Research On The Relation Between The BER Gene MUTYH And The Susceptibility Of Colorectal Cancer

DNA oxidative damage is one the most frequent causes of mutation in cells. Reactive Oxygen Species(ROS),a series of metabolic byproducts which can also be caused by exogenous oxidations due to physical or chemical reasons,accounts for over 10⁴ of DNA damages per cell per day.The moment that the antioxidant defense system become over-loaded,the accumulated ROS may cause permanent damages to nuclear and mitochondrial DNA,such as base mismatch,modification,site formation of depurination or depyrimidination,DNA fragmentation,as well as DNA-Protein cross-linking.ROS is certain degenerative diseases such as aging,cancer,immune system decline,nerve degeneration,cataracts,and so the cause.Oxidative damages to DNA mainly result in the product of 8-oxo-deoxylguanine (8-oxo-dG) and 2-OH-Adenine(2-OH-A),and the former is more stable and significant. The mechanism of the damage brought out by 8-oxo-dG is either direct oxidation of Guanine(G) on the template strand or oxidation of free Guanosine in the dNTP pool in bacterial and yeast.As combining rate of DNA-polymerase with 8-oxo-dG:A is 5～200 times higher than that of 8-oxo-dG:C,in the condition that the mismatch is not replaced, thus eventually leading to further mismatches in daughter strands during replication,a transversion of G:C→A:T will build up in genes which may consequently change their functions and,in some cases,the formation of tumor.Base Excision Repair(BER) system runs a conservative process in cells that plays an significant role in the mutation build-up caused by oxidative damages.This system includes a series of genes:NUDT1,located on chromosome 7p22,of which the protein it codes for can hydrolyze oxidized dGTP in dNTP pool,and prevent their entry into DNA replication process;OGG1,located on chromosome 3p26.2,of which the protein it codes for can trigger the BER system during replication by identifying and excising 8-oxo-dG from 8-oxodG:C pairings;MUTYH,located on chromosome 1p32.1 to p34.3, codes for a specific Adenine DNA glycolase which can remove the mismatched Adenine from 8-oxo-dG:A on the daughter strand after replication.Transversion of G:C to A:T during replication will accumulate when MUTYH glycolase is deactivated. Researches have shown that an increased mutation rate in oncogenes and tumor-suppression genes due to defects of BER system leads to the formation of tumor.In cases reported of multiple Adenomatous Polyposis Coli(APC) and colorectal cancer,high-frequency mutation from G:C to A:T in somatic cells of APC genes is related to germline mutations on MUTYH alleles.This type of diseases caused by the mutations of Y165C,G382D,466delE,E466X and Y90X are reported in Caucasian, Indians,Pakistanis and other ethic groups.The mutation rate of Y165C and G382D is around 80%in Caucasian patients.Mutations from Y165C toY165C or G382D to G382D which form homozygote,as well as mulriple mutations from Y165C to G382D which form heterozygote,accounts for most of the cases while rarely detected in Eastern Asians including Japanese,which indicates that the two types are ethnic-specific allele mutation.Based on that,we deduced that the mutations on Y165C and G382 are of low dominant rate and its susceptibility are similar to previous cases involving APC and CHEK2 mutations.Therefore,we inspected APC and MUTYH gene samples from 3 Japanese MAP suspected families and found one new Single Nucleotide Polymorphism(SNP) site.We carried out SNP detections in 283 colorectal cancer cases and 307 control cases and found significant relation between IVS1-5 A＞C, the MUTYH SNP sites in Japanese population,and colorectal cancer.The results have demonstrated that IVS1-5 A＞C,the MUTYH allele mutation site,may prove to be susceptive to colorectal cancer in Japanese population. Materials＆methods1.Materials(1) family casesBlood samples from 3 suspected MAP examined family patients were furnished by the laboratory department of the affiliated hospital of Hamamatsu University,School of Medicine,with the consent of the patients and their families to conduct gene screening to the blood sample;diagnosis were done by the Department of Pathology and all patient files were provided by the In-patient Department.(2) Blood sample and cell linesBlood samples of 283 colorectal cancer patients were furnished by the laboratory department of the affiliated hospital of Hamamatsu University,School of Medicine,and diagnosis were done by the Department of Pathology.307 control samples were taken from volunteers in Fukuoka,aged from 20 to 74,with no familial adenomatous Polyposis or inflammatory intestinal diseases,and did not suffer colorectal cancer or underwent any relative surgery.70 tumor cell lines were provided by Hamamatsu University,School of Medicine.2.Methods(1) DNA extraction5ml venous blood was taken from each examinee;DNA samples were collected using QIAamp(?) DNA Blood Maxi Kit from the whole blood.(2) RNA extraction5ml venous blood was taken from each examinee;RNA samples were collected using PAXgene Blood RNA Kit from the whole blood.(3) DNA extraction of the cell linesDNeasy(?) Tissue Kit was used to extract DNA from 70 human tumor cell lines.(4) Design and synthesis of primersThe total sequence of APC(NM₀00038.2) and MUTYH(NM₀12222.1) genes were found in Genebank;Oligo@SIGMA GENOSYS was used to design the primers for all exons of APC and MUTYH gene respectively.(5) PCR reaction system:20μl PCR reaction system consisted of 0.2u Hotstar Taq as DNA polymerase,2μl 10×PCR buffer(including 15mM MgCl₂),1.6μl dNTP (200μM),0.2μl primer(0.25μM),1μl DNA template,and double distilled water adding to 20μl.The reaction condition:initial denaturation at 95℃for 15 min,denaturation at 94℃for 30s,annealing for 30s and elongation at 72℃for 1 min,totaling in 37 cycles and the last elongation period was 10 min.(6) Restriction enzyme reaction system20μl reaction system consisted of 15μl PCR amplified products,2μl 10×buffer, 0.2μl 100×BSA and different types of restriction enzyme,incubated overnight at 37℃.(7) Analysis of single strand conformation polymorphism(SSCP)17μl PCR amplified products was mixed with FEXB(95%formamide,0.02M EDTA and 0.05%bromophenol blue) as the proportion of 1:1.5,and then denature at 96℃,then ice-bath for 15 min.10μl of each sample underwent 8%PAGE electrophoresis at 4 conditions(room temperature,RT+5%glycerol,4℃, 4℃+5%glycerol),150v for 3.5h at RT and 4.5h at 4℃.The gel underwent silver dyeing. Generally,the bands on the gel that differ from the control samples,which means migration,extra bands of wider bands,shall be regarded as abnormal and thus indicate possible mutations,and further DNA sequencing is required.(8) DNA sequencingAs the abnormal bands were detected by PCR-SSCP,the relative PCR products were sequenced by the Chain Termination Method.The apparatus used here included ABI PRISM 3100(for sequencing),and QIAquick PCR Purification Kit(for purification of PCR products before sequencing),and Nanodrop ND-1000 Spectrophotometer(for quantifying the DNA).ABI BigDye(?) Terminater v3.1 Cycle Sequence Kit was used in sequencing and the sequence analysis was done with GENETYX 8. (9) PCR with confronting two-pair primers(PCR-CTPP)Two-pair primers were designed for the mutation site IVS1-5 A＞C,and were used in PCR,annealing at 60℃for 40 cycles.(10) Reverse transcription-PCR(RT-PCR)RNA samples were extracted from examinee's whole blood,and were reverse-transcribed to cDNA in the SuperScript^TM First-Strand Synthesis System respectively.(11) Real-time fluorescence quantitative PCRPrimers targeting the mutation site IVS1-5 A＞C on MUTYH gene was designed. The 20μl reaction system consisted of 5μ1 0.2×cDNA sample,10μl 2×SYBR Master (including 2.5mM MgCl₂),1μl 0.5μM primer of each kind,and 3μl RNase-free water. Amplification was conducted using LightCycler 3.0 Fluorescence Real-time Quantitative PCR system:initial denaturation at 95℃for 15min denaturation at 94℃for 15s,annealing at 61℃for 20s and elongation at 72℃for 10s,totaling 50 cycles. Signal collection was carried out at 81℃and the Melting curve analysis was carried out in such condition:cooling at 20℃/s from 95℃to 65℃;after incubation at 65℃for 10min,heating at 0.1℃/s to 95℃.Results1.Results of PCR-SSCP and DNA sequencing of APC and MUTYH gene of 3 suspected familial MAP patientsPCR-SSCP and DNA sequencing were used to inspect all exons of APC gene and MUTYH gene of 3 suspected familial MAP patients,and 11 SNP sites were found in APC gene,3 in MUTYH gene.5 sites among the former and 1 site among the later were reported for the first time.2.The selection of SNP site and the inspection using PCR-CTPPThe scientific community already had insight into the mechanism of the formation and proliferation of human colorectal cancer and FAP involving APC gene.Our research focused on the relation between the MUTYH gene and human colorectal cancer and FAP.Although 5 unreported SNP sites among 11 SNP sites in APC gene were discovered,we chose MUTYH gene instead of APC which already attracted attention,thus targeting IVS1-5 A＞C,the unreported SNP site on MUTYH gene,as the mutation site.We inspected the SNP mutation sites IVS1-5 A＞C in blood samples from colorectal cancer patients and control group in Hamamatsu and Fukouka in Japan, using PCR-CTPP,finding 11 and 2 patients with colorectal cancer had mutation on MUTYH IVS1-5 A/C and MUTYH IVS1-5 C/C respectively among the 283 patients;4 in 307 control group were found with mutant MUTYH IVS1-5 A/C,and none were found homozygote of mutant MUTYH IVS1-5 C/C.3.Analysis of MUTYH IVS1-5 A＞C polymorphism(1) General data of the subjectAll subjects in this research were residents of Hamamatsu and Fukouka in Japan, aged 20-74,average age of the study group 62.15±6.16,average age of the control group 58.81±6.78,thus statistically different(U=6.27,P＜0.001).the proportion of males in the two groups were 61.10%and 52.10%respectively,thus statistically different by chi squar test(χ²=4.87,P=0.027).(2) Distribution of genotypesAmong 307 peoples in the control group,303(98.70%) had wild-type(A/A) MUTYH IVS1-5,and 4(1.30%) were heterozygote(A/C) of mutant MUTYH IVS1-5; among the patients in the study group,the proportion of wild-type(A/A),heterozygote (A/C) and homozygote(C/C)of mutant MUTYH IVS1-5 were 95.41%,3.89%and 0.70%respectively.The gene frequency of allele A/C on MUTYH IVS1-5 was 0.65% in the control group while that of A/C and C/C allele was 2.65%,thus statistically different by chi squar test(χ² =7.43,P=0.006).(3) The relation between MUTYH IVS1-5 A＞C polymorphism and colorectal carcinoma susceptibility in JapaneseTaking MUTYH IVS1-5 wild-type(A/A) carriers as the control group,after adjusted for age,gender role,those who carry at least one MUTYH IVS1-5 alleles(A/C and C/C),have higher risk of suffering from colorectal carcinoma.The adjusted OR of MUTYH IVS1-5 genotype polymorphism in colorectal carcinoma patients are 3.60.4.Results of RT-PCR and Real-time PCRWe had extracted RNA from each blood sample and reverse transcribed them into cDNA.After amplification,gel electrophoresis,and imaging,the genome DNA in PCR-CTPP did not form any bands and cDNA samples from examinee 1;examinee 2; intestinal cancer cell lines and intestinal cancer patients did not show any difference on the gel either.Then we inspected the relative expression level of MUTYH IVS1-5 A＞C in cDNA samples from intestinal cancer cell lines,non-patients,and examined family patients using Real-time PCR,and found significantly higher expression level in the last than that in the former two.5.Results of inspection of DNA from 70 cancer cell lines using PCR-CTPPInspection of DNA from 70 human cancer cell lines using PCR-CTPP resulted in negative detection of heterozygote(A/C) and homozygote(C/C) of mutant MUTYH IVS1-5.DiscussionColorectal cancer,one of the high grade tumors most commonly seen in human which brought out 945,000 new cases in the year 2002 alone,takes nearly 500,000 lives a year,only subject to lung cancer and breast carcinoma as the most popular malignant tumors.It also prevails in China,ranking the 4^th in morbidity rate of tumors which is climbing at present,and had already drawn attention for the public.The formation of colorectal cancer is a complicated process with multi-factors, multi-stages and multi-genetic abnormities.It may be caused by accumulating mutations in oncogenes and tumor-suppression genes,and defects or malfunctions of DNA damage repair systems also lead to increasing rate mutation and tumor formation. DNA damage repair systems serves as a major defense against DNA damages caused by both exogenous and endogenous factors.Defects or low functioning rate of it will increase the hazards of gene mutation and tumor formation.The research has shown a higher susceptibility to tumors in the population with compromised function of DNA repair system than the average level,therefore individual differences in DNA repair may lay the foundation of genetic susceptibility to tumor.It is known that SNP exists in many genes related to DNA repair with a growing number of studies concluding that SNA which featuring alteration of amino acids may as well alter the activity of corresponding enzymes.Thus,SNP,in genes related to DNA repair system,especially the SNP that located in coding regions and/or regulatory genes weighs heavily against individual difference in DNA repair function.Genetic polymorphism of DNA repair system can lead to higher risk of cancer in individual being.DNA repair system in human consists of Base Excision Repair,Nucleotide Excision Repair and Mismatch Repair,and MUTYH gene is responsible for the tertiary defense in BER.MUTYH gene is located on the short(p) arm of chromosome 1 between positions 32.1 and 34.3,with 7.1kb and 16 exons,and codes for an enzyme constituted by 535 amino acids.MUTYH glycosylase,located in nucleus and mitochondria,scans the daughter strand and excise the Adenine mismatched with 8-oxo-dG of the parent strand shortly after replication.Transversion from G:C to A:T as the MUTYH glycosylase with lower of non-function will promote tumor formation.Several SNP sites have been found in MUTYH gene,in which Y165C and G382D are studied in depth.The mutation rate of Y165C and G382D is around 80%in Caucasian patients.Mutations from Y165C toY165C or G382D to G382D which form homozygote and mulriple mutations from Y165C to G382D which form heterozygote account for most of the cases.In Asia,Y90X and E466X,mutation sites in MUTYH gene,were found in India,Y90X in Pakistan,and R231C and IVS10-2 A＞G in Japan, of which the former mutates on both alleles to form a homozygote and later mutates on single allele and can be excised.Mutations of MUTYH gene also appear in family genetic gastric cancer:IVS10-2 A＞G was found in excision site in Japanese population, and two new mutation sites,Pro18Leu and Gly25Asp,were found in Chinese population.As to mutations on Y165C and G382D which are relatively common among Caucasian population have not been detected in Eastern Asians,and thus indicates specific mutations of MTUYH gene in different ethnic groups.Our research for the first time reported MUTYH IVS1-5 A＞C,a new SNP mutation site in MUTYH gene among Japanese population,and analyzed the relation between its polymorphism and the susceptibility to colorectal cancer among Japanese population.The result gave 3.64 times higher risks of individual beings with heterozygote(A/C) or homozygote (C/C) of a mutant-type MUTYH IVS1-5 suffering from colorectal cancer than those with homozygote(A/A) of wild-type MUTYH IVS1-5.With confounding factors like age and gender being adjusted,the result OR value remained 3.60,which indicates that the gene polymorphism of MUTYH IVS1-5 must play a role in the formation of colorectal cancer among Japanese population.Some had done function analysis of the mutations on MUTYH gene and found a decreased expression level of MutY gene in E.Coli that had mutations on Y165C and G382D gene.They also had done affinity analysis of enzyme and substrate with FA (2-fluoro-2-deoxyadenosine),a substrate analogue,and concluded that those two mutations strongly reduced the ability of MUTYH gene's coding products to identify 8-oxo-G from Guanine.Perhaps the mutation of MUTYH IVS1-5 A＞C exert the same effect.In addition,IVS1-5 A＞C,the mutation site on MUTYH,is also where Replication Protein A(RPA) combines with MUTYH gene,and RPA plays an important role in initiation and elongation period during replication by binding the single stand and unwinding the double helix.In that process,the phosphorylation of RPA can effect on its activity of DNA binding and interactions with proteins concerned, and thus regulate DNA replication.Other than normal DNA replication,RPA also participate stress reactions of DNA damages including nucleotide excision repair, mismatch repair,homologous recombination and Non-homologous End Joining,which may raised the level of susceptibility to colorectal cancer of those who carries the mutant type of MUTYH IVS1-5.Details in the mechanism remained unknown.We had inspected the MUTYH IVS1-5 A＞C,the SNP mutation site,in 70 tumor cell lines with PCR-CTPP,resulting no mutations on MUTYH gene detected.It resembles that reported by Parker et al,although part of the micro-satellite stable cell lines had defects of mismatch repair of 8-oxo-G and A and increased level of 8-oxo-G, no mutations on MUTYH gene was detected,which mainly because the lack of phosphorylation of adenine DNA glycolase,the MTUYH gene's coding product,in those cell lines.We had done RT-PCR inspection to cDNA samples from Examinee 1,Examinee 2, intestinal cancer cell line KATOâ…¢and intestinal cancer patients,no visual differences were found in their gel-electrophoresis images,which indicates the influence that the MUTYH IVS1-5 A＞C mutation has on susceptibly to colorectal cancer is not to change the expression level of the adjacent MUTYH gene's No.2 exon.After inspecting the relative expression level of MUTYH IVS1-5 A＞C of cDNA samples from intestinal cell line,non-colorectal cancer population and the examined family patients,we found significantly higher expression level in the last than that in the former two.The higher expression level of MUTYH IVS1-5 A＞C in examined family patients contradicted to the tumor-suppressive effect of DNA damage repair genes,but enlighten us that the position of SNP mutation site on MUTYH IVS1-5 A＞C may reduced its binding rate with RPA and increased the mutation rate of APC gene,thus increasing the susceptibility to colorectal cancer in the carriers of mutant MUTYH IVS1-5.Limited by the small number of samples inspected with Real-time PCR, further inspection of more samples shall be carried out for confirmation. ConclusionOur research discovered a new SNP mutation site,IVS1-5 A＞C,on the MUTYH gene which is related to DNA damage repair,and analyzed the influence of its polymorphism on the formation of and genetic susceptibility to colorectal cancer in Japanese population.It helps us look into the mechanism of the formation of colorectal cancer and can facilitate the screening of the high risk population.