A total of 51African women from Burkina Faso affected by breast cancer have been selected by genetic counseling at CERBA/LABIOGENE laboratory of University of Ouagadougou (Burkina Faso) to perform a genetic testing for the screening of BRCA1 and BRCA2 genes. The molecular analyses were carried out at the Medical Genetics Laboratory of University of Rome Tor Vergata.
All patients had an age at diagnosis less than 40 years. The mean age of patients at diagnosis was 34.8 ± 4.14. Ninety-four percent (94%) of patients had an invasive ductal type of breast carcinoma, and only 24% (12/51) reported to have family history of breast cancer.
The NGS analysis produced an output of 644 entries containing the variant code, chromosomal, coding and amino acidic position. Only 30 of them have been found just one time in a single patient. Of these 30 unique variants (exonic and intronic), 4 are pathogenic (13%), 2 are variants of uncertain significance (VUS) (7%), 2 are novel (7%) and 22 are benign variants (73%).
Overall, on the total of 51 patients analyzed we found 8 carriers of a pathogenic variant (16%), 2 carriers of a variant of uncertain significance (VUS) (4%) and 2 carriers of a new undescribed variant (4%) (Fig. 1). We observed that the median age of patients carrying pathogenic variants was lower (33.25 ± 3.77) compared to the median age of patients carrying only benign variants (35.16 ± 4.16), although this difference was not statistically significant (p value = 0.25). The identified pathogenetic variants were in total 6 (4 unique and 2 recurrent in more than one patient). Three variants were in BRCA1 gene and 3 in BRCA2 gene. The BRCA1 mutation carriers had a mean age of (32.4 ± 3.78) years, BRCA2 mutations carriers had a mean age of (34.67 ± 4.04). The mutation prevalence was evaluated in our cohort and resulted to be 15.7% (95% CI: 5.7–25.7%) for the two BRCA genes, in particular 9.8% (95% CI: 1.6–18.0%) for BRCA1 and 5.9% (95% CI: 0.2–11.4%) for BRCA2. The mutations included missense, nonsense, small deletion and intronic variants (Table 1). In particular, in BRCA1 gene we identified the presence of 2 recurrent pathogenic variants in 4 unrelated patients. Specifically, 2 patients were affected by the same mutation in BRCA1 gene, c.4088C > G, (p.Ser1363*). This is a nonsense mutation which causes the substitution at amino acid 1363 from Serin to a stop codon. Patients carrying this mutation had an undifferentiated and ductal type of breast carcinoma at ages of 37 and 34 years, respectively, and both had a family history of breast cancer. Other 2 patients shared the intronic mutation c.4986 + 6T > C ( LRG_292t1:c.4986 + 6T > C) in BRCA1 gene. This variant has a severe impact on splicing because leads to activation of a downstream cryptic splice donor site, which results in an aberrant RNA transcript and a truncated protein. These patients were affected by a breast ductal carcinoma, diagnosed at early ages of 28 and 29 years, respectively. One patient reported to have family history of cancer, while the other patient did not. Moreover we identified 2 frameshift mutations in BRCA2 gene, the c.6445_6446del, (p.Ile2149*) and the c.6757_6758del, (p.Leu2253Phefs*7) and 1 frameshift mutation in BRCA1 gene, c.5177_5180del,(p.Arg1726Lysfs*3) .Two of the frameshift variants identified were in women with no family history of breast cancer. The only missense mutation identified, c.8009C > T, (p. Ser2670Leu), was in BRCA2 gene. The variant was present in a 37-years-old woman with a medullary breast carcinoma and a family history for breast cancer. Interestingly, the pathogenetic variants frequency detected in our cohort is statistically different respect to the frequencies listed in the GnomAD database for the African population (p-value < 0.05), with the two variants: the c.6445_6446del, (p.Ile2149*) and the c.8009C > T, (p.Ser2670Leu), which are not even present in GnomAD database (Table 1). In addition to the pathogenetic alleles we identified two missense variants of uncertain significance (VUS); one in BRCA1 gene (c.5348T > C, p.Met1783Thr) and one in BRCA2 gene (c.7504C > T, p.Arg2502Cys) with an allelic frequency not statistically different respect to the frequencies listed in the GnomAD database for the African population (p-value > 0.05) (Table 1). Both these variants are in a functional domain of the BRCA1 and BRCA2 protein, respectively. The variant Met1783Thr is in the BRCT2 domain of BRCA1, while the variant Arg2502Cys is in the BRCA2 helical domain. To predict the potential impact of these variants on the protein we used different tools (Mutation Taster and PolyPhen-22). The in silico analysis predicted a damaging role for the BRCA1 variant (Mutation Taster: disease causing; PolyPhen-2: Probably damaging, with a score of 1.000); moreover the sequence alignment of the BRCA1 protein with its orthologous proteins showed that the wild type residue seemed to be moderately preserved in species, implying a role for this residue in the protein function. The in silico analyses for the BRCA2 variant Arg2502Cys gave a benign computational effect on the protein (Mutation Taster: polymorphism; PolyPhen-2: benign, with a score of 0.022). In this case the sequence alignment of BRCA2 protein with its orthologous proteins showed that the wild type residue is poorly conserved among species implying an irrelevant functional or structural role of this residue on the protein.
Finally, we have identified 2 unclassified variants (4%) in BRCA1 gene: 1 missense variant, c.2359G > A, (p.Glu787Lys) (rs1288796003) and 1 nonsense variant, c.872T > A, (p.Leu291*). We used in silico tools (Mutation Taster and PolyPhen-22) to evaluate the potential impact of these variants on BRCA1 protein.
The missense variant c.2359G > A causes a non-conservative amino acid change, the substitution of Glutamic Acid 787 to Lysine. This variant is present on dbSNP database (rs1288796003) but is not present on any clinical databases (ClinVar, LOVD) and is not reported on GnomAD. The in silico analysis with different software gave conflicting verdicts.
The variant c.872T > A, (p.Leu291*) cause the changing of amino acid 291 from Leucine to stop codon thus breeding a truncated protein that comes shorter of 6916 amino acids. In accordance with the American College of Medical Genetics (ACMG) this variant is classified likely-pathogenic-class 4 [15 ].