Genetic Diversity and Structuring of the BRAF gene in Breast Tumors

The International Agency for Research on Cancer (IARC) estimates an increase in breast cancer worldwide. In Senegal, the most common malignant tumour in women is breast cancer. But also in Senegal as in most African countries, benign tumours occupy an important place in mammary pathologies. To better understand the impact of nucleotide variability and genetic instability of benign and malignant breast tumours, we used the BRAF gene, which is a nuclear gene. This work allowed us to compare the polymorphism, diversity, structure and genetic evolution of exon 15 of the BRAF gene between patients with benign tumours, malignant breast tumours and control subjects. The analysis at the identification of mutation of exon 15 of the BRAF gene led us to conclude that BRAF was mutated in (1.5%) of cases. We observed a synonymous mutation A598A in malignant tumours. Our results showed that somatic mutations of this BRAF gene were common in Senegalese patients with both benign and malignant breast tumours. So our results allowed us to conclude that the BRAF gene is involved in breast tumours.


Introduction
BRAF is a proto-oncogene involved in the KRAS MAP-kinase intracellular signaling pathway. The activation of membrane receptors by binding with their specific ligands induces a cascade activation of this pathway and the stimulation of several cellular functions. This occurs in many types of cells.
BRAF is normally activated by RAS proteins. It can also be activated by mutations called "function gain" or "activators"; the proto-oncogene then becomes an oncogene. Activating mutations of the BRAF gene were first detected in human tumors about eighteen years ago [1], and their frequency varies greatly according to tumour type. Mutations generally appear during the early phases of oncogenesis. BRAF mutations are mainly located in the exon 15 activator segment, i.e. acquired, somatic and non-germinal mutations [2]. The absence of germline mutation can be explained by molecular genetic experiments in mice: BRAF mutations induce embryonic lethality [2]. The most frequent mutation is a localized punctually mutation at exon 15 (thymine [T] 1799 has been transformed into adenine [A] -gTg/ gAg) substituting a valine (V) into glutamic acid at position 600 of the protein (V600E); this mutation is found in the vast majority of cancers with a mutated BRAF form [1]. The mechanisms of acquisition of the V600E mutation in the protein are probably linked to alternative mechanisms that have not yet been identified. The mutated protein BRAF V600E has 500 times more kinase activity than the wild form of BRAF, which stimulates ERK protein phosphorylation and cell signaling in a disproportionate way [1]. Apart from the V600E mutation, other somatic mutations of BRAF have been described in human melanomas: In mutated melanomas for BRAF, 74 to 90% are V600E and 16 to 29% are V600K. Depending on the studies, the proportions of one type of mutation compared to another are slightly different. BRAF's amino acid V600 is located in the kinase activation domain, near the threonine 599 and serine 602 residues on which phosphorylation induces kinase activity. The V600E mutation could thus simulate the phosphorylation of threonine 599 and serine 602. Another hypothesis of the uncontrolled activation mechanism is the increase in exposure of the activation segment when a small hydrophobic amino acid (valine) is replaced by a hydrophilic residue (glutamic acid), [3]. The BRAF gene is mutated in the majority of patients with melanoma and a minority of patients with breast, colon and lung cancer [4]. In this study, the hypothesis of the existence of nucleotide mutations involved in breast tumors in Senegalese women has been put forward. For this purpose, exon 15 of the BRAF gene, which is a nuclear gene, was chosen to test this hypothesis.

Methodology Patients and samples
The study involves sixty-six (66) surgical samples composed of malignant and benign tumors and twelve (12) blood samples from patients who are managed at the Aristide Le Dantec Hospital Cancer Institute. Samples were collected from these patients after informed and written consent in a standardized form.

Molecular analyses
The exon 15 sequences of the BRAF gene, from the three groups (malignant, benign and control), are carefully verified, corrected and aligned with Bio Edit software version 7.0.8 [5]. Alignment is indeed an important step in data analysis. It In addition to the universality of the DNA molecule in the living world, there is its variability. This variability results in random changes in the DNA sequence (mutations) that can affect the cellular activity and the entire body. Therefore, to estimate the genetic diversity of the BRAF gene, we determined the number of variable and invariable sites, the number of informative sites, the total number of mutations, the number of haplotypes (h), the average number of nucleotide differences (k), the haplotypic (HD) and nucleotide (π) diversity, using the DnaSP software version 5.10 [6]. Nucleotide frequencies, nature of mutations (% transitions and transversions) and molecular distances with the Kimura 2 Parameter (K2P) model were calculated with the MEGA program version 6.06 [7]. Nucleotide frequencies and molecular distances were also calculated at each codon position.
The nucleotide sequences of the BRAF gene are transformed into amino acid sequences using MEGA software version 6.06 [7], using the best reading frame. The level of significance of amino acid frequency variations between the three groups (controls, benign tumors, and malignant tumors) was demonstrated by the chi2 test with a level of significance (P-value) of0.05.
Genetic distances between controls vs TB, controls vs TM and TB vs TM at the intra-and inter-individual level were explained by Nei's genetic distance using the MEGA software version 6.06 [7].
We conducted demogenetic tests that compare the level of adjustment between diversity to the three groups and expected theoretical values. Among these tests: the D of Tajima [8], the D*and F* of Fu and Li [9] and the H of Fay and Wu [10] and the R2 of Ramos [11]. These different estimators are obtained with the DnaSP version 5.10 programs [6] and Harlequin version 3.5.1.3 [12]. By choosing as a starting hypothesis that exon 15 of the BRAF gene is under positive selection, the existence of any selection has been apprehended by a positivity (dN>dS) thanks to the MEGA 6 software with dN is the non-synonymous substitution rate and dS is the synonymous substitution rate. This test was performed using the Nei-Gojobori model and the pairwise deletion method. A value of P < 0.05 was considered significant with a bootstrap value of 10000 replications. After the demogenetic tests, we determined the analysis of the distribution disparity (Mismatch distribution), which is the graphical representation of the distribution of genetic distances existing between individuals. Mismatch's analysis is accompanied by two indices that test the quality of adjustment of the distribution. These indices are the SSD (sum of squares of deviations) and the Rag (irregularity index). The graphs are built with DnaSP software version 5.10 [6]. The SSD and Rag indices were obtained with Harlequin software version 3.5.1.3 [12].

Results
A total of 86 sequences (12 for controls, 31 for benign tumors and 35 for malignant tumors) were sequenced, aligned and analyzed.

Nature and frequency of mutations
In benign tumors, we find the mutations L588H and  Table 1.
We also observe a synonymous mutation A598A, in malignant tumors.

Genetic diversity
The analysis in Table 2 shows that the BRAF gene is more diverse in terms of malignant tumors and TB than in controls. However, we note a great diversity among TMs. This increased diversity in malignant tumors is reflected in a high number of variable sites (20) compared to TB (17) and controls (8). We also note a higher total number of mutations (26) for TMs compared to TB (22) and controls (10). The average number of nucleotide differences (k) is higher at the TM levels (3.615) compared to TB (3.009) and controls (2.045).We find that the percentages of transversions are higher than those of transitions in the three groups. However, we note that in the percentages of transversions they are higher in controls and benign tumors respectively (73.42%) and (78.82%) compared to the TM (53.16%).
In the nucleotide frequency diagram, we see that bases A and T are the most dominant with respectively 27% and 35% for all three groups compared to bases C and G respectively 17.5% and 20%. And we also notice a predominance of (A +T) in all three groups with a percentage of 62% compared to (C+G) 38%.

Variability of BRAF amino acids
The analysis of inter-tissue protein diversity (Table 3) by highlighting amino acid variations indicates that, despite slight variations in amino acid frequencies between the three groups, no significant difference could be determined.

Genetic differentiation
The genetic distance values (d) at the intra and inter tissue level and the degree of genetic differentiation (Fst) between controls and benign tumors and between controls and malignant tumors as well as benign and malignant tumors are recorded in Table 4. The analysis of genetic distances between controls and malignant tumors and benign and malignant tumors revealed a low respective genetic diversity (d=0.0159), (0.0186), but great-er than that between controls and benign tumors (d=0.0139).
We note that within malignant and benign tumors the values of the genetic distance are equal (d=0.02), higher than those of the controls (d=0.01). We also find that the degree of genetic differentiation shows an Fst that is equal to 0 and not signifi-

Mismatch distribution analysis
The disparity of distribution (Mismatch distribution), base pairs for exon 15 of the BRAF gene between the three groups, shows the expected and observed frequencies (solid and dotted line respectively) of the differences per pair between the samples (Figure 3). Under the assumption of a constant population and an expanding population, we have a unimodal distribution for controls. However, the distribution is multimodal for benign and malignant tumors.     It has been shown by [14] that BRAF activating mutations are common in some benign tumors, such as scalloped colonic polyps, where their frequency reaches 51%, and melanocytic nevi.
We observe a synonymous mutation A598A in malignant tumors. These results are different from the substitution of BRAF V600E resulting in the substitution of glutamic acid by valine, which accounts for 80% of mutations and is thought to be involved in 66% of malignant melanomas [3], and also involved in several other cancers [15].  between controls and benign tumors, controls and malignant tumors as well as between benign and malignant tumors. We also found that within malignant and benign tumors, the genetic distance values are equal (d=0.02), higher than those of the controls (d=0.01). This shows that cancer cells have a different property from healthy cells [19], and it may also explain that during carcinogenesis, cancer-related changes the internal structures of the cells but also their environment. Cancer cells have been shown to be less rigid than normal cells due to a reorganization of the cytoskeleton [20]. Breast cancer cells deform more than non-cancerous cells [21].
The analysis of inter-tissue protein diversity by highlighting amino acid variations indicates that, despite slight variations in amino acid frequencies between the three groups, no significant difference could be determined. This can be explained by the aspect of the nuclear DNA gene that has less replication than the mtDNA gene.

Conclusion
Genetic mutations and molecular pathway activation play a vital role in tumour formation. The analysis at the identification of mutation of exon 15 of the BRAF gene led us to conclude that BRAF was mutated