To the Editor,

It is well known that the pathogenic mutations in BRCA1 or BRCA2 genes are detected in 5–10% of total breast (BC) and ovarian cancer (OC) and in 20–30% of BC/OC found in families with strong history of cancer [1]. However, the incidence of the BRCA1 and BRCA2 pathogenic mutations depends on the criteria adopted to select the families to be studied [2], and the mutation spectrum varies considerable due to the influence of the ethnic groups [3]. In addition, ethnicity could substantiate the appearance of founder mutations coming from an old ancestor such as the recurrent mutations detected among Ashkenazim [4, 5].

The Program of Genetic Counselling in Cancer of Valencia Community was launched in March 2005 and we reported the results of the first 147 nonrelated families included, describing the preliminary mutational spectrum [6] and novel mutations [7]. However, since then, the number of subjects studied for BRCA1 and BRCA2 mutations has increased considerably having reached the 704 families at the end of 2008. This large number of individuals prompted us to update our series and to establish a consistent mutational spectrum of the population of Eastern Spain identifying the recurrent mutations and relevance of the novel mutations.

We studied 704 index patients (IP) (689 females and 15 males) who enrolled in the Program of Genetic Counselling in Cancer of the Valencian Community from March 2005 to December 2008. The IPs were selected by the Units of Genetic Counselling in Cancer (UGCC) according to the inclusion criteria established in the Program of Genetic Counselling in Cancer for familial BC/OC [8, 9]. All the individuals signed a written consent elaborated by the Consellería de Sanitat (Valencia Community) in accordance with the Helsinki Declaration (1964, amended in 1975 and 1983) [10].

BRCA1 and BRCA2 mutations were detected by amplifying all the exons and the exon–intron boundaries of both genes by PCR using the primer pairs and the PCR conditions reported in the Breast Cancer Information Core (BIC) [11]. To identify the genetic variations, we carried out a pre-screening of the heteroduplexes of the PCR products by conformation sensitive gel electrophoresis (CSGE) [12] followed by the sequencing of the PCR products in which heteroduplexes were identified.

In BRCA1, we found 26 different deleterious mutations in 62 IPs (17 frameshift, 3 splicing, 4 nonsense, and 2 missense mutations; Table 1); 16 of these mutations have been included in our previous reports [6, 7]. The mutation with the highest recurrence was c.5242C>A missense mutation in exon 18, detected in nine families, followed by the frameshift mutations c.2080delA of exon 11 and c.187_188delAG of exon 2 found in seven and six families, respectively. These three mutations represent 35.5% (22/62) of all the mutations detected in this gene. If to these we add the missense c.330A>G and the splicing c.331+1G>A mutations, both in exon 5, each found in three families, the five mutations represent the 45.2% (28/62) of the mutations in this gene.

Table 1 Pathogenic mutations detected in BRCA1
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Besides, we found two novel frameshift pathogenic mutations c.4424_4425delCT and c.4476delG of exon 13, both present in single families (Table 1). The first one causes a stop at codon 1439, while the c.4476delG mutation causes the loss of the last nucleotide (G) of exon 13 and a stop at codon 1455. The FruitFly software [16] predicts that the latter mutation presumably affects the donor-splicing site.

In BRCA2, we found 36 different deleterious mutations in 71 IPs (30 frameshift, 1 splicing, and 5 nonsense; Table 2); 13 of these mutations had been previously reported [6, 7]. The recurrent mutations with highest incidence were all located in exon 23 such as the two frameshift mutations c.9254_9258delATCAT and c.9206_9219del14 found in 14 and 6 families, respectively, and a nonsense mutation c.9246C>A found in five families. Likewise, the c.3492insT mutation of exon 11 showed a high recurrence and was identified in five families. These four mutations cover 42.2% (30/71) of the families with the pathogenic mutations detected in this gene.

Table 2 Pathogenic mutations detected in BRCA2
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Besides in BRCA2, we found three novel mutations, two frameshift mutations c.1874_1877delAGGA in exon 10 and c.6118delA in exon 11 and a splicing mutation c.8860+2T>G in intron 20 (Table 2). The c.1874_1877delAGGA creates a stop at codon 556 with the consequent truncation of the translation of the BRCA2 protein. The c.6118delA causes the interruption of the synthesis of the BRCA2 protein at codon 2003. The splicing mutation c.8860+2T>G affects the second nucleotide of intron 20, suppressing the donor site as predicted by the FruitFly software [16].

The current mutational spectrum of our population has broadened with regard to our previous report; recurrent mutations have emerged and five novel mutations have been identified. Thus, the mutational spectrum for BRCA1 and BRCA2 increased from the 16 and 13 mutations in our previous reports [6, 7] to 26 and 36 mutations, respectively, in the present report.

Eight of the mutations detected in BRCA1 were also observed in recent studies carried out in the Spanish population [13–15]. However, our results are not as concordant as those expected with the larger study carried out in the Spanish population [13]. Only 7 of the 35 different mutations (20%) observed in the mentioned study, c.187_188delAG, c.330A>G, c.331+1G>A, c.2080delA, c.5242C>A, c.5272+5G>A, and c.5273-1G>A, were detected in our population. These mutations represent 48% (30/62) of the pathogenic mutations detected in our study. However, only two of them, c.187_188delAG and c.330A>G, were recurrent in both the studies.

For BRCA2, there is a coincidence of 11 mutations with other reports performed in the Spanish population [13–15]. The 2003 report of Diez et al. [13] is concordant with our study in 10 out of the 22 different mutations (45%), c.1538_1541delAAGA, c.3036_3039delACAA, c.3492insT, c.4150G>T, c.5804_5807delTTAA, c.6503_6504delTT, c.6857_6858delAA, c.9206_9219del14, c.9246C>A, and c.9254_9258delATCAT, which represent 70% (37/53) of the families reported by that study [13] and 58% of the families (41/71) of this study. The frameshift mutation c.9254_9258delATCAT, the most recurrent mutation of this study, also showed high incidence in the report of Diez et al. [13]. This recurrent mutation is considered characteristic of Eastern Spain. However, for most of BRCA2 recurrent mutations, no correspondence was observed between both the studies.

We observed greater diversity and higher incidence for BRCA2 mutations than for BRCA1 (50.7% in BRCA2 vs. 41.9% in BRCA1) in correspondence with our previous reports [6], and with other studies performed in the Spanish population [14–17], but in disagreement with the other one [13]. We think that the larger incidence of mutations in BRCA2 could be a trait of the population of certain geographical areas of Spain, not related with the presence of the c.187_188delAG BRCA1 Ashkenazim mutation, as suggested by Infante et al. [15], since in this study, we observed larger incidence of BRCA2 mutations in spite of having high incidence of this mutation.

We describe five novel mutations (four frameshift and one splicing) in BRCA1 and BRCA2, which in addition to the 10 novel pathogenic mutations of our previous reports [6, 7] represent a relevant percentage of 24% of novel mutations (18 novel mutations out of 62 different mutations), which attributes to the great heterogeneity of the Spanish population.

In summary, the population of Eastern Spain showed a great heterogeneity in the BRCA1 and BRCA2 mutations with slightly higher mutation incidence and heterogeneity for BRCA2. For BRCA1, we observed three major recurrent mutations—c.5242C>A, c.2080delA, and c.187_188delAG—which were present in 35% (22/62) of the families. For BRCA2, c.9254_9258delATCAT, c.9206_9219del14, and c.9246C>A are the major recurrent mutations present in 35% of the families (25/71). We detected in our population a total of 15 novel mutations, which represent the 24% of the different mutations. All these results attribute to the singularity of the population of Eastern Spain.