Autosomal SNP Mutation Analysis Of Breast Cancer And Gynecological Tumor Tissues

Objective: In the forensic examination,the tumor tissue is a special biomaterial,perhaps the only reference samples,compared with the routine ones such as blood,blood stain,hair,saliva,etc.As SNP,different from STR,is characterized by low mutation rate and easy detection,it has potential value for the identification of tumor tissues.But there is still a lack of systematic study on the stability of SNP in tumor tissues at home and abroad.The study explored the SNP mutation pattern about tissues of gynecological tumor and breast cancer,and analyzed the effect of tumor types,tissue types,differentiation degrees,and clinical stages on SNP mutation rates.Then we assessed practical value of the SNPs on the two kinds of tumor tissues and established efficient and accurate SNP genotyping method to deal with biological specimens from tumors,in order to provide a reference for forensic testing on this kind of case.Methods:1 Sample collection: Samples used in this experiment were collected by our department including 63 breast cancer specimens,62 gynecologic cancer specimens and matched paracarcinoma tissues,10 gynecologic benign tumor tissues and normal tissues for control.Correspondingly patients' ages,tumor types,histological types,clinical stages,degrees of differentiation,and other information were noted.2 DNA extraction: TissueGen DNA Kit and TIANamp FFPE DNA Kit were used to extract DNA from the above samples.Then the DNA was quantified with ND-1000.3 SNP genotype: SNapShot multiplex kit was applied to amplify 55 autosomal SNP loci,and then PCR product purification,single base extension reaction,purification,electrophoresis genotyping by ABI 3130 genetic analyzer were carried out in turn.GeneMapper ? v3.2 was adopted to analyze results in order to obtain the genotyping of the tumor tissue and normal tissue.After matching the results,we obtained the SNP loci and types of mutation.4 Allele-specific polymerase chain reaction4.1 Primer design of allele-specific polymerase chain reaction: 9 mutational SNPs and 9 SNPs without mutation were selected from 55 loci.According to the reference sequences of http://www.ncbi.nlm.nih.gov/snp/,two allele specific primers and a universal primer were designed by Primer Premier 6.0 and Oligo 7.0,and then the primer sequences and human genomic DNA sequences were aligned using NCBI BLAST in order to search amplified products.An artificially mismatch base was introduced into two alleles specific primers respectively to increase the specificity.In addition,a nucleotide sequence unmatched with the template was added to the 5'end of one allele specific primer to convert the sequence difference to length difference,which facilitates the following detection by capillary electrophoresis(CE).4.2 Single locus allele-specific polymerase chain reactionIn order to verify the accuracy of AS-PCR system,4 known SNP genotype DNA samples of healthy individuals were detected by AS-PCR using Q5 High-Fidelity DNA Polymerase and AmpliTaq Gold DNA Polymerase.The SNP genotype was obtained after the PCR product was tested by ABI 3130 genetic analyzer,whose data were collected by Data Collection V3.0,and analyzed by Genemapper V3.2.5 Verification of SNP mutationThe verification of samples of mutation by SNapShot was repeated several times,and some of the samples were tested by Sanger sequencing and single locus allele-specific polymerase chain reaction.6 Statistical analysisStatistical analysis was performed by Excel and SPSS v21.0.The ?~2 test was used to investigate the difference of SNP mutation rate in the tumor types,tissue types,the degrees of differentiation in breast cancer and gynecological tumor tissue,and compared the differences of SNP mutation rate between breast cancer and gynecological tumor tissue.Rank sum test was used to compare the differences of SNP mutation rate in different clinical stages in the two tumor tissues.Results:1 Construction of AS-PCR system and accuracy validation The AS-PCR primers of 18 SNP loci were designed,with their lengths ranging from 18 bp to 32 bp,and the PCR product lengths from 107 bp to 217 bp.4 known SNP genotype DNA samples were experimented by AS-PCR,which showed that the products with Q5 High-Fidelity DNA Polymerase were inconsistent with the known genotype because of non-specific amplification;however,the SNP genotype with AmpliTaq Gold DNA Polymerase was completely consistent with the known genotype.Six alleles of 6 loci were selected to verify the accuracy of AS-PCR by Sanger sequencing,suggesting that rs1109037,rs740598,rs10092491 were not successfully sequenced,and the allele sequences of rs2342747,rs6444724,rs159606 were the same as the standard sequences provided by NCBI.2 SNP mutation of gynecologic tumor tissuesThere was no mutation in the 10 gynecological benign tumor tissues.Mutation was found in 5 SNPs from the 5 gynecologic malignancy tissues by SNapShot method,rs13218440 of sample 9 and sample 29,rs1523537 of sample 16,rs1498553 of sample 18,rs315791 of sample 28.The SNP alleles of samples 9,18 and 29 were analyzed by AS-PCR,and rs13218440 of sample 9 and sample 29,rs1498553 of sample 18,rs315791 of sample 28 were sequenced by Sanger sequencing.The rs1523537 of sample 16 was not verified by AS-PCR and Sanger sequencing because the genotype of three SNapShot experiments was consistent.Finally,there were 3 cases(9,16,28)with a total of 3 SNP(rs13218440,rs1523537 and rs315791)presenting mutation,and the mutation types were complete loss of heterozygosity.The ?~2 tests showed that there was no significant difference of the SNP mutation rate between benign and malignant gynecologic tumor tissues(P>0.05),and no significant difference in different tumor types,histological types,differentiation degrees of gynecological tumor tissues.The rank sun test showed that the difference of mutation rate of SNP in different clinical stages of gynecological tumor tissues was not statistically significant.Comparison of SNP with preliminary STR results of gynecological tumor tissues showed that the mutation rate of SNP was significantly lower than that of STR(P<0.05).3 SNP mutation of breast cancerAfter genotyping of 63 cases of breast cancer by SNapShot,mutation was found in 26 SNP loci of three samples,rs445251 of sample 2,rs2270529 of sample 40 and 24 SNPs of sample 7.15 SNPs of sample 7 tumor tissue were selected for Sanger sequencing,and 7 SNP loci were verified by AS-PCR.According to the above experimental results,a total of 21 SNP loci of 3 breast cancer samples have mutations,of which 15 SNP mutation types were loss of heterozygosity,2 were homozygous into heterozygote,3 were base conversion,and 1 was base transversion.The rank sum test results showed that there was no significant difference of SNP mutation rate between different clinical stages of breast cancer.The ?~2 test results showed that there was no significant difference of SNP mutation rate between highly and poorly differentiated individuals.Comparing SNP with preliminary STR mutation rates of breast cancer,SNP mutation rates were significantly lower than STR mutation rates in the number of individual and locus(P<0.05).4 Comparation of SNP mutation rates between gynecological tumor and breast tumorThe ?~2 test indicated the difference of mutation rate between gynecological tumors and breast cancer was not significant in the number of individual,while the number of mutation SNPs of breast cancer was significantly higher than that of gynecologic tumors(P<0.05).Conclusion:1 SNP mutation rate is less than the STR in breast cancer and gynecological tumor tissues,so the stable SNP system is expected to be obtained,which can be applied to tumor tissues in forensic examination.2 AS-PCR is an accurate,simple typing method for SNP of tumor tissues,providing an effective detection method for forensic genetics.