Introduction

It is currently estimated that 5–10% of all breast cancers are hereditary and a proportion of them attributable to mutations in several highly penetrant susceptibility genes. Mutations in the tumor-suppressor genes BRCA1 (MIM#113705) and BRCA2 (MIM#600185) confer an increased risk of developing breast cancer and ovarian cancer [1, 2].

Family history is essential for identifying individuals at risk for mutations in the BRCA genes in the clinics. Hallmark characteristics include a personal or family history of early onset breast cancer, multiple family members diagnosed with breast or ovarian cancers, multiple generations affected, breast cancer bilaterality, the occurrence of male breast cancer, and ethnicity (descent from a population with high incidence of founder mutations, such as Ashkenazi Jewish or Icelandic ancestry).

A negative family history, however, does not exclude the presence of a germ line mutation in BRCA1 or BRCA2 genes. Indeed, it may reflect a small family size, predominance of males in the pedigree, incomplete penetrance, premature death of at risk women, or inadequacy of the cancer history information [3]. In patients without a positive family history, mutation testing of these genes may be considered when early onset, multifocality, bilaterality, or other features suggestive of a hereditary component are present. Furthermore, recent studies show that tumor phenotype, such as the so-called triple-negative (absence of estrogen and progesterone receptors and HER2-neu expression) breast cancer, is an indicator that may help to identify candidates for BRCA1 mutation testing, especially among young women without family history [4], who otherwise would not have been eligible.

Since the identification of BRCA1 and BRCA2, more than 3,600 distinct genetic variants (including deleterious mutations, polymorphisms, and unclassified variants) have been reported for these genes (BIC database) [5]. Despite the high number of mutations identified and the large number of studies carried out world wide, to our knowledge there is only one published report of a de novo mutation in BRCA1 [6] and three in BRCA2 [7–9].

During the course of the molecular analysis of BRCA1 and BRCA2 on hereditary breast/ovarian cancer families and women diagnosed with breast cancer at young ages, we identified a novel de novo BRCA2 mutation located in exon 2 in a Spanish patient with early onset bilateral breast cancer without a family history of breast/ovarian cancer. Furthermore, we ascertained the presence of the mutation in the paternal allele.

Personal and family history

The proband was a 38-year-old woman who had been diagnosed at the ages of 27 and 37 with bilateral infiltrating ductal carcinomas of the breast. The first tumor was estrogen and progesterone receptor negative, and the second one was estrogen and progesterone receptor positive, and HER2 negative.

No other breast or ovarian cancers were present in the pedigree (Fig. 1). Following genetic counseling, blood and tumor samples were collected from the proband for mutation screening.

Fig. 1
figure 1

Family pedigree: BC breast cancer; CR colorectal cancer. Between parenthesis: age at diagnosis. The BRCA2 −26G/A and wt/279inA genotypes are indicated

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Mutation identification

Genomic DNA was prepared from 10 ml EDTA blood samples according to standard procedures. Analysis of the entire coding and flanking BRCA1/2 sequences were carried out by direct sequencing (Abi Prism 3130 DNA Sequencer, Applied Biosystems).

Mutation screening of the BRCA2 gene in the proband indicated in exon 2 the presence of 1 bp duplication (c.51dupA, according to HGVH nomenclature) (c.279_280insA according to BIC nomenclature) (BRCA2 GenBank entry: NM_000059). The insertion results in a frame shift and a premature termination of translation at codon 30 in exon 3 of BRCA2. As far as we know this mutation has not been previously reported by the Breast Cancer Information Core or another database, and it has not been published before.

After identification of the mutation, the analysis was then carried out on DNA samples obtained from the proband’s mother, father, and siblings. Surprisingly, neither of her parents was found to carry the mutation, suggesting that this germ line alteration had occurred de novo. The presence of the mutation in the proband was confirmed on an independent DNA sample obtained from peripheral blood leukocytes, and in DNA extracted from micro dissected archival breast cancer tissue. However, no apparent LOH was identified in the tumor.

Paternity confirmation

To confirm paternity, a set of highly polymorphic short tandem repeat (STR) markers was analyzed by QF-PCR analysis using the ANEUFAST Kit (Genomed Ltd). The markers included in the kit were D21S1414, D21S1446, D21S1411, D21S1435, D13S631, D13S305, D13S634, D13S258, D18S525, D18S391, D18S386, D18S390, AMXY, X22, HPRT, DXYS218, and SRY. PCR amplification and fragment analysis were performed according to the manufacturer’s instructions. Markers were analyzed on an ABI Prism 3130 DNA Sequencer (Applied Biosystems).

All markers proved to be highly informative, consistent with the reported paternity with high probability. This result confirmed that the duplication identified in the proband was a de novo germ line mutation in BRCA2 that occurred during parental germ-cell gametogenesis.

Parental origin of the mutation

To determine whether the mutation had occurred on the maternal or paternal allele, we analyzed a polymorphism located near the site of mutation in the 5′UTR part of the BRCA2 gene (−26G > A according to HGVH numbering, −203G > A according to BIC). This single nucleotide polymorphism was fully informative because the father was homozygous GG, and the mother was homozygous AA (Fig. 1).

An allele specific PCR was performed using primers designed to distinguish between the two SNP alleles, and therefore the allele carrying the mutation from the wild-type allele. Specific primers were designed using the software Primer3 in combination with MFE primer to search specificity and SNP blast alignments.

The forward primer sets were designed to distinguish the −26A and the −26G allele with the last nucleotide: 5′-TCTGTTTTGCAGACTTATTTACCAAA/G-3′, respectively. The primer reverse was 5′-GTTTCCTCTTTATCCACTAGACGTTC-3′. Primer sets were tested on the samples from both the father and the mother, used as homozygote controls. PCR was carried out using standard conditions (available upon request).

The −26G allele was present in cis with the mutated c.51dupA allele. In accordance, the mutation was absent in the −26A allele sequence (Fig. 2). These data indicate that the BRCA2 c.51dupA mutation was on the father’s allele and also further suggest that this mutation probably arose as a de novo event in a testicular germ cell.

Fig. 2
figure 2

Electropherograms of reverse sequences corresponding to the two alleles specifically amplified from the DNA of the BRCA2 mutation carrier. The sequence of the paternal allele shows the presence of 5′UTR-26G and c.279insA genotype. The sequence of the maternal allele shows the presence of 5′UTR-26A and the wild-type (wt) genotype

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Discussion

We describe the identification of the BRCA2 c.51dupA mutation in a woman affected by bilateral early onset breast cancer at the ages of 27 and 37 and no family history of the disease. No one else in the family was identified to carry this mutation, and paternity was confirmed with the analysis of polymorphic markers. We therefore conclude that the identified novel mutation in the proband is a de novo mutation.

The presence of the alteration in the paternal allele was determined by the analysis of a polymorphism located near the mutation (−26G > A), taking advantage that the father was homozygous GG, and the mother was homozygous AA for this polymorphism. The result suggests a de novo event in a testicular germ cell, although an occurrence in the zygote can not be discarded. This is in agreement with previous findings in de novo mutations in BRCA1 and BRCA2, in which the paternal origin could be ascertained [5, 8], and also in other hereditary cancer syndromes (neurofibromatosis type 1, MEN2B and retinoblastoma) [10, 11]. It has been suggested that the origin of new mutations may be influenced by both genomic imprinting effects and the increased number of cell divisions in spermatogenesis compared with oogenesis. However, other familiar cancer syndromes do not exhibit a marked bias for a paternal origin of new mutations [12].

Although this specific mutation has not previously been described, several other presumably deleterious alterations have been reported in its vicinity in exon 2 (BIC). Examples include c.245delAA (reported three times), c.254delC (reported seven times), c.264insT, c.264delT, c.265G > T, and c.265insT (reported once each), c.279delAC (reported five times), and c.291delA (reported twice), suggesting that this area of the gene could be prone to mutations.

The observation that a previous known recurrent mutation in BRCA genes may appear as a de novo alteration suggest the existence of hot spots for small alterations. As an example, the BRCA2 c.3036_3039del4 mutation may have arisen on many separate occasions in various ethnic groups, according to the results of haplotype studies, and it has also been identified as a de novo mutation [8]. The authors suggested that these occurrences could be related with tandemly repeated sequences, which favor misalignments during DNA replication.

Although all germ line mutations started as a de novo mutation in some ancestor, the proportion of de novo mutations among gene carriers seems to be low. To our knowledge, there is only one published report of a proven de novo mutation in the BRCA1 gene and three in the BRCA2 gene, and there is only one previous report of a novel de novo mutation in BRCA2.

Haplotype studies of founder mutations in BRCA1 and BRCA2 demonstrated that they remain present in the population for many generations, probably due to the fact that cancer usually occurs after the reproductive capacity has been fulfilled. However, BRCA mutation studies are usually performed in families with multiple cases of breast/ovarian cancer, although the de novo mutations could be particularly expected in early onset breast cancer patients with no family history of the disease. One may hypothesize whether prevalence of de novo mutations would increase in case BRCA genetic testing was offered to unselected patients with early onset breast cancer.

There are a number of reports of BRCA1 and BRCA2 germ line mutations detected in early onset breast cancer patients without family history of the disease. In a population-based series of Spanish women diagnosed with breast cancer before age 40, the prevalence of BRCA1 and BRCA2 germ line mutations was 11.6% [13]. Family history of breast cancer and ovarian cancer was absent or not suggestive of hereditary condition in one-fourth of mutation carriers. Lidereau et al. [14] found that 6 of 70 women (9%) with breast cancer diagnosed at age 35 or below unselected for family history carried a BRCA1 mutation. One of them did not have any family history of breast/ovarian cancer. In a study of 254 white women from the United Kingdom, diagnosed with breast cancer before age 36, a germ line BRCA1 or BRCA2 mutation was identified in 15 women (6%), and only one had a family history [15]. Another recent study identified six mutations (11%) in 54 women diagnosed with high-grade, triple-negative invasive breast cancer at or before age 40, and little or no family history of breast or ovarian cancer [4]. The authors concluded that women with early onset triple-negative breast cancer are candidates for genetic testing for BRCA1, even in the absence of a family history of breast or ovarian cancer.

These studies underline the importance of mutation screening of the BRCA1 and BRCA2 genes in women with early onset breast/ovarian cancer or males with breast cancer, without a strong family history of the disease. The absence of family history might be related to non-informative kindreds (few women who lived until ages at risk of developing breast or ovarian cancer) or, albeit infrequent, the presence of a de novo mutation. Bearing in mind this possibility, the two parents of a new mutation carrier should be tested for the mutation to ascertain its origin. This information could be useful when considering the incidence of de novo mutations of the BRCA1 and BRCA2 genes in genetic epidemiological studies.