CYTOCHROME B AND BETA-FIBRINOGEN IMPLICATION IN THE GENETIC EVOLUTION OF BREAST MALIGNANT TUMOUR IN SENEGALESE WOMEN

Ahmed Mohamed Mze 1 , Fatimata Mbaye 1 , Binta Keneme 1 and Mbacke Sembene 1,2 . 1. Departement de Biologie Animale, Faculte des Sciences et Techniques, Université C.A. Diop, B.P. 5005 Dakar, Senegal. 2. Centre de Biologie des Populations AnimalesSahéloSoudaniennes (BIOPASS), UMR 022, Institut de Recherche pour le développement, IRD/Bel-Air, Senegal. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The International Cancer Centre (CIRC) stated that there is an important increase of breast cancer throughout the world. In Senegal, breast cancer is known as the malicious tumour more developed among women. From thirty (30) Senegalese women were tested, the notice is that a mitochondrial (Cytochrome b) and a nuclear (Beta-fibrinogen) gene evaluated the level of genetic diversity, genetic differentiation and demographic evolution. Our results showed a real diversity of Cytochrome b compared to Beta-fibrinogen. A significant difference has been found between normal tissues and cancerous ones within Cytochrome b. these results showed an implication of mitochondrial DNA compared to nuclear DNA.

…………………………………………………………………………………………………….... Introduction:-
Cancer is a sickness characterized by the proliferation of an abnormal cell within a normal tissue of a body. These cells come from all from a same clone, the initiator cell of cancer that has acquired some characteristics that permit it to divide itself and to be able to form metastases (Bertram, 2001). There are two types of tumour: benign tumour and malignant ones. In the first case, the tumour is drowned into conjunctive tissue. This encapsulation permits the considerable slowdown of neoplasm's growth, making them less dangerous. Beauty spots and warts are examples of this type of tumour easily eliminable by surgical intervention. In the second case, malignant tumours include no capsulated neoplasm. Tumour mass can, then, increase limitlessly what facilitates its infiltration into tissues as well as its invasion by other organs. Contrary to benign tumours, malignant tumours can kill (Voet, 2002). The International Research Cancer Centre (CIRC) valued the new cases of cancer to 14, 1 thousand (CIRC, 2013). It notices an increase of breast cancer throughout the world and with 1,7 thousand of women diagnosed every year. These last years, incidence and mortality have increased respectively of 20% and 14% in the world (OMS, 2013). Breast cancer is the most frequent cause of women death by cancer and the most frequent cancer diagnosed among women in the world. It still be the most serious public health problem, particularly among women of less under 35 year's old where it is aggressive (Axelrod et al., 2008). In Senegal, breast cancer is the second from all the feminine cancers (Dem et al., 2008). Only 5 to 10% of cancers are hereditary ones (Claus et Risch, 1991) attributable, in majority, to BRCA1 and BRCA2 (DumitrescuetCotarla, 2005). Mitochondria have been suspected for a long time as playing an important role in the development and the progression of cancers. Many associated mitochondrial alteration have been identified and described in literature. These alterations include modification of mitochondrial genes, structural or quantitative mitochondrial abnormalities, or abnormalities of enzymatic components of Cyt b and FGB genetic diversity:-In order to study the genetic diversity of cancerous tissues in the level of each gene, we determined the number of variable and invariable sites, the number of informative sites, the total number of mutations, the number of haplotypes, the average number of various nucleotides, haplotypical (h) and nucleotide (π) diversityusing to DnaSP 5.10 (LibradoetRozas, 2009) software. Nucleotide frequencies, the nature of mutations (% of transitions and tranversions) and molecular distances with Kimura 2 parameter (K2P) model were executed in MEGA version 6.00 (Tamura et al., 2013). Nucleotide frequencies and molecular distances were only calculated for Cyt b which is a coding gene in various positions of the codon.

Study of Cyt b amino acids variability:-
Cyt b nucleotides sequences are transformed into amino acids sequences using to MEGA software version 6.06 5 (Tamura et al., 2013)with the best reading frame. The frequencies of amino acids were stand out for normal and cancerous tissue. Chi2 test was realized to see the amino acids that presented significant differences.
Cyt b and FGB genetic structure:-Genetic distances between sane and cancerous tissues at intra and inter individual level for the two genes were explained by the genetic distance of Nei (Nei,1978) using MEGAsoftware version 6.06 version software (Tamura et al., 2013).

Signature selection test:-
We have made demo-genetic tests that compare the level of adjustment between the two genes diversity and theoretical expected values under the hypothesis of the evolution under a neutralist model (to mutation-derive 505 balance). We have, among these tests Tajima D (Tajima, 1989), Fs of Fu (Fu, 1997) and R2 of Ramos (Ramos, 2002). These three tests were realized on FGB. These various estimators are obtained usingDnaSPversion 5.10 (LibradoetRozas, 2009) and Arlequin version 3.5.1.3 (Excoffieret al., 2010). As for Cyt b, the existence of any selection was apprehended by report dN/dS thanks to MEGA 6 softwarewith using Kimura model. dN is the substitution rate not synonymous and dS the substitution rate synonymous. The level of significance was held to 5% and a bootstrap value of 1000 replications. The distribution disparityanalysis (Mismatch Distribution) that is the graphic representation of distances genetic distribution existing between individuals was also determined. Mismatch analysis is accompanied by two indices that test the quality of distribution adjustment. These indices are SSD (sqarred sums of deviation) and Rag (irregularity indices). The graphs are built with DnaSPsoftware version 5.

Cyt b Amino acids variability:-
We remark that the frequency of Cyt b amino acid is lightly difference between sane tissues and cancerous ones without any significant statistic (table 2). However, we observe a significant value on glutamine. 506

Distribution disparity analysis (Mismatch distribution):-
Distribution disparity of base pairs for both genes shows the expected and observed frequencies (full of dotted lines respectively) differences by pairs between samples. Results testify multimodal distributions for both genes (figure 1).

Discussion:-
In this study, Cyt b which is a mitochondrial gene and FGB which is a nuclear gene were analyzed among 30 Senegalese patients suffering from cancer breast. We proceeded to the study of genetic variability, of genetic differentiation and genetic evolution in order to compare cancerous tissues sequences of Cytb and FGB for the purpose of determining the implication of both genes in the mammary carcinogenesis.
Our results revealed a strong variability of Cytbcompared to FGB. This strong variability of Cyt b is in mutual agreement with the works by Mbayeet al., (2014). Several types of explanations can be considered.
It can be mentioned: a sparsely regular replication (Kunkel & Loeb, 1981), mitochondrial polymerase would be less regular than that of the core, a deficiency or an absence of systems of correction and reparation and apparent absence of recombination as well as a rate of renewal and so, of more important replication than that of nuclear DNA (Brown et al., 1982).

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Cyt b number of variability (217) and FGB (11) as well as that the substitutions of R, Cyt b (1.453), FGB (0.637) lead to nucleotide variability rate of DNAmt. That rate of substitutions was noticed by (Tan et al., 2002) while examining the presence of DNAmt mutation in breast cancer, of which 58% were the substitutions of gene Cyt b and the no coding area (D-Loop).Among mammal it was accurately estimated, that rate is higher than that described for only nuclear sequence and of a multiplicative factor of 5 or 10 (Brown et al.,1982). That rate is variable following genome area considered but remains globally higher.
DNAmt general characteristics of mutations are transitions C-T and A-G (Beckman & Ames, 1997) that are in correlation with our results with 60% of substitutions that are transitions among Cyt b. They declared that the main reason of DNAmt mutations in tumour is the high level of ROS. On contrast, among FGBtransversions types mutations (58.9%) are high with respect to that of transitions (41.06%). These transversiontypes substitutions wouldn't have not effect since they happened in an intronic area of FGB.
Our results reveal that each amino acid frequency deriving from cytochrome b sequences are weakly differentiated between sane tissues and cancerous ones without a statistic signification except glutamine. Most of cancers like breast cancer depend on a high rate of aerobic glycolysis for their growth and survival. Paradoxically, some cells lineages of cancer present also the dependence to glutamine in spite of the fact that glutamine is a no essential amino acid that can be synthesized from glucose. The high absorption rate of glutamine by exposed cells depending on glutamine seemed not coming only from its role as nitrogen donor in nucleotides and amino acids of biosynthesis. Instead of that, glutamine plays a role in the required absorption of amino acid essential in the maintaining and the activation of the TOR kinase. Furthermore, in many cancerous cells, glutamine is the primary mitochondrial substrate as well as the support to maintain the potential of the mitochondrial membrane and integrity as well as the support of NADPH production necessary for oxydoreduction control and macromolecular synthesis (Levine etPuzio-Kuter, 2010).
In this present work, we have made a comparative study of genetic distance intra and inter sane and cancerous tissues of both genes. As for Cyt b, inside cancerous tissues the value of genetic distance (d= 0.112) is higher than that of sane tissues (d= 0.093). This can be explained by the fact that, cancerous cells are no longer under the control of cellular division regular mechanisms. One of the characteristics of cancer is the rapid proliferation of abnormal cells (OMS, 2012). And as for FGB, values don't nearly present a difference inside sane tissues (0.006) and inside cancerous tissues (0.000). Thisis explained by the aspect of nuclear DNA gene which has a less important replication than that of DNA mt (Brown et al., 1982). On contrat, the weak distance of FGB (0.003), confirms the characteristic of nuclear DNA which has a mutation rate less important than that of DNAmt. Methionine is under positive selection for Cyt b gene (dS = 1.618; dN = 8.561. dN -dS = 6.943 with a value of p= 0.048). In other words, it is advantageous to tumour evolution. Contrary to sane cells, most of tumour cells need a exogenous contribution of Methionine, an essential amino acid (Durandoet al., 2008). Molecular mechanisms that help explain their dependence to Methionine are numerous. In vivo, various approaches were realized in order to lack in Methionine. As the main source of Methionine is food, synthetic diet plan lacked in Met have been widely used. Other alternatives were to use metabolism inhibitors of Methionine or anenzymaticdegradation thanks to Methioninase. Among animal, deficiency in Methionine permit to limit tumour growth and reduce the height of some tumours. However, some studies also showed a limited effect over time with an upturn of the tumour growth after the interruption of the deficiency. These different modifications suggested the use of a deficiency in Methionine in tumour cells in association with conventional chemotherapy. Many pre-clinical studies showed a synergic effect of a deficiency association in Met and various cytotoxic agents. Currently, little clinical investigations were realized in order to explore this therapeutic strategy.
The observed number of differences between haplotypes doubly taken produced a multimodal distribution. The amount of variances squared SSD, Cyt b (0.00336; p ˃ 0.9), FGB (0.02400; p ˃ 0.1) are positive and aren't significant for both gene. As for FGB, respective values: Fs of Fu (-6.917; P = 0) and R2 (0.162; P = 0) of Ramos are significantly negative and positive. However, Tajima D (-0.56656; P = ˃ 0.3) is not significantly negative. These positive and negative values of neutrality tests are due to bare mutations. These results suggest a constant height of cancerous cells population for both genes, as show multimodal distribution (Ramos-OnsisnsetRozas, 2002).

Conclusion:-
Cyt b implication in cancers, in particular in breast malignant tumour, is no more demonstrated and is more and more studied. However, FGB is less implicated in breast malignant tumour than Cyt b. As the role of mitochondria 509 is known in the apoptosis, the presence of these mutations could contribute to alter cellular response to anticancerous agents. A significant difference of glutamine frequency between normal tissues and cancerous tissues was obtained. These results from Senegalese women suffering from breast cancer should be confirmed in quantifying the level of twenty amino acids from serum or plasma of these patients unpaired to subject of control. Cancerous cells need glucoses for their development and glutamine is a no essential amino acid that can be synthesised from glucose. As glucose comes from carbohydrates, to eliminate carbohydrates, replaced by proteins and sane greases, would be less ananticancerous treatment without medicines. Methionine is necessary in carcinogenic processes. So, their inhibition is a means of reducing the growth of cancerous tumour.