In this study, we have reported the prevalence of BRCA1-2 germline mutations in patients with a positive family history of breast and/or ovarian cancer from Sardinia, whose population shows genetic peculiarity due to geographical isolation and strong genetic drift [27, 28]. Prevalence of BRCA1-2 mutations may indeed vary among distinct populations due to concurrence of different environmental factors and genetic backgrounds; in other words, patients origin may strongly account for different mutation rates in candidate genes.
In the present study, a germline pathogenic mutation in either BRCA1 or BRCA2 was identified in 10% of screened breast-ovarian cancer families (BRCA1 mutations were detected in about 3% of cases, while BRCA2 mutations were identified in about 7% of families). BRCA positivity reached 17.5% when considering the "high risk" families, 29% in families with probands diagnosed at age ≤40 years and 31% in the presence of ovarian cancer in the family. Prevalence rate in "high risk" families was higher than that of families with 3 or more affected member regardless of age at onset (11.7%). These results confirm that clinical characteristics such as ovarian cancer in the family, age at diagnosis and number of cases are good predictors for the likelihood to be a BRCA mutation carrier.
The overall prevalence of BRCA1-2 mutations among breast cancer patients from the entire Sardinia was quite similar to that previously reported for breast-ovarian cancer families originating from the Northern part of the island (for both studies, 15% BRCA -positive carriers were observed in breast-ovarian cancer families [14, 18]. However prevalence of BRCA deleterious mutations was higher in Middle Sardinia (38% vs 11% in the North. and 19% in the South). When compared to many other Italian and European studies, the frequency is relatively low [10–13, 29–32]. Since the sensitivity of mutation detection methods is not complete, some mutations may have remained undetected in the present study. Large genomic deletions, which do escape detection by both DHPLC and direct sequencing may account for a fraction of mutation-negative breast and ovarian cancer families in Sardinia. Overall and even considering a lack of sensitivity of the screening approach used in the present study, the prevalence of BRCA mutations in Sardinian families remains low. The selection criterion of two close relatives with breast cancer before the age of 50 years, used in this study, may somehow explain this low prevalence rate, though it is not possible to exclude that a specific genetic background may play a role on breast cancer susceptibility among Sardinian population. Noteworthy, a similar low prevalence of BRCA mutations was reported in the Finnish population which has genetic features comparable to the Sardinian one, where the historical, cultural and geographical isolation may have selected specific genetic variants as susceptibility genes for breast cancer .
The geographical origin of the families positive for deleterious BRCA1-2 mutations is shown in Figure 1. The BRCA2 c.8764_8765delAG and c.3950_3952delTAGinsAT variants were previously described as founder mutations in North and Middle Sardinia, respectively. In particular, cases carrying the BRCA2 c.8764_8765delAG mutation belonged to unrelated families originating from different villages in the northern part of the island [11, 14]; most of families genotyped with markers flanking the BRCA2 gene at 13q12-q13 locus were demonstrated to share a large haplotype, not found in control chromosomes from the same geographical area . Conversely, the BRCA2 c.3950_3952delTAGinsAT, which was previously reported as a founder mutation, was instead running in families belonging to a single extended pedigree confined to a small village of the central part of Sardinia . The BRCA1 variants occurred in families originating from different areas of the island without a defined geographical clustering, though about half of the cases carrying the BRCA1 mutations originated from the South Sardinia. Interestingly, in three unrelated families from the South and Middle Sardinia we identified carriers of the BRCA1 c.916_917delTT mutation that has been reported in breast-ovarian cancer families from South Italy . Altogether, the geographical distribution of the genetic variants in the island, suggests that BRCA1-2 mutations are related to the specific three large areas of Sardinia, which reflects its ancient history: a) the North area, delimited by the mountain chain crossing Sardinia and linguistically different from the rest of the island (where the c.8764_8765delAG variant acts as founder mutation); b) the Middle area, land of the ancient Sardinian population and domain of pastoral culture (where BRCA2 mutations are still more common than BRCA1 mutations); and c) the South-West area, with many Phoenician and Carthaginian archeological sites (where BRCA1 mutations are prevalent).
Majority of BRCA1-2 germline mutations identified in Sardinian families is unique: most of the variants were detected in single families and 6 were novel mutations.
The frequencies of BRCA1 or BRCA2 mutations among breast-ovarian cancer families varies widely between populations world-wide: Icelandic breast cancer families present almost exclusively BRCA2 mutations , roughly equal numbers of BRCA1 and BRCA2 mutations have been described in French Canadian or British breast cancer families [35, 36], while a clear prevalence of BRCA1 mutations has been observed in the United States [37, 38]. In this study BRCA2 mutations were more frequent than BRCA1 mutations, although it should be taken into account that a high proportion of families are carrying the BRCA2 c.8764_8765delAG founder mutation. Mutations in BRCA2 gene have been detected in up to 40% of male breast cancers in Iceland ; in our isolated population, we detected a BRCA2 mutation in 1/14 (7%) men with breast cancer with a frequency similar to that reported for male breast cancers in USA (4%) . Again, these findings suggest that other environmental and/or genetic factors are contributing to the susceptibility to male breast cancer in Sardinia.
Specific pathological features have been reported in hereditary breast cancer with differences between BRCA1 and BRCA2 associated tumors . In our series, the analysis of clinico-pathological characteristics showed some differences between BRCA1-2 wild type and mutation carriers. Though no correlation with the stage of disease (the main prognostic factor for breast cancer patients) was observed, the presence of BRCA1-2 mutations were significantly associated with some pathological characteristics (higher tumor grading, lack of expression of estrogen/progesterone receptors) which are recognized to have a negative impact on prognosis. Unfortunately, data from mutation screening of BRCA1 and BRCA2 were combined for the statistical analysis due to the low number of positive cases for each single gene in our series, although distinct differences in both molecular pathology and histopathology have been shown according to the specific carrier status.