Identification of BRCA1/2 p.Ser1613Gly, p.Pro871Leu, p.Lys1183Arg, p.Glu1038Gly, p.Ser1140Gly, p.Ala2466Val, p.His2440Arg variants in women under 45 years old with breast nodules suspected of having breast cancer in Burkina Faso

Introduction

Breast cancer is the most common cancer in women, especially in those over 50 years old for whom it is the leading cause of death worldwide. The global trend in the epidemiology of breast cancer in African countries is not far from that observed in the western countries of the world. Breast cancer is the second most frequent cancer both in the totality of African countries and in the specific area of Western Africa (which comprises Burkina Faso) accounting for the 25 to 30% of cases of cancers [1]. Nevertheless, its incidence is higher in developed than in developing countries (64.2/100,00 vs 23.1/100,000 women) [2].

Breast cancer is a malignant tumor of the mammary gland. This cancer is most common in women, with 89 cases per 100,000. 5 to 10% of these cancers have a clear genetic basis, whereas 85-90% of cases (called sporadic or non-hereditary) have a complex etiology still poorly understood [3, 4, 5, 6]. In Burkina Faso (one of the west African countries) breast cancer is being responsible for 17.7% of women’s deaths [7]. A recent study provide preliminary information about breast cancer risk decreasing with multiparity among women in Burkina Faso [8, 9]. Genetic and non-genetic factors are involved in the etiology of breast cancer.

The genetics of breast cancer in African countries is generally undetermined. A genetic test may reveal the risk to women from a family where there is a history of mutations in a specific gene [10, 11]. The lack of data on the genetics of breast cancer in African countries might be due to lack of diagnostic tools in molecular biology, preventing women from performing this test [2, 12, 13, 14, 15]. Two predisposing genes for breast cancer have been identified [16, 17] so far: BRCA1 on chromosome 17 (more than 500 mutations or sequence variations have been identified) and BRCA2 on chromosome 13 (over 100 different mutations have been reported). Women who have inherited mutations in BRCA1 (17q21, chromosome 17: base pairs 43,044,294 to 43,125,482) or BRCA2 (13q12.3, chromosome 13: base pairs 32,315,479 to 32,399,671) have an increased risk of developing breast and ovarian cancers [11, 17]. The probability of developing breast cancer in a carrier of a BRCA1 mutation is about 65% before the age of 70 and 45% for carriers of mutations in the BRCA2 [3, 18, 19]{Couch, 1997 #2200;Whittemore, 1997 #2219;Mote, 2004 #2212;Whittemore, 1997 #2219}. In a study by Rebbeck et al, among BRCA1 mutation carriers, 46% of women were diagnosed with breast cancer, 12% with ovarian cancer, 5% with breast and ovarian cancer, and 37% without cancer. And among BRCA2 mutation carriers, 52% of women were diagnosed with breast cancer, 6% with ovarian cancer, 2% with breast and ovarian cancer, and 40% without cancer [20].

The available information about breast cancer in Burkina Faso, regarding the histo-pathological characteristics of tumors and parity risk factors, is limited [8, 9], with few specific records about genetic features [8, 9, 21].

The aim of this study was to provide the first ever cancer related genetic information for a West African country. To achieve this aim, these specific objectives were targeted: i) identify women with breast nodules suspected of being breast cancer; ii) determine genetic variants associated with breast cancer in Burkina Faso by PCR.

Materials and methods

Results

Mutation screening

After loading the PCR products on 2% agarose gel (3 mL) they were visualized under UV light as reported in Fig 1 (examples of BRCA1 and BRCA2 profiles). An example of the sequencing profile of an heterozygous specimen is reported in Fig 2 in exon 10 of BRCA1: a non-synonymous and heterozygous change at the 6^th base pair is present

Figure 1

BRCA2, Exon 21 and 22 PCR product profile in agar gel 2%. Legend: L: Fermentas GeneRulerTM DNA Ladder #SM0313; N: Negative control; Number 1 to 9: specimens.

Figure 2

Sequencing profile of a DNA. Legend: variation heterozygous change at the 6th base pair (T/T+C).

in the figure corresponding to the change c.2622C>T; p.Leu871Pro, rs799917), where a clear double peak of C/T could be identified in the lower panel. The upper panels shows a non-synonymous change but in homozygous state. All specimens had a non-synonymous change at this site.

Discussion

BRCA1 and BRCA2 genes have been screened in young women with nodules, suspected of being breast cancer, and some variants have been found. Each specimen in this study had at least one variant. All variants identified are benign. However, others populations have high proportions of oncogenic variants. For example, in the case of Ashkenazi families, a high frequency of causative mutations of the BRCA1 and BRCA2 genes, as insertions and deletions, were observed [22, 23]. A single variation 3’UTR was found in the last exon (BRCA1 exon 23) with reported SNP rs3092995, it is possible that this variation may alter the mRNA structure in this specimen [24, 25]. All identified variants were located between c.2612C>T and c.4837A>G regions for BRCA1 and between c.7319A>G and c.7397C>T regions for BRCA2. These regions are not located in the breast cancer cluster regions identified by Rebbeck and Collaborators in 2015, but some of these are in the ovarian cancer cluster region (Exon 11 of BRCA1) [20]. This revealed that breast cancer may have non-genetic predictor factors as well as varied genetic factors. The variants identified in this study are found mostly in cases of benign tumors. According to a study published in 2008, non-cancerous breast diseases may promote the subsequent occurrence of cancer [26]. Some of the variants we identified may be predisposing factors. In general, variants may not cause cancer. Some variants, oncogenic in other countries may not be in Africa because the genotype may be influenced by the environment [27]. Some mutations can act synergistically to cause cancer [28], especially since in this study there were patients with family history of cancer.

Among our study population, we identified the same variation (regarding the amino acid change c.2622C>T corresponding to the protein change p.Leu871Pro) that like other variations found in our specimens, had been reported previously in dbSNP database. This common variation is on the BRCA1 gene (SNP rs799917). Some studies have found that BRCA1 rs799917 polymorphism may contribute to the risk of cervical cancer in Chinese populations, and further validation in other populations would be warranted [29]. Recently in Burkina Faso, Zoure et al. targeted exon2, exon5 and exon11A for BRCA1 sequencing [21]. These exons were described as likely to contain causative mutations of cancer in northern Africa and some west African countries’ [30, 31]. But they did not detected mutations in any of the 15 women diagnosed with family breast cancer history in Burkina Faso [21]. In our study, we sequenced the whole BRCA1 and BRCA2 gene. This is the first reported study which screened these genes in their entirety to detect mutations in a Burkina Faso population. It is worth noting that our specimen size was very small and selected mostly based on the age of presentation. Also, in the familial cases of breast cancer, the proportion of BRCA1 and BRCA2 carrier is relatively low (5%) [3, 4, 32] and this could explain why known oncogenic variants have not yet been determined that confer an increased risk for breast cancer in this study.

Indeed few studies concerning BRCA1 and BRCA2 in the West African population have been published. We can consider the study in Yoruban populations where the BRCA1 Y101X mutation (1.1%) has been identified [33]. A study carried out in Algeria observed that 9% of sporadic cases of early-onset breast cancer and 36% of family cases have been associated with BRCA1 mutations [34]. In Ghana another high proportion (61%) of mutation was found among patients with breast cancer [35]. Zeng and collaborators performed a study involving Africans, in which no association between identified SNPs and breast cancer risk was identified [36]. The limited data may indicate that the west African families do not have the same access to screening for mutations in target genes compared to other countries [22, 23]. The detection of BRCA mutations within a family member would have predicted hereditary breast cancer [3, 18, 19]. For those at risk, also for other family members, active surveillance through early detection would be necessary. In addition, mutations may vary among individual racial and ethnic groups [22, 37] and early studies suggested that BRCA mutations were relatively rare among Africans [14, 33].

The variant c.4837A>G (corresponding to the protein substitution p.Ser1613Gly) has been found to be most frequent in South Asian (50%), East Asian (37%), European (36%), American (37%) and Africans (23%) populations’. About 77% of the alleles found in the African population studies are ancestral allele T (dbSNP). This proportion is similar to those found in the African sub-populations with variant c.2612C>T rs799917 SNP (88% of ancestral allele A), c.3113A>G rs16941 SNP (84% of ancestral allele T), c.3548A>G rs2227945 SNP (96% of ancestral allele T), c.7397C>T rs169547 SNP (91% of ancestral allele C), c.7319A>G rs4986860 SNP (96% of ancestral allele A), c.4837A>G rs1799966 SNP (77% of ancestral allele T). However, the rs16942 SNP, 22% of the alleles found in the African population studies, are ancestral alleles (C).

Mutations in several other genes remain to be identified, even if AKT1, TP53 and PTEN, ATM variants have been associated with hereditary breast cancer [38]. Further work is required and include additional analyses of these genes known to be involved in breast cancer. Also, some studies reported the whole spectrum of hereditary breast cancer is not summarized by only BRCA1 and BRCA2 mutations [39, 40, 41, 42, 43]. A study in a Japanese population free of BRCA mutation revealed some large deletions in RAD51C [43]. So RAD51C, RAD51D or even PALB2 could be explored. Esteller et al., have found a high rate of loss of heterozygosity at the BRCA1 locus that accounts for the vast majority of BRCA1 biallelic inactivation, in most tumors from BRCA1 families [41]. Therefore, another perspective could focus on epigenetics in tumors.

Conclusions

This study describe some variants found in BRCA1 and BRCA2 genes, in young women with nodules and suspected breast cancer in Burkina Faso. Breast cancer in Burkina Faso can have a genetic basis because some of variants that we identified may be predisposing, but there is need to have a large number of specimen and more specific genetic testing to confirm if this can be considered as conferring an increased risk for breast cancer in this West African population.