| Background:Breast cancer（BC）,among many other cancers,represents the major cause of death in women and one of the most diverse and complex diseases.The number of people carrying BC increased significantly during the past few years.According to the Global Cancer Statistics 2020,BC was the most commonly diagnosed cancer with2,261,419 new cases（11.7%of the total cancer cases）in both sexes combined and the primary cause of cancer-related mortality among women（684,000 deaths）.Its incidence was estimated to be around 8.3%in 2020in Africa and is projected to grow over the years.In Burkina Faso,BC is the primary cause of cancer-related mortality in both sexes combined and among women,thus becoming a public health issue.Recent advances in genetic screening techniques have made it possible to diagnose breast cancer at an early stage,which has greatly increased the likelihood of survival.Yet,the absence of information on the genes involved in BC in certain populations makes genetic testing difficult to perform.A family history of BC is a major risk factor for the development of the disease.Mutations in tumor suppressor genes BRCA1 and BRCA2account for the vast majority of cases of hereditary BC syndrome.Indeed,women with a BRCA mutation have a 40-85%chance of developing BC in their lifetime;in addition,5-10%of breast cancer cases in women are caused by a germline mutation in one of these genes.Thus,genetic screening for the identification of these mutations has been shown to hold important clinical significance.The BRCA1/2 gene mutations are used for cancer risk evaluation and prevention approaches and can help in making treatment decisions.The mutation spectrum and prevalence of BRCA1 and BRCA2 vary between populations;hence,adequate knowledge must be accessible in a particular population before establishing effective procedures for genetic testing.However,data regarding these genes involved in hereditary breast cancer are still unknown in the Burkina Faso population.In addition,only a few works were conducted in Sub-Saharan African populations,and only one of those studies included unselected BC patients as well as both the BRCA1/2 genes.In previous research,it has been established that cancer is caused by mutations in the DNA of cancer cells.Recent genome analyses have revealed that TP53 and PIK3CA,along with PTEN and AKT1,are the most commonly mutated genes in primary BC.In the COSMIC database,somatic variants in PIK3CA,TP53,PTEN,and AKT1 were found at a high frequency in breast cancer,with PIK3CA 26.4%,TP53 24.7%,PTEN 3.8%,and AKT1 2.8%.They have significant clinical implications for cancer prevention,classification,and treatment because of the subgroup specificity of their mutation spectrum.Therefore,the study of these mutations in different populations will aid in the understanding of breast cancer pathogenesis and the development of better preventive,screening,and treatment options.The high mortality rate of BC,especially in developing countries is mainly due to late diagnosis.Most cancer cases are detected at a late stage or after the tumor has already formed.However,genetic indicators can be recognized in the earliest stages of cancer,even before the cancer cell is formed.Consequently,nucleic acid analysis can be used to enhance cancer screening and early diagnosis.Identification of altered genetic material,particularly single-nucleotide mutations,in oncogenes is crucial for cancer outcome prediction and targeted treatment.DNA sequencing,SSCP,PCR-RFLP,ARMS-PCR,Gene Chip,and real-time PCR have been widely utilized for the discrimination of mutant DNA.However,these methods have a number of drawbacks,such as the ease of sample contamination,the difficulty of sample handling,and especially the high cost of the equipment,which make them inaccessible for most people in resource-poor setting countries.Due to their high sensitivity,selectivity,ease of use,and low cost,numerous biosensing platforms have been designed and developed for the detection of biological molecules.Especially,the colorimetric method has garnered a lot of interest because of its practicality,affordability,rapidity,and ability to produce accurate results consistently.In the field of clinical diagnostics,colorimetric techniques have seen widespread application,particularly in the detection of nucleic acids,protein biomarkers,infectious bacteria,viruses,and metal ions,among other things.It is a technique that involves evaluating the color that results from the interaction of smart material with the target biological component.The color shift is visible to the naked eye without needing an expensive signal readout instrument.Objectives:This work aimed to establish a comprehensive mutation profiling of BRCA1,BRCA2,TP53,PIK3CA,PTEN,and AKT1 genes in Burkina Faso BC patients.To identify and compare the recurrent pathogenic mutations found in our population with results in Chinese and other populations.To examine the possible connections between TP53,PIK3CA,PTEN,and AKT1 mutations and several clinicopathological and demographic variables.Finally,to propose an affordable colorimetric biosensing technology for the detection of the recurrent mutations observed to lessen the burden of the cost of genetic testing.Methods:The second chapter"BRCA1,BRCA2,TP53,PIK3CA,PTEN and AKT1 genes mutations in Burkina Faso breast cancer patients:prevalence and spectrum":Tackled the genetic aspect of BC in Burkina Faso population.Our investigation was conducted in Burkina Faso and China.The Pietro Annigoni Biomolecular Research Center（CERBA）and the Molecular Biology and Genetics Laboratory（LABIOGENE）of the University of Ouaga I Pr Joseph KI-ZERBO in Burkina Faso served as sample processing and storing place,while in China,the Laboratory of Biochemistry and molecular biology of the school of Life sciences in Central south university was used for molecular and cellular analysis.Between June 2019 and May 2020,samples were collected from 133women previously diagnosed histologically by a pathologist for having breast cancer.Blood samples were collected for germline variants detection in BRCA1/2 genes and tumor samples for somatic variants screening in TP53,PIK3CA,PTEN,and AKT1 genes.The patients were recruited from four different hospitals in Ouagadougou（Burkina Faso）.They were not chosen based on their family history or age upon diagnosis;they were all18 or older and signed a consent form to participate in the study.Clinical,paraclinical,demographic,lifestyle and other data were collected using a standardized questionnaire.By analyzing previous works conducted on Asian,African,American,and European populations,we identified the hotspot regions of BRCA1 and BRCA2 genes susceptible to carrying pathogenic variants.Around 90-100%of the mutations were around those regions.Ten sets of primers for BRCA1 and 13 sets for BRCA2 were constructed using the NCBI genome browser and primer3 software to cover the hotspot regions analyzed in this study,BRCA1（exon 2,7,11,12,20）and BRCA2（exon 9,10,11,17,23,24）.Afterwards,we designed 8primer pairs to cover the hotspot regions of TP53（exons 4,5,6,7,and 8）,PIK3CA（exons 9 and 20）,PTEN（exons 5 and 7）,and AKT1（exon 3）.PCR amplification followed by mutation screening using direct Sanger sequencing was then performed.The raw data obtained after sequencing were analyzed using Snap Gene 5.0.4（GSL Biotech LLC,USA）,and Multi Align software was used for sequence alignment.Variants annotations and significances were obtained from NCBI Clin Var,COSMIC,LOVD,BRCA Exchange,db SNP,UMD,and Exome Aggregation Consortium databases.All variants were classified according to the American College of Medical Genetics,and Genomics recommendations and the Human Genome Variation Society（HGVS）rules for gene variants nomenclature.Variants referred to as"Pathogenic"in NCBI Clin Var,or any other previously cited databases were also labeled pathogenic in our study.In-Silico analysis tools SIFT and Poly Phen-2 were used to predict the functional effects of new mutations and variants of uncertain clinical significance（VUS）.Mean and Standard Deviation were employed to quantify and interpret continuous data.The correlation between the categorical variables was calculated using contingency tables and Fisher’s exact test.Statistical significance was assumed at P values<0.05.SPSS23.0 was used for all statistical analyses（IBM,USA）.In genetic diseases,it has become common to identify novel variant by using sequencing technologies,but it is considerably more difficult to determine their pathogenicity.Although it is hard to verify that a particular gene mutation has a pathogenic effect,a probability of error in the assessment of pathogenicity can be determined.The evaluation of the potential pathogenicity of a novel variant or variant of uncertain significance necessitates the collection of specific data,including phenotype features,functional analyses,the number of reported patients,and the number of families harboring the same mutation.In our case,we hypothesized that the novel TP53（c.312G>T;p.Q104H）variant might disrupt p53 protein function（tumor suppressor role played by p53 through the regulation of apoptosis,angiogenesis,and genome stability）or expression due to its location（exon 4,region known to be encoding the DNA binding domain）and high frequency of recurrence（17 cases,or 12.78%in BC subjects in our study population）.Therefore,we proceeded to an intensive bioinformatic analysis using multiple in silico prediction tools（Mutation3D,SIFT,Poly Phen2,SNAP2,SNPs&Go and Ph D-SNP,Mut Pred2）to assess and analyze the deleterious effect,the functional significance,a possible disease association,the stability changes,and the structural conformation conservation associated to the novel TP53 variant.We also constructed a plasmid with the mutant TP53 sequence which was then transfected in MDA-MB-231 breast cancer cells to study the effect of the mutation in the growth and proliferation of the cells using the CCK-8assay.The third chapter"Novel colorimetric detection of single-nucleotide mutation using RCA and G-quadruplex-based DNAyme/TMB":We tried to propose an affordable colorimetric biosensing strategy for the detection of single nucleotide mutations.DNA oligonucleotides were custom-designed using NCBI genome browser and primer3 software and chemically synthesized and purified through HPLC by Sangon Biotech,Shanghai,China.The primers,padlock probes,and wild-type/mutant type DNAs used to detect the different point mutations were designed based on the results found in the previous chapter through Sanger sequencing BRCA1（c.3331C>T）,BRCA2（c.5635G>T）,PIK3CA（c.1634A>C）and TP53（c.312G>T）.Padlock probe DNAs that recognize corresponding single nucleotide mutation and forward PCR primers were modified with phosphate at the 5′-terminal end to facilitate the digestion by lambda exonuclease,allowing for the generation of ss DNA target and subsequent ligation into a circular RCA template.The same DNA samples used in the previous chapter collected from Burkina Faso breast cancer patients were used for this assay.The detection procedure was divided into four steps:a)PCR amplification of the target DNA,b)lambda-exonuclease digestion of the PCR product to generate single-stranded DNA,c)G-quadruplex-generating RCA through isothermal amplification,and d)DNAzyme complex formation by addition of hemin followed by colorimetric detection through H2O2-mediated oxidation of TMB catalyzed by the DNAzyme.Results:The second chapter:Among the 133 samples screened,one patient（0.75%）was found to have a pathogenic germline mutation in exon11 of BRCA1 gene（c.3331C>T）,and one patient（0.75%）was found to have a pathogenic germline mutation in exon 11 of BRCA2 gene（c.5635G>T）.None of the patients in whom the pathogenic mutations were found had a history of breast cancer in their families.In the hotspot areas screened for variants in this work,the overall prevalence of BRCA1 and BRCA2deleterious mutations was 1.5%（2/133）,which was lower than the prevalence rates found in other populations.There was a total of six VUS found in our study,4 in BRCA1（c.1137T>G,c.2368A>G,c.3600G>C,c.5232A>T）and 2 in BRCA2（c.1400A>G,c.3010A>G）.Eleven out of the133 subjects carried a VUS,one patient had 2 BRCA1 VUS and 3 VUS were recurrent.The overall prevalence rate of BRCA1 and BRCA2 VUS in the hotspot locations chosen for this study was 8.55%（6.3%BRCA1 and2.25%BRCA2）.All six VUS found in this work were missense mutations that had been reported by other studies.The in-silico analysis tool Poly Phen-2 was used to help clarify and predict the functional effect of the VUS.Two VUS in BRCA1（c.1137T>G,c.2368A>G）and one VUS in BRCA2（c.3010A>G）were therefore predicted to be probably or possibly damaging（prevalence of 3%）.Regarding TP53,PIK3CA,PTEN and AKT1,the results of the screening revealed that 52 subjects（39.09%;52/133）had PIK3CA mutations,17 subjects（12.78%;17/133）had TP53 mutations,and 0patients had PTEN or AKT1 mutations（0%;0/133）.8 individuals had mutations in both PIK3CA and TP53 genes,while 72 patients（54.14%,72/133）were non-carriers.Two missense variants（c.1634A>C and c.1658G>C）and 1 frameshift deletion mutation（c.1658 1659del GTins C）were found in the PIK3CA gene.A co-mutation c.1634A>C（;）1658＿1659del GTins C was detected in 35 patients with 26.31%（When the two variants are determined to be on the same allele,the co-mutation is linked to a disease known as Cowden Syndrome 5）.Specifically,All the variants were found in a single exon（exon 9）known to code for the helical domain（PIK helical domain）.Increased lipid kinase activity and carcinogenesis have both been linked to mutations in this region.In 17 subjects（12.78%,17/133）,a new missense variant（c.312G>T）not reported in any databases or previous research was found in the TP53gene.According to computational modeling in silico analysis,it was predicted to be"tolerated"by SIFT and"benign"by Poly Phen-2.In 8individuals,we found a TP53 co-mutation c.215C>G（;）312G>T.Exon 4,where the protein’s DNA-binding domain is encoded was the sole site for TP53 mutations.Overall,we found a PIK3CA mutation frequency of 39.09%in our study population.This frequency was significantly higher in the Burkina Faso population compared to other countries’populations（Singapore,France,Germany,Australia,China,India…）evaluated for the same gene and exons in BC patients.Interestingly,we discovered that the spectrum of PIK3CA pathogenic somatic variants in our population was different from what is typically found at higher frequencies in other populations.We identified the c.1634A>C（p.E545A）variant in 38.34%of the subjects;this variant is typically observed at a far lower frequency in any other population according to our literature research and is linked to a wide variety of malignancies.On the other hand,we did not find the most common PIK3CA variants（c.3140A>G,p.H1047R,and c.1633G>A,p.E545K）in our study population.In terms of the relationship between clinicopathological-demographic characteristics and mutation status,we found that PIK3CA mutation,TP53 mutation,and PIK3CA/TP53 co-mutation carriers were more common in Her2-negative BC and in subjects who had undergone chemotherapy and surgery,but the association was not statistically significant.We successfully overexpressed the wild-type TP53 and mutant TP53（c.312G>T;p.Q104H）and the CCK-8 assay showed no statistically significant difference in cell proliferation and morphology between the two.These results confirmed the in-silico analyses in which the novel variant was predicted to be benign and not harmful.Therefore,no further molecular analyses were performed.However,given the high recurrence of the variant,a more specific study with wider clinicopathological data involving the 17 patients and their close relatives must be conducted in the future to truly assess the role played by this variant in breast cancer pathogenicity.We can also speculate that the variant may be a result of a certain drug or treatment just like in the case of the TP53（c.215C>G,p.P72R）variants which have been reported as a benign mutation occurring due to a drug response according to multiple studies recorded in Clin Var database.The third chapter:The feasibility and selectivity of the method were demonstrated by proceeding to the assay with two different DNA targets,a wild type and a mutant strand（containing a single nucleotide mutation）.The RCA products were analyzed using electrophoresis gel.The G-quadruplexes generation through RCA was examined by the addition of hemin to trigger the formation of a DNAzyme complex（G-quadruplex/hemin）.The color change from colorless TMB into colored ox TMB for the solution with the mutant target after the addition of H2O2was due to the DNAzyme complex that catalyzes the oxidation of TMB to ox TMB.The colorless state of the WT solution indicated the absence of the formation of DNAzyme to catalyze the oxidation of TMB due to the non-generation of G-quadruplexes.Therefore,the color change could be used to indicate the successful generation of G-quadruplexes and by that the presence of a single nucleotide mutation.The OD measured at 450 nm for both solutions（WT and mutant）using a microplate reader showed a high absorbance for the mutant solution and hardly any absorbance was recorded for the WT.The method’s sensitivity was tested using various concentrations of mutant DNA target sequence ranging from 0 to 1000 n M（0,0.01 n M,0.1 n M,0.5 n M,1n M,10 n M,50 n M,100 n M,500 n M,1000n M）.It has been observed that the absorbance and the color intensity gradually increased with the target DNA concentration showing a good linear correlation（R2=0.9770）,with the lowest concentration recorded being 180 p M.However,a change of color was observable by the naked eye for a target mutant DNA concentration of 10 p M.Therefore,10 p M was chosen as the limit of detection（LOD）for visual detection.12 DNA samples randomly selected from the patients in which a mutation was observed previously by Sanger sequencing 1 for BRCA1（c.3331C>T）,1 for BRCA2（c.5635G>T）,5 for PIK3CA（c.1634A>C）and5 for TP53（c.312G>T）were used to demonstrate the practical use of the method in clinical samples.A PCR was performed for each 4 mutations using a 5’phosphorylated Forward Primer to allow lambda exonuclease to digest the 5’-phosphorylated strand in the 5’to 3’direction in order to generate an ss DNA.The successful generation of the targeted DNA sequences and the RCA products was verified by agarose gel electrophoresis which clearly shows the bands at their corresponding sizes.The colorimetric detection was carried out in a 96-well plate after the addition of hemin,H2O2,TMB,and H2SO4.Except for the control wells（5）,a color change was visible with the naked eye in all other wells containing mutation-positive samples（12）.The results obtained were 100%similar to the results obtained previously by using Sanger sequencing on the same samples.These data indicate that our assay for colorimetric detection of DNA point mutation in genes related to breast cancer has great potential in early diagnosis in the case of hereditary BC for the establishment of individualized therapy and prognosis analysis.By using colorimetric biosensing,we significantly reduced the cost and complexity of the signal readout,the presence of a mutation was simply observable through naked eyes.The only instruments needed were a Thermal Cycler for PCR amplification,an UV light and a water bath.These are all the instruments that can be easily found even in poor resource setting countries.Compared to other methods,our approach has proven to be more affordable and its sensitivity and selectivity were also very great.Here are several advantages of our approach:a)good visual LOD of 10 p M and relatively short detection time,b)possibility to detect any single nucleotide mutations just by using the appropriate PCR primers and padlock probe DNAs,c)easily applicable to real samples thanks to the generation of ss DNA using lambda exonuclease with a readout visible by naked eyes,d)no heavy or expensive equipment required.Conclusions:We provided a comprehensive mutation profiling of BRCA1,BRCA2,TP53,PIK3CA,PTEN and AKT1 genes in Burkina Faso BC patients.Our results showed a relatively low prevalence of BRCA mutations and a high percentage of PIK3CA somatic mutations with an unusual spectrum.The most notable was the PIK3CA c.1634A>C（p.E545A）variant found in 38.34%of the patients;this variant is typically observed at a far lower frequency in any other population according to our literature research,and is linked to a wide variety of malignancies.A novel recurrent TP53 variant c.312G>T（17 cases）was also registered;further molecular（gene cloning,CCK-8 assay）and bioinformatic（in silico analysis）studies suggested that the variant is likely not pathogenic.Finally,we proposed a novel affordable colorimetric biosensing method for easy and affordable single nucleotide mutation detection.The method took advantage of PCR,GQ-RCA,and G-quadruplex/hemin DNAzyme complex formation catalyzing the oxidation of TMB which gives a color detectable by the naked eye.Our work will provide new strategy for the establishment of efficient molecular diagnosis and the creation of personalized therapeutic methods fitting our populations.There are 31 figures,28 tables and 315 references... |
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