNA repair function, it is not evident that it has reduced penetrance with respect to cancer susceptibility. Collectively, these findings together with observations from genetically engineered mouse models (9) are strongly suggestive that viability and tumor suppression phenotypes are not completely concordant among BRCA1 mutant alleles.

Apart from the biological implications, the findings in this study have importance to the interpretation of genetic variants. Variants of uncertain significance (VUS) are a common finding in genetic testing for inherited cancer syndromes and pose challenges in counseling and management (27). Co-occurrence of a VUS in trans with a known deleterious BRCA1 mutation is felt to be a strong indication that the VUS is not clinically important (28). Our findings suggest the presence of a BRCA1 VUS in trans with an established deleterious BRCA1 mutation should not be considered as definitive evidence against pathogenicity. This work also highlights the importance of examining multiple distinct lines of evidence when interpreting a VUS, including clinical phenotype. This lesson is particularly pertinent in the era of massively parallel DNA sequencing, as a large number of VUS will be identified using this methodology and caution will be needed in interpreting these for clinical use.

METHODS

Supplementary Material