Introduction

Breast cancer (BrCa) in western countries affects about one in ten women, with only 5–10% of cases presenting a strong inherited component. Germline mutations in the high penetrance BRCA1 and BRCA2 (BRCA1/2) genes, responsible for the Hereditary Breast and Ovarian Cancer syndrome (HBOC), account for 20–30% of these cases [1]. In families with BRCA1/2 mutations, carriers are known to be at higher risk for BrCa, which is observed at an earlier age compared with sporadic cases. However, BrCa is also observed among non-carrier relatives, with a frequency of about 10–20% in retrospective and up to 6% in prospective studies [2–6].

These women are considered phenocopies and raise difficult counselling issues concerning the first selection of the index case, in particular for those affected with premenopausal (<50 years) BrCa, and the estimate of the potential residual cancer risks in negative members of families with a strong BrCa history.

To date only a few studies have investigated the presence of potential genetic susceptibility factors underlying the occurrence of phenocopies in BRCA1/2 families, mainly focussing on BrCa-associated single-nucleotide polymorphisms (SNPs) [2, 5].

We otherwise hypothesized that, as for other Mendelian diseases, a spontaneous correction of a pathogenic mutation, leading to somatic mosaicism, might occur also in BRCA pedigrees, thus determining a greater BrCa risk compared with truly negative cases. This phenomenon, named revertant somatic mosaicism, was observed in patients affected with diverse haematological (including Fanconi anaemia) and non-haematological genetic diseases, mainly affecting the skin, and recent studies suggest that it is not a rare event [7–11]. It is characterised by a complete or partial recovery of somatic cells to the wild type genotype through different mechanisms, which include intragenic crossover, gene conversion, back mutation or second-site mutation events [11]. As consequence, the revertant somatic mosaicism leads to a somatic mosaicism occurring in multiple cell lineages. Evidences of revertant mosaicism in HBOC have not been reported to date and genetic modifiers, which might modulate the penetrance in high risk families, are regarded as the most likely predisposing factors for the development of breast cancer in a fraction of non-carriers. Nevertheless, mosaicism in non-carrier relatives has never been specifically investigated and a putative tissue-specific contribution of the familial mutation to the development of cancer has not been ruled out in these patients.

It is, in fact, conceivable that the somatic mosaicism could be undetected. The BRCA1/2 genetic testing is indeed routinely performed only on DNA from one tissue type, mainly peripheral blood leukocytes (PBLs), and in non-carriers BrCa-affected the family mutation could not be identified due to corrective events occurring in blood cells. In addition, the Sanger sequencing approach could not be enough sensitive to detect low-level mosaicisms.

Based on these considerations, the somatic revertant mosaicism could explain the absence of BRCA1/2 mutations in the BrCa affected, belonging to positive BRCA 1/2 families. The presence in an individual of the mutation only in a minor subset of cells, not detectable with standard sequencing approach (i.e.: Sanger sequencing), could still represent a cause underlying the disease in such cases. Moreover, even if at low-level, a putative somatic mosaicism would raise important concerns, especially in relation to the residual risk of developing breast and ovarian cancer as well as of transmitting the mutation to the offspring.

In the present study, we addressed for the first time this issue by evaluating the presence of the family mutation, with a mosaicism-sensitive approach, in 16 patients considered phenocopies based on both clinical and genetic assessment.

Materials and methods

Patients and samples

We included in this study 16 non-carrier women belonging to 15 families with heterozygous BRCA1/2 mutations (10 BRCA1 and 5 BRCA2) who developed BrCa before the age of 50 (mean age at diagnosis of first BrCa 43,3 years; median 44,5 years). The only exception is patient 4-e, who was included because affected at the age of 58 with two synchronous tumours belonging to the clinical spectrum of BRCA mutations (BrCa and pancreatic cancer). In addition to patient 4-e, three other phenocopies developed more than one type of tumour: patient 1-b developed a yolk sac tumour at age 20 and BrCa at age 45, patient 14-o developed a non-Hodgkin Lymphoma at age 18, a cervical cancer at age 33 and BrCa at age 43, patient 15-p developed BrCa at age 48 and a melanoma at the age of 50. The pedigrees are shown in S1 Fig.

We evaluated medical history of phenocopies and relatives up to the 4th degree. All the families included in the study resulted eligible for the BRCA1/2 mutation screening, according to the criteria in use at our institution [12].

All the patients were classified as phenocopies type A, consistent with the definition of Smith and colleagues [2], with the exception of patient 2-c, for whom information about the paternal side of the family was lacking. Moreover, all the phenocopies were first-degree relatives of a proven mutation carrier (type A1 phenocopies), with the exception of patient 7-h, 13-n and 15-p, who were second-degree relatives (type A2 phenocopies).

A significant breast or ovarian cancer history could be identified in either the paternal or the maternal side of the family, with the exception of family 8. In fact, both the phenocopy 8-i and her sister, who was found to carry a BRCA2 mutation, developed premenopausal BrCa and reported no other BrCa or ovarian cancers cases in both sides of the family. In order to investigate a possible de novo origin of the identified mutation, blood samples were collected also from both healthy parents.

For the diagnostic purposes, the investigation of the family mutation was performed by direct sequencing on blood DNA in all patients. In addition, in four of them, who developed BrCa before the age of 40 (2-c, 7-h and 13-n) or bilateral BrCa (10-k), the sequence analysis was extended to the whole BRCA1/2 coding sequence followed by Multiplex Ligation-dependent Probe Amplification (MLPA). The analyses did not reveal any pathogenic variant in the BRCA genes. Breast tumour features, family mutations and type of the analysed biological sample are shown in Table 1.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Characteristics of the BRCA1/2 phenocopies analysed in the study.

https://doi.org/10.1371/journal.pone.0171663.t001

Results

The MALDI TOF-MS analysis (~5% mutation detection threshold) was performed on the 16 phenocopies.

In 7 cases (from families 1–6) we were able to retrieve for the analysis breast tumour samples, in which a putative mosaicism for a BRCA1/2 mutation should be detected at a higher frequency compared with other tissues. In patients 7-h and 8-i the analysis was performed on PBLs, buccal mucosa and urinary tract cells. The other 7 patients (from families 9–15), for whom additional tissues were not available, MALDI TOF-MS was carried out on PBLs DNA only. In all the investigated phenocopies, the respective family mutations were not detected in any of the analysed tissues.

Fig 1 shows the mass spectra of families 1–8, in which the analysis was performed on tumour samples or other ecto/endodermal tissues of the affected phenocopies, and on PBLs DNA of heterozygous BRCA1/2 mutation carriers from each family. Mass spectra of the other seven patients from families 9–15, for whom the analysis was carried out on PBLs DNA only, are shown in S2 Fig.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Mass spectra of phenocopies (white arrows) and mutation carriers (black arrows) from families 1–8.

Mutant (mut) and wild type (wt) alleles are discriminated by the difference in size. Mass spectra of patients a-g (Families 1–7) refer to DNA extracted from breast tumour; for patients h-i (Families 7–8) the results shown refer to the analysis on DNA extracted from urine samples. The analysis of the father (♂) and mother (♀) of patient 8-i was carried out on blood DNA.

https://doi.org/10.1371/journal.pone.0171663.g001

Additional studies were performed in family 8, due to the presence of healthy parents and, with the exception of the proband and her sister, no other breast and ovarian cancer cases in both sides of the family (Fig 2). The MALDI-TOF-MS analysis of the phenocopy (8-i) excluded the presence of the family mutation in PBLs, buccal mucosa and urinary tract cells. The same analysis, extended to PBLs DNA of the parents, showed that the mutation was not present in either of the two.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Pedigree of family 8.

The white arrow indicates the phenocopy 8-i; the black arrowhead indicates the family proband (i.e. the first tested family member), carrier of the de novo BRCA2 mutation c.771_775delTCAAA (p.Asn257Lysfs*17). Genetic analysis results (MUT, mutation carrier; WT, wild-type) are reported beneath tested individuals; neoplastic diseases and age at diagnosis in years (y) are reported beneath affected individuals. BrCa, breast cancer; CNS, central nervous system;?, uncertain data.

https://doi.org/10.1371/journal.pone.0171663.g002

In order to investigate a non-paternity, we performed a STRs segregation analysis of 17 loci in both affected sisters and their parents. The analysis confirmed the presence of the paternal alleles in both the proband and the phenocopy, thus excluding a non-paternity (data available upon request).

In order to further investigate the presence of somatic mosaicism in patient 8-i using an additional more sensitive technique, we performed a quantitative genotyping assay by real-time PCR (~1% mutation detection threshold) of the BRCA2 c.771_775delTCAAA mutation, which was not detected in PBLs and buccal mucosa cells of the phenocopy. A putative somatic mosaicism in the patient’s parents was also investigated through the real-time PCR approach, and neither parent resulted to carry the mutation even at a low frequency. These results, on the whole, support the hypothesis of a de novo occurrence of the mutation identified in the family proband.

Discussion

Whether or not individuals testing negative, from BRCA families, are at increased risk for BrCa is still debated. A first retrospective study [2] of selected high-risk families documented a five-time higher risk in non-carriers, compared with the general population, and reported a two-fold risk even in a prospective data subset. Several subsequent studies, though, did not confirm the presence of a significantly increased risk [3, 6, 14–17]. More recently, it has been tested if some non-carrier relatives from high-risk families might display a higher risk due to the effect of genetic modifiers, such as SNPs associated with an increased risk for BrCa either in the general population or in BRCA mutation carriers. However, this association was recorded only in BRCA2 families [5].

In this study, we addressed for the first time whether the family mutation, in a mosaic state undetected by conventional screening methods, could underlie the development of BrCa in relatives testing negative. This could be explained by revertant somatic mosaicism, a phenomenon described at high frequency in hematological conditions, in particular primary immunodeficiencies and Fanconi’s anemia, and also observed in non-hematological disorders, such as epidermolysis bullosa, Duchenne muscular dystrophy and tyrosinemia [11]. Notably, revertant mosaicism predominantly involves tissues with high cell proliferation rates, as the hematopoietic system.

Following this hypothesis, in order to increase the chances of identifying the family mutation, we selected BRCA1/2 families with non-carriers who were unexpectedly affected at an early age, most of them classified as type A phenocopies according to Smith et al [2]. By using techniques sensitive to low-level mosaicisms, we performed a mutation screening in blood and other available tissues. Of note, in six cases the analysis was carried out even on tumour samples, which ought to harbour the mutation if it were the primary causal factor. Our results confirmed the absence of the family mutation in all the analysed phenocopies, thus suggesting that a mutation reversion event is not expected to occur in such cases.

Interestingly, we observed that among phenocopies only three patients developed a high-grade BrCa, whereas most displayed low-intermediate hormone receptor positive BrCa (Table 1). This datum is noteworthy, since low-intermediate grade tumours are not frequently found in BRCA1/2 families and are considered to be negative predictors of the BRCA mutation status [18]. In line with our results, this observation further points out that different pathomechanisms might underlie these cases. Moreover, in our cohort, four phenocopies also developed other tumours, which are not usually associated with the BRCA1/2 phenotype. The presence of multiple primary tumours might be due to chance, or to the preceding use of genotoxic therapies, or might further indicate that a different type of susceptibility affect these individuals. The presence of a putative somatic mosaicism, in fact, is not expected to result in a phenotype more severe than usually observed in carriers of constitutional mutations. Notwithstanding, the inclusion of such cases in our study should not undermine our conclusions, as we avoided a selection biased towards specific phenotypes by including all the type A phenocopies in our series who developed BrCa below 50 years of age (with the exception of patient 4-e). Since BrCa is a very common disease in the general population, with most cases being sporadic, the age threshold was set in order to reduce the chances of including potential sporadic cases. This strategy was conceived based on the ratio of the age-specific BrCa rates in BRCA1/2 carriers compared with the general population, which is estimated 20:1 in the age range 40–49 years, but drops down to 6:1 between 50–59 years and further decreases to 4:1 in the range 60–80 years [5]. Nevertheless, we can’t exclude that the selected phenocopies are actually a chance finding of naturally occurring sporadic cases in these families.

Although the BrCa risk for non-carrier relatives is not completely defined, the high phenocopy rate reported by many authors raises highly relevant issues. Primarily, phenocopies could be chosen as index cases and offered genetic testing before other affected relatives, who are mutation carriers. Nielsen and colleagues noted that in 3% of all families in their cohort, a phenocopy was tested as the first person in the family [6]. In addition, a study from the German Consortium for HBOC reported that among families tested negative at the first analysis, in whom another relative was offered BRCA1/2 testing, about 5% (8/169) of second tests identified a mutation [4]. The incorrect choice of the first individual to be tested, inevitably leads to overlook pathogenic mutations, underestimate the disease penetrance in such families, and erroneously estimate the breast and ovarian cancer risks in all the relatives. To overcome this eventuality, we usually offer testing to a second family member if, without taking into account the first tested patient, the family still fulfils our selection criteria for the BRCA1/2 analysis [12]. Notably though, if the patient from our family 8, who was the youngest affected individual, had been available for testing at the time of the first analysis, following our guidelines we would have missed the de novo BRCA2 mutation identified in her sister.

Moreover, even if a BRCA1/2 mutation is already identified within a family, the presence of early onset BrCa-affected non-carriers hampers accurate risk estimates for both mutation carriers and other negative family members. These considerations, on the whole, emphasize the importance of identifying the causal factors underlying BRCA phenocopies.

The de novo occurrence of the mutation identified in family 8 adds further complexity to both the risk assessment and genetic counseling, in particular when the family history is not informative. Reports of de novo mutations affecting the BRCA genes are extremely rare, to date only 11 cases have been described in the literature (five in BRCA1 and six in BRCA2) [19–29]. Previous studies suggested that this event might be especially expected in early onset BrCa patients without family history of the disease. Therefore, due to the presence of premenopausal BrCa in both the proband and her sister, this finding was particularly surprising in our family 8.

Some authors suggested that the overall frequency of de novo events is estimated to be low, also due to a possible fertility advantage provided by BRCA mutations that survive selection pressure [30]. However, as usually probands’ parents are not routinely tested, it is important to underline that an undefined number of de novo mutations might have been overlooked. The correct identification of these cases would have major implications for the offer of genetic testing to the relatives of mutation carriers.

Finally, a recent study documented for the first time, through a next generation sequencing approach, a low-level constitutional mosaicism of a de novo BRCA1 mutation, in a woman who developed a triple-negative BrCa at the age of 43 [28]. This remarkable finding not only demonstrated that mosaics for BRCA1/2 mutations can occur, but it also highlighted that, in a subset of cases, a pathogenic mutation could remain undetected by a standard testing approach.

Conclusions

This is the first study to investigate the presence of mosaicism for the family mutation in BRCA1/2 phenocopies. Our results suggest that revertant somatic mosaicism is not likely to be a common phenomenon in BRCA1/2 families.

As our families were selected for testing based on high-risk selection criteria, further studies are needed in order to discover other genetic factors potentially involved in the pathogenesis of most of these cases. The identification of causal factors in BRCA1/2 phenocopies will have a significant impact on risk assessment and genetic counselling for both BRCA1/2 mutation carriers and their relatives. Furthermore, the identification of a de novo BRCA2 mutation in our cohort highlights that, although rare, this event should be taken into account in the evaluation of high-risk families.

Supporting Information

Acknowledgments

The study was financed by funds from Italian citizens who allocated the 5 x 1000 share of their tax payment in support of the Fondazione IRCCS Istituto Nazionale dei Tumori, according to Italian laws (INT-Institutional strategic projects “5 x 1000”) and to the initiative “Prove Aperte” (Filarmonica della Scala of Milan in collaboration with Unicredit Foundation) to SM. We thank Dr. Daniela Zaffaroni for her support as data manager in the study.