STUDY OF NUCLEOTIDE VARIABILITY WITH THE MITOCHONDRIAL COX I GENE FROM CANCEROUS TISSUES IN SENEGALESE PATIENTS WITH CERVICAL CANCER

Background: Cervical cancer is the first gynecological cancer in Senegal with 1,195 new cases per year and an annual mortality of around 66% (LISCA, 2019). Mitochondrial involvement in the process of apoptosis and tumorigenesis has been analyzed previously from different cancer, and from analyses, 21 sites polymorphisms of the Cox genes (including CoxI, CoxII and CoxIII) contributed to dysfunction of mitochondrial respiratory function and have been associated with sensitivity to cancers such as prostate cancer(Green, 1998)(Cavali and Liang, 1998). These data stimulated interest in examining the potential role of mtDNA mutation of host inprogression and maintenance to cancer stades.The aim of thisstudy is to analyse the polymorphism of COXI gene from biopsies of cervical cancer in Senegalese subjects. Methods: In this study, polymorphisms of mitochondrial Cox1 gene were highlighted in 65 patients with cervical cancer women admitted to Aristide Hospital Le Dantec-Julio Discussion and Conclusion: Genetic events leading to transformation from a normal cell to a cancer phenotypeappear to be multiple. Human papilloma viruses (HPV) are probably involved in the initiation of cancer and perhaps in the maintenance of the malignant state. However, for the sake of controlling this cancer, it is necessary to identify different variants within the host cell (the woman) which could be responsible for maintaining the malignant state and the occurrence of this cancer. Our study reveals a high variability of Cox1 gene, and could be useful for an establishment of a sensitive and rapid genetic screening test. were obtained through DnaSP version 5.10.01 (Rozas et al., 2010), Arlequin Version 3.1 (Excoffier et al., 2005)and MEGA 7.0.26 (Kumar et al., 2016) softwares. The evolution of the mutations will be done by neutrality tests which will be carried out in order to know the nature of the selection. The different parameters to be highlighted are the D of Tajima (Tajima, 1989), the D and F of Fu and Li (Fu, 1997) as well as the H of Fay and Wu are used to test the deviation from the hypothesis neutrality using DnaSP version 5.10.01 software (Rozas et al., 2010) and Arlequin software version 3.5.1.3 (Excoffier and Lischer, 2010). Values of P less than 0.05 are considered significant at a 5% confidence interval. malignant state and the occurrence of this cancer for the establishment a sensitive and rapid genetic screening test. Our results showed a high variability of the Cox1 gene.


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Discussion and Conclusion: Genetic events leading to transformation from a normal cell to a cancer phenotypeappear to be multiple. Human papilloma viruses (HPV) are probably involved in the initiation of cancer and perhaps in the maintenance of the malignant state. However, for the sake of controlling this cancer, it is necessary to identify different variants within the host cell (the woman) which could be responsible for maintaining the malignant state and the occurrence of this cancer. Our study reveals a high variability of Cox1 gene, and could be useful for an establishment of a sensitive and rapid genetic screening test. Until recently, research has mainly focused on alterations in nuclear DNA in various cancers. However, alterations in mtDNA in tumors have received much less attention, despite the fact that the mutation rate of mtDNA is at least 10 times higher than nuclear DNA (Wallace, 1994). It is not surprising that mtDNA is the preferred target of chemical carcinogens and that the damage caused by these compounds is persistent.
MtDNA replicates independently of nuclear DNA, throughout cell life. It is likely that single-stranded DNA is particularly sensitive to the effects of carcinogens.
MtDNA is combined with few proteins, while nuclear DNA is protected to some extent by chromatin proteins. DNA repair systems in the mitochondria are ineffective, so that damage caused by chemical agents can persist during successive replications as long as the damaged molecules have retained their replication capacity. Mitochondrial involvement in apoptosis (Green, 1998), and also in tumorigenesisprocessus (Cavali and Liang, 1998), stimulated interest in examining the potential role of mtDNA mutation in development and the maintenance of cancers.
The complete Human mtDNA is16.569bpin size, and includes 37 genes, whose the structural genes for 13 of the protein subunits of the oxidative phosphorylation system, the 12S and 16S rRNA genes and 22 tRNAs (Anderson et al., 1981). MtDNA has a high number of copies (103 to 104) per cell (Anderson et al., 1981).
Recently, somatic mutations of mtDNA have been reported in several of the female cancers, including ovarian and breast cancer. These mutations may play a role in cancer formation by increasing the production of reactive oxygen species (ROS) during mitochondrial oxidative phosphorylation. The resulting ROS are mitogenic and therefore have functional relevance in the proliferation of cancer (Diwanjiand Bergmann,2017). Mitochondrial mutations can bring about to increased production of ROS, which in turn can lead to tumorigenesis and increased tumor growth. In addition, an increase in intracellular ROS caused by mtDNA mutations can lead to tumor metastasis in vivo (Ishikawa et al., 2008). Consequently, human populations, from discrete maternal lines, harbor unique sets of mononucleotide polymorphisms (SNPs) of mtDNA which define a specific genetic history called haplogroups (Torroni et al., 1996).
SNPs in protein coding regions are common in the human genome and lead missense or silent mutations (Ng and Henikoff, 2006).
The COX genes encode three subunits of the respiratory complex IV, a key enzyme as the third and last enzyme in the electron transport chain of mitochondrial oxidative phosphorylation in aerobic metabolism. 21 sites polymorphisms of the COX genes (including COXI, COXII and COXIII) have contributed to dysfunction of mitochondrial respiratory function and have been associated with sensitivity to prostate cancer.
In this studywe investigate and analysed the polymorphism of mitochondrial COXI gene. Hence we hypothesized that CoxI mutations influence the risk of progression of the cervix cancer or are simply transient.Then objectives are to screen the mutations of cox 1 sequences in cervical cancer patients and to analyze the impact of thesese mutations on tumor process.

Materials and Methods:-Ethical Clearence:
Objectives and benefits of study were explained clearly to subjects using local dialect before inclusion. And written informed consent was obtained from adult or legal representatives of participants. Our protocol has been reviewed according to the rules issued by the National Committee for 188 Ethics for Health Research (

Cervical Cancer Cohort study (Tableau 1):
The study group consisted of black Senegalesewomenbornindividualswhose parents and grandparentswereborn in Senegal. Cervix Cancer patients wereenrolledfromparticipating and correspondingsubjectshospitalized in Juliot Curie Centre atHospital Aristide LeDantec. In brief, a total of 64 women with cervical cancer were sampled. Age, ethnicity and marital status of each patient were recorded for each patient, a sample of cancerous tissue (biopsy) is taken during surgery from Joliot Curie Institute team at Aristide le Dantec hospital. They are immediately sent to the Laboratory at the Center for Immunophysiopathology and Infectious Diseases of the Institut Pasteur in Dakar. The samples are stored in the refrigerator (80°C) in Nunc alcohol-free tubes.

DNA extraction, PCR and Sequencing:
The total DNA of the tissues was extracted using the Standard Qiagen method (Kit QiagenDneasy Tissue), and subsequently used as template for polymerase chain reaction (PCR). Themitochondrial cytochrome c oxidase subunit 1 gene (cox1) was amplified with primers: F (CATTTTGCTGCCGGTCARCAYATGTTYTGRTTTTTTGG) and R (CCTTTGTCGATACTGCCAAARTAATGCATDGGRAA) and previously designed used reference cox gene (Ref….) PCR was carried out in a total volume of 25 μl reaction containing 5 μl of template DNA, 12 μl of Master Mix, 4.5 μl of water and 1.5 μl of each primer. PCR amplification was performed as follows: 95 °C for 5 min, 35 cycles: 94 °C for 30 s, 58 °C for 30 s and 72 °C for 1 min 30s, followed by 10min at 72°C. The sequencing was carried out,determining the nucleotide sequence of a DNA fragment. Point mutations such as SNP, insertions and deletions, were highlighted by comparing the cox 1 sequences of different individuals. The method used proposed by F. Sanger (1977), is based on the use of specific nucleotides called dideoxyribonucleotides which block the synthesis of DNA by DNA polymerases after their incorporation. In other words, it is a particular PCR reaction using, in addition to the usual compounds (template DNA, polymerase, primers, dNTP, Mg2 +), modified nucleotides: dideoxyribonucleotides (ddNTP). These ddNTPs have the particularity of being coupled to fluorochromes: ddATP-green, ddTTP-red, ddCTP-blue and ddGTP-yellow (in black on the electropherogram). This blockage is due to the impossibility of these nucleotides to form a phosphodiester bond with another nucleotide due to the absence of the hydroxyl group on carbon 3 '.

Genetic analyzes:
Genetic analysis was performed in 64 patients at all. In order to determine the polymorphisms of COX 1gene, the raw sequencing data were submitted to Genalys software (Takahashi et al.,2003). This program compare the raw sequences data with genomic DNA of a reference COX1 available in NCBI. The sequences obtained and chromatograms were thoroughly double checked, cleaned, and aligned to identify homologies among sites. Using BioEdit version 8.0.5 software (Hall, 1997) and ClustalW algorithm (Thompson et al., 1994). The 64 sequences werecarried out. At the end<, the Cox1 sequences were submitted to the MITOMAP database in order to double check thoroughly the mutations observed at first.The sequences were submitted to a fully genetic diversity analysis. And many parameters,such as nucleotide frequencies, nature and the rate of mutationswas carried out using the software MEGA 6 version 6.05 (Tamura, 2013). Other parameters such as the number of polymorphic sites, the haplotype diversity (Hd), the nucleotide diversity (Pi) vs. average number of nucleotide difference (K) (Nei, 1987) were obtained through DnaSP version 5. 10 ., 2016) softwares. The evolution of the mutations will be done by neutrality tests which will be carried out in order to know the nature of the selection. The different parameters to be highlighted are the D of Tajima (Tajima, 1989), the D and F of Fu and Li (Fu, 1997) as well as the H of Fay and Wu are used to test the deviation from the hypothesis neutrality using DnaSP version 5.10.01 software (Rozas et al., 2010) and Arlequin software version 3.5.1.3 (Excoffier and Lischer, 2010). Values of P less than 0.05 are considered significant at a 5% confidence interval.

Results:-
Socio-demographiccharacteristicsof study group: Table Isummarizes the demographiccharacteristics of 65 patients included in the study. In summary, the range of patient ageisfrom27 yearsto 82 years, with an averageage of 51.33years. The age group with the largestnumber of participants was the 45-55agebracketwith48.43%enrolled patients. Regarding marital status, 87.5% of the patients were on a polygamousregime and 12.5 on a monogamousregime.

Genetic diversity of Cox I gene:
The analysis of the genetic diversity of the study group is summarized in TableIII 58 transitions sites and 41 transversions sites of CoxI were recored. The ratio transitions / transversions was estimated at 1.38. In this gene, it appears that the haplotypic diversity is higher (Hd = 0.9931 +/-0.048) than that of the nucleotide (Pi) which has a value equal to 0.09227 +/-0.045. The average number of nucleotide differences (k) is 29,236 (

Intra-specific neutrality tests for cancer tissues:
The statistical values (Table 4) of Tajima's D are significantly negative for the CoxI (D = -1.99104 P <0.05). Whereas D * and F * indices are not significant. Analysis of the mismatch distribution curves (Fig 1) of cancerous tissues under the assumption of an expanding population gives a multimodal appearance.

Discussion:-
The relationships between variation in mitochondrial DNA and oncogenesis have been demonstrated in many types of tumors (Jeronimo et al., 2001 andCarew, 2002). A nucleotide variability analysis of cancer tissues was performed in Senegalese patients with cervical cancer in cytochrome oxidase I (Cox I). We used the population genetics approach applied to DNA samples taken from tumor tissue. The tissue is considered a population, exhibiting genetic diversity when cancer is present. The majority of these tissues analyzed Our study population showed high haplotypic diversity (Hd = 0.9931+ / -0.048) and low nucleotide diversity (Pi = 0.09227 + / -0.045). This suggests that cells in the cancerous tissues of the cervix undergo rapid growth from an original, small clone. These results follow the Darwinian mode of development of cancer as it was explicitly formulated by Nawell in (1976). According to this etiological model, a neoplasia originates from a single cell which is the target of mutations which free it from physiological mechanisms limiting its proliferation. Thus, the succession of mutations conferring a selective advantage followed by periods of clonal expansion, leads to the formation of a malignant tumor (Nowell, 1976). So this result might suggest that these host mutations may have an impact on carcinogenesis of the cervix in Senegal because in the majority of cases, cancer of the cervix is due to persistent infection with a virus: the human papillomavirus (HPV). The long-term presence of this virus can sometimes affect cells in the lining of the cervix and initiate a multi-step process that can lead to the development of cancer. So these mutations can participate in this process.
Allelic frequency deviations were tested under neutrality for Cox I. The indices of D of Tajima, of D * and F * of Fu and Li are negative, but only the D of Tajima is Significant. This may suggest an excess of rare alleles in cancer tissue due to a sudden expansion of the mutations. This shows that the variations noted at the level of the polymorphic sites and at the level of the singleton sites are fixed under the influence of environmental effects of the random genetic drift. Indeed, Chinnery et al. (2002) showed that the random genetic drift was powerful enough to explain the fixation of rare mtDNA mutations in tumor tissue. This expansion was confirmed by the mismatch distribution curves.