In the present study, we evaluated the frequency and mutational spectrum of BRCA1/2 in a series of patients or putative carriers, unselected for age or family history. No obligation of fulfilling criteria of mutation probability methods molecular screenig was applied. In our cohort, 12% (156/1,267) of the patients carried a deleterious germline BRCA mutation (93 BRCA1 and 63 BRCA2), being the vast majority very rare, found in only one or two individuals. Particularly, the most frequent BRCA1 mutation, accounting for 20,8% of all BRCA1 mutations, was the c.5266dupC (p.Gln1756Profs*74), while the BRCA2 c.2808_2811delACAA (p.Ala938Profs*21), accounting for 12,7%, was the most frequent of all BRCA2 mutations.
The BRCA1 c.5266dupC (p.Gln1756Profs*74) was found in 20 individuals and among all geographical regions in Brazil. This mutation, also known as 5382insC or 5385insC, is a known founder mutation in individuals of Ashkenazi Jewish ancestry23. Noteworthy, only four individuals self-identify as Ashkenazi Jewish /East European descent between those with the BRCA1 c.5266dupC mutation. The median age at breast cancer diagnosis in this series was 35 years-old, and five individuals were discriminated as mutation carriers without breast, ovarian or any other type of cancer. All carriers had a family history of breast or ovarian cancer in at least one first- and second-degree relatives at young age. Unilateral triple negative breast cancer was seen in 7/20 and only one patient presented bilateral breast cancer.
The BRCA2 c2808_2811delACAA (p.Ala938Profs*21) was found in 8 individuals from the Southern and Southeastern regions of Brazil. Five women with unilateral breast cancer harboring this specific mutation were detected in this series. The median age at breast cancer diagnosis in these cases was 35 years-old, which include one male carrier at the age of 40, with family history of hereditary breast and ovarian cancer syndrome, and two females’ carriers at the age of 23 and 61. The 23 years-old female has a family history that included one case of a male breast cancer at the age of 30. An additional high burden of other types of cancers has been observed in the families with BRCA2 c2808_2811delACAA carriers, in particular, an enrichment of prostate cancer. It is relevant to mention that among all BRCA2 mutations a few were seen in several populations in different ethnic groups, but the c.2808_2811delACAA is in the top 10 most frequent mutations detected between all ethnicities24. Recently, the BRCA2 c2808_2811delACAA was described as the second most common BRCA2 variant in a Brazilian populational study19.
Notably, considering the most frequent P/LP variants in this dataset (Fig. 2), the three most common are in accordance with a recent worldwide BRCA1/2 mutational spectrum report, which listed BRCA1 c.5266dupC, BRCA1 c.3331_3334delCAAG and BRCA2 c.2808_2811delACAA, among the top 5 BRCA1/2 mutations in Brazil24. With particular interest, we highlight a BRCA2 mutation in our cohort that are strongly associated with the African American population. The c.6405_6409delCTAA p.Asn2135Lysfs*3 was the second most common BRCA2 mutation, and it was present in six individuals from the Southern and Southeastern regions of Brazil. Four women with unilateral breast cancer, two triple negative and a 60 years-old patient with ovarian cancer harbor this pathogenic variant. Within this group there was also a female carrier at the age of 22 with family history of hereditary breast cancer. Of note, the c.6405_6409delCTAA was found among the 10 most common mutations in Brazil, African American and South/Central America24. It is worth mentioning that the geographical distribution of BRCA1/2 mutations in Brazil, as shown in Fig. 2 and Supplementary Tables 2 and 3, highlights the unequal frequency of patients referred to genetic testing and access of supplementary health in the country. The Brazilian Society of Medical Genetics (https://www.sbgm.org.br/) points out that one third of the country`s geneticists are located in the state of São Paulo, which certainly influences molecular investigation and treatment of the patients.
Nearly all P/LP in BRCA1/2 described here were previously reported, except for three novel variants in BRCA2: (i) a frame shift mutation starting at codon Asn1201 [c.3601_3602delAAinsT; p.(Asn1201Serfs*8)] detected in a 64 years-old woman with breast cancer, and family history for breast and prostate cancer; (ii) a 1 bp duplication in exon 11 that interrupts the reading frame prematurely at position 1220[c.3659dupA; p. (Tyr1220*)], detected in a 60 years-old woman, African descent with triple negative breast cancer status, and no family history for breast, ovarian or other cancer type; and (iii) a 1bp deletion in exon 21 leading to a frameshift [c.8682; p.(Val2895PhefsTer14)] effect in a 28 years-old Italian descendant woman with bilateral triple negative breast cancer and familial history of Hereditary breast, ovarian and colon cancer.
Regarding the frequency of VUS, this class of variant represent only 2% (31/1,267) in our cohort. VUS impose a challenge relating to the management and surveillance of carriers as well as in risk assessment. To categorize these variants, six different in silico tools were applied (i.e., phyloP; Grantham dist; Align GVGD; SIFT and Mutation Taster), and their classification was also based on consulted databases and complementary sources as previously described. A total of 31 unique VUS were identified tin the patients (14 and 17 in BRCA1 and BRCA2, respectively), and none of them was detected neither at carriers of BRCA1/2 P/LP variants nor present more than once. Out of the 14 BRCA1 VUS, 11 are reported on ClinVar database to have unknown clinical significance, while the remaining 3 have no records on public databases (BRCA1 c.4893T > A, c.4934G > C, and c.3975G > T) (Table 1). Interestingly, we identified one non-coding VUS in BRCA1 localized in the 3´UTR region, the c*291 C > T, which was detected in a 57 years-old woman with unilateral breast cancer and family history of breast cancer. A functional luciferase assay previously showed that the c*291 C > T increased BRCA1 3'UTR activity25. Although there is an increasing number of data associating germline non-coding variants with higher cancer risk, a co-segregation analysis was not possible to be made because the VUS was reported only in this woman. It is noteworthy that this non-coding variant was only described in breast cancer cases26. Among the 17 BRCA2 VUS, 14 are reported on ClinVar database to have unknown clinical significance, while 3 of them have no records on public databases (BRCA2 c.67 + 25T > C, c.3045G > T and c.8755G > T) (Table 2). In particular, the BRCA2 c.8755G > T variant was previously described in a Brazilian series where it also was provisionally classified as VUS18; in silico tools predicts that this variant exerts a possible effect nearest the splice site. Considering that there is a degree of inconsistency between these in silico predictions tools, it is impossible to draw any conclusions on the pathogenicity of VUS. Nonetheless, an integrated strategy which will include co-segregation analysis, tumour pathology data, as well as functional assays is needed to complete a comprehensive assessment of pathogenicity of these variants.
The characteristics of BRCA1/2 mutation carriers in our cohort (i.e., the overall mutation prevalence was higher in patients diagnosed with cancer > 35 years old, and with a family history of cancer) were similar to other studies. Although it was expected that patients with a BRCA1/2 mutation were more likely to have bilateral breast cancer, in the current study unilateral breast cancer represent the largest number of cases. Nonetheless, patients with P/LP variants in BRCA1/2 have approximately 3 times more bilateral breast cancer when compared to those with VUS. Different definitions of familial breast cancer and genetic testing methods for BRCA1/2 mutations between the studies may influence the results. Currently, the National Comprehensive Cancer Network (NCCN) guidelines are the most widely used criteria for testing and inform insurance coverage decisions even though recent data suggest that expansion of testing may be appropriate. The mutation detection rate in patients with breast cancer tested based on NCCN guidelines varied widely depending on the type and number of criteria met. In general, BRCA detection rates were significantly increased when the reason for testing was age ≤ 45 at time of diagnosis or having a known family history of a BRCA mutation, but specific clinical scenarios such as triple negative breast cancer status also have been associated with a high risk for BRCA mutations. We used a low stringency criteria for BRCA genetic testing referral in our patient cohort, which may explain the high diagnostic yield (12%) when compared to other studies where unselected patients were investigated.
In summary, the present data expand our knowledge of the frequency and BRCA1/2 germline mutational spectrum in Brazil, being a valuable clinical resource for genetic counseling and cancer management programs in the country. Also, based on the increase interest for BRCA genetic testing among individuals who are at high risk of carrying a mutation, our data may help provide guidelines in the future for patients with breast cancer who should undergo molecular screening.