Detection of mutations in hereditary breast and ovarian cancer related BRCA1 and BRCA2 genes is an effective method of cancer prevention, early detection, and treatment. Mutations in the highly penetrant BRCA genes explain around a quarter of these cases . The frequency of germline mutations identified on both genes varies depending on the geographic and ethnic distributions. In some populations, a wide spectrum of different mutations is present, whereas in other groups specific recurrent BRCA mutations have been reported that may be due to the founder mutation effect [27, 28].
In Tunisia, so far, only 18 deleterious BRCA mutations have been reported. In the current study, 16 BRCA mutations, including 11 novel variations, have been identified in a cohort of 354 Tunisian breast and ovarian cancer patients. For breast cancer cases, high fractions of young patients (31.94%), cases with family history of breast cancer (35.24%), Triple negative breast cancer (24.31%) and high tumor grade (47.41%) have been observed. As reported in previous studies, the high fractions of early onset, triple negative cases and also the presence of family history of breast cancer may be associated with germline BRCA mutations [29, 30]. Indeed, it is now well documented that breast cancer patients in North Africa are almost 10 years younger than patients from western countries . In Tunisia, around 11% of breast cancer cases are under 35 years old . In fact, at a young age, the human organism usually functions as well as it ever will. However, interactions between some genetic and environmental factors (GxE) may cause a physiological decline of some organism systems leading to early disease presentation. Therefore, the influence of specific genetic background, differences in variant penetrance and frequency between populations along with environmental factors may explain this early onset of the disease. Large cohorts of young breast cancer patients should be studied to elucidate these GxE factors.
For ovarian cancer cases, the mean age at diagnosis was 52.62 years and the majority presented with serous ovarian carcinoma. Previous studies have shown that among all patients diagnosed with serous ovarian carcinoma, which is the most common subtype, over 15% will have germline BRCA mutations .
Among the 16 distinct deleterious mutations that have been observed c.19_47del, c.668dupA, c.915T > A, c.1612C > T, c.2418dupA, c.2433delC, c.3049G > T and c.5030_5033delCTAA in BRCA1 and c.-227-?_7805+? (Del exons 1–16), c.249delG, c.632-1G > A, c.1389_1390delAG and in BRCA2, are reported for the first time in the Tunisian population. We have also identified an inframe deletion reported to have a conflicting interpretation of pathogenicity effect in early onset bilateral breast cancer patient BRCA1_c.5017_5019delCAC. This variation has been described in multiple breast and ovarian cancer cases, with some families showing incomplete co-segregation of the variation [34, 35, 36] .
Among BRCA mutations identified in breast cancer patients BRCA1_c.211dupA, BRCA1_c.5266dupC,BRCA2_c.-227-?_7805+?,BRCA2_1310_1313delAAGA, BRCA2_c.1389_1390delAG and BRCA2_c.7654dupA occurred in BCCRs that are considered to be associated with an increased likelihood of breast cancer compared to ovarian cancer. Considering BRCA mutations identified in ovarian cancer patients BRCA1_c.1612C > T and BRCA1_3049G > T arose in OCCRs. Other mutations, namely c.19_47del, c.668dupA, c.915T > A, c.2418dupA, c.2433delC, c.5030_5033delCTAA in BRCA1 and c.249delG, c.632-1G > A in BRCA2 do not overlap with previously reported breast or ovarian cancer cluster regions. This could be explained by ethnic differences in BRCA mutation spectrum or it may indicate shared cluster regions for both breast and ovarian cancer.
In the BRCA1 gene, the c.19_47del mutation was identified in one breast cancer patient. This mutation was previously described only in the Algerian population . The c.2433delC mutation was described in Korean breast and ovarian patients [38, 39], and in Mexican patients [38, 39]. The pathogenic c.1612C > T mutation was identified in 4 breast and ovarian cancer patients. This mutation has been identified in Brazilian population , in ovarian cancer patients from Israeli population  and in Macedonian population . We also detected the c.668dupA mutation in one patient. This latter has not been reported in previous studies neither in Tunisia nor in other populations. Nevertheless, it is already listed and classified as pathogenic in ClinVar and predicted to result in the substitution of Alanine to Glycine (p.Ala224Glyfs) which leads to BRCA1 protein truncation. Another new mutation was identified in BRCA1 gene, c.2418dupA, that was reported by our group for the first time in the Tunisian population and was not reported previously in other populations . c.3049G > T has been identified in one ovarian cancer patient. This mutation has been reported in Thai patients with non-mucinous epithelial ovarian cancer . The c.5030_5033delCTAA mutation was identified among one patient with breast and ovarian cancers and it is reported in Brazilian population . The c.915T > A mutation is novel and not described in public databases.
In addition to the identification of rare and novel BRCA1 mutations, other mutations seem to be recurrent and/or were described in previous Tunisian reports. The c.211dupA mutation was shared by 6 patients belonging to the same geographical origin. This mutation has so far been reported only in hereditary breast/ovarian cancer families of Tunisian origin, particularly in the North-East region, suggesting a founder effect. In order to unravel the genetic specificities of this mutation and to trace its origin a haplotype analysis has been conducted by our group on the North Eastern region . Results have determined the founder haplotype segregating with this mutation and have revealed that it arose in the period of colonization approximately 130 years ago.
The c.5266dupC mutation has been identified among two families. This mutation was previously described in 8 Tunisian breast cancer families [10, 15, 16, 19]. It was originally described as an Ashkenazi founder mutation. Haplotype analysis has shown that this mutation arose approximately 1800 years ago in Northern Europe . Then, it has been reported in several other populations such as, Italian, Russian Slovenian and Greek .
Interestingly for BRCA2 gene, 6 breast cancer patients were double heterozygous carrying the two deleterious mutations c.632-1G > A and c.1310_1313delAAGA, and 4 other unrelated patients carried only the c.1310_1313delAAGA mutation including one male breast cancer (MBC). c.632-1G > A mutation appears to be rare in other populations since it was only reported in one patient with prostate cancer in the UK . However, c.1310_1313delAAGA seems to be a founder mutation in Maghrebin countries [15, 16, 50, 51]. It has been also identified in patients with Lebanese, European [53–56], African , Asian  and Latino ancestry  as well as in Caribbean cohorts [60, 61]. These results show the genetic heterogeneity of breast and ovarian cancers in Tunisian patients and the admixed origins of BRCA mutations in Tunisia.
Five male breast cancer cases were investigated in the current study among which 2 carried BRCA2 mutations ( c.1310_1313delAAGA and c.1389_1390delAG). Male breast cancer is a rare disease accounting for less than 1% of all breast cancer cases and it was previously shown that nearly 90% of MBC arising in BRCA mutation carriers are found to harbor a BRCA2 mutation . Unfortunately, being a man with “a women's disease” makes MBC a disease surrounded by social taboo and lack of awareness especially in underdeveloped countries. Indeed, the treatment of MBC has been extrapolated from the knowledge of female breast cancer, despite the multiple differences in the pathogenesis, biology and genetics of these two disease entities. These evidence make MBC a gender issue that requires more attention from the scientific community.
The introduction of the c.1310_1313delAAGA mutation, that have been encountered in diverse populations, in the Tunisian population could be explained by the immigration of Andalusians in Tunisia which has been intensified after the fall of Granada in 1492 and lasted for two centuries before the total expulsion of all Andalusian Moriscos from the Iberian Peninsula in 1610. The diverse geographical distribution of this mutation may further suggest independent origins as shown for the 4184del4 BRCA1 mutation reported to have at least three independent origins in the study of Neuhausen et al. . The c.7654dupA BRCA2 gene mutation which was identified in a unique family with a strong family history of breast and ovarian cancer is reported previously and exclusively in Algerian population , and could be therefore specific to North African countries.
Through this report and despite the identification of novel mutations in Tunisian population, it is clear that the genetic susceptibility to breast cancer is explained in a vast majority of cases by recurrent mutations. Indeed, more than 44.44% of carriers harbor BRCA1-c.211dupA or BRCA2-1310_1313deAAGA mutations which highlights the importance of screening these mutations in the treatment workflow of cases with early onset or strong family history of breast cancer. In fact, identifying germline BRCA1 and BRCA2 pathogenic mutations is a crucial component in the medical management of affected patients. Regular surveillance and/or prophylactic mastectomy of the second breast or prophylactic salpingo oophorectomies, which have been shown to reduce the risk of developing cancer, are recommended to these carriers. Moreover, relatives who test positive for a germline BRCA pathogenic mutation may take appropriate action to prevent cancer or have cancer diagnosed as early as possible for better treatment options .
In addition, mutations in the BRCA genes and their associations with clinico-pathological features were reported in several studies [65–67]. However, in Tunisia this aspect was not previously investigated. This point was raised in the present study and our results showed that patients with BRCA1 and BRCA2 mutations were similar with regard to several epidemiological and clinico-pathological parameters. Nevertheless, BRCA1 carriers were more likely to be triple negative breast cancer compared to BRCA2 carriers (p = 0.0001) and BRCA2 carriers were more likely to be luminal B breast cancer tumors (p = 0.001). Consistent with our findings, various previous studies reported that there is a much higher rate of TNBC among BRCA1 mutation carriers [68, 69] and BRCA2-related breast cancer is often luminal . Additionally, positive ER was significantly associated with BRCA2 + tumors (p = 0.000056). PR status was significantly different between BRCA1 and BRCA2 mutation carriers; BRCA2 carriers are more likely to develop progesterone receptor (PR) positive tumors and PR-negative breast cancer are associated with BRCA1 mutation carriers (p = 0.000084). It was reported that the ER positivity was predominantly seen in BRCA2 mutation carriers, which is consistent with our findings [65, 71]. Furthermore, a previous report has found that BRCA2-associated cancers are mainly PR positive . Other studies have raised some pathological differences between BRCA1/2 mutation carriers and BRCAx patients. In our study, BRCA carriers seem to be younger than BRCA-negative patients (p = 0.049). Furthermore, patients with a positive family history of ovarian cancer are more likely to be BRCA positive (p = 0.004). We also observed a significant predominance of SBR grade III tumors among BRCA1/2 mutations carriers (p = 0.025). These findings are in line with previous literature [29, 73, 74].
Furthermore, we have assessed disease outcomes in BRCA carriers, and we have observed a relatively high proportion of contralateral breast cancer and ovarian cancer occurrence that were more frequently observed in BRCA1 carriers. Previous reports have demonstrated that women carrying a pathogenic mutation in the BRCA1 or BRCA2 genes have an increased risk of developing a second primary cancer in the contralateral breast. The cumulative risk 20 years after breast cancer diagnosis was estimated to 40% for BRCA1 carriers and about 26% for BRCA2 carriers . In accordance with our findings, it was shown also that the occurrence of both breast and ovarian cancer in a woman is associated with a high likelihood of a germline BRCA1 mutation .
Besides the BRCA genetic mutations that have been identified in our study, mutations on other high to moderate breast cancer genes such as TP53, ATM, BLM and CHEK2 have been also identified for the first time in North Africa populations (data not shown). All these findings reflect the genetic heterogeneity of cancer predisposition in Tunisia and highlights the importance of the use of NGS to identify clinically actionable genetic variants that have a crucial role in disease management. Therefore, technological advances in terms of array and DNA sequencing technologies made the route towards the examination of genetic risk largely clear. However, practical challenges related to marked population-specific differences still exist. In this context, Manolio and colleagues conveniently classified LRRK2 as a high penetrant gene associated with Parkinson disease  with G2019S mutation being the main cause of Parkinson familial cases. Recently the international LRRK2 consortium reported a worldwide frequency of 1% of LRRK2 G2019S, 30–40% in Arab patients from North Africa and 10–30% in
Ashkenazi Jews, but is very rare in Asians [78, 79]. As a variant´s frequency has a direct impact on its penetrance, this example shows the ethnic-dependent penetrance of some important variants involved in complex diseases and the role of consanguinity and endogamy in shaping the genetic susceptibility to these diseases. Therefore, the same reflection can be applied on high and low penetrant breast cancer variants in order to review their penetrance in underrepresented populations such as North Africans. A disproportionate distribution of the identified mutations is observed between the Northern and Southern regions of Tunisia (Fig. 1), with the vast majority are found in North. This can be explained by a selection bias because most of the recruited participants come from Northern governorates but it can also be explained by the very high consanguinity rates in the South that reaches 98% in some cities and that may have an impact on BRCA mutations frequency and prevalence.
Clearly, the prevalence assessment of BRCA1 and BRCA2 mutations rely on the quality of both cohort selection criteria and mutation ascertainment methods. The identification of novel BRCA mutations and the assessment of their penetrance in a specific population will help to implement more affordable and cost effective targeted genetic testing strategies.
Finally, up until now, most data on BRCA1/2 mutations associated with high risk for hereditary breast and ovarian cancer do not cover the North African populations. Accordingly, the novel mutations identified in this study will help to improve knowledge on the genetic component of hereditary breast and ovarian cancer in the North African region and will lead to a better clinical management of cancer patients. In addition, we are aiming to share genetic and phenotypic data with larger multi-ethnic Consortia of BRCA1/2 carriers such as the CIMBA consortium . This will make our findings more broadly useful and will give us a global overview of the similarities and differences that the Tunisian population has compared to other ethnicities.