Mutation Analysis Of Autosomal And X Chromosomal STR In Breast Cancer And Gynecologic Cancer

Objective: tumor tissues may sometimes be the only source of biologicalmaterial for forensic investigation. However, STR mutation in tumor tissuesmakes genetic typing difference between tumor tissue and normal tissue fromthe same individual. The study aims to explore the types and rule of autosomaland X chromosomal STR mutation in gynecologic cancer and breast cancertissue for application of such tumor tissues in forensic practice.Methods:1Samples collection:63breast cancer specimens,62gynecologic cancerspecimens and10gynecologic benign tumor tissues with clear pathologicaldiagnosis and corresponding normal tissues were investigated.40gynecologiccancer specimens include corresponding peripheral blood. The patient’s age,tumor type, tissue type, clinical stage and degree of differentiation wererecorded.2DNA extraction: DNA were extracted from gynecologic cancer andbreast cancer tissues and corresponding normal tissues using TissueGen DNAKit and TIANamp FFPE DNA Kit respectively. And genome DNA in bloodwere extracted using BloodGen Mini Kit. Then DNA were quantified withND-1000.3STR genotype: DNA were amplified using Powerplex21System Kitand Investigator Argus X-12Kit including33loci. Capillary electrophoresis ofPCR products was carried out on an ABI3130Genetic Analyzer. The data wasanalyzed by GeneMapper v3.2software to obtain STR genotype of tumortissue, normal tissue and blood. Contrast genotype between normal tissue andblood, between tumor tissue and normal tissue to record the locus and type ofSTR mutation.4verification of STR mutation: STR mutation was verified by repeat detect, different kits and single locus amplification. The mechanism of STRmutation was made sure by single allele sequencing.5Microdissection: Some of the gynecologic tumor tissues with STRmutation were microdissected inorder to distinguish tumor cells and normolstromal cells. Microdissected DNA was extracted and amplified, PCR productswere separated by3130Genetic Analyzer. Genotypes of tumor cells, normalcells, tumor tissue and normal tissue were contrasted respectively.6Statistical analysis: Statistical analysis was performed using Excell andSPSS v16.0software. Counting method was used to analyse STR mutationrate of individual locus of various mutation types. The χ2tests were used toinvestigate the difference of STR mutation in different tumor types, tissuetypes, clinical stage and degree of differentiation and the difference of STRmutation in breast cancer and gynecologic cancer.Results:1STR mutation in gynecologic cancer tissueThe same STR type of blood sample and normal tissue were found bycontrasting genotype of the two samples from gynecologic cancer patients andsuggested that there was no STR mutation in peripheral blood.4kinds of STR mutation types were found in gynecologic cancer tissueincluding additional alleles, new alleles, complete loss of heterozygosity andpartial loss of heterozygosity. The first three mutations make the genotypechange and result in mistake. The same results were obtained by repeat tests,different STR-typing kits and sequencing of single locus in some samples withSTR mutation. Most of mutations come from the change of repeat unit number,generally full step mutation, including increase and decrease steps, and keep inline with replicatin slippage mechanism.The results of statistical analysis showed that the detection rate of eachmutation type was different. There was no significant difference of individualdetection rate in three mutation types. It shows significant difference on locusdetection rate of autosomal STRs and the whole33STRs.The range of mutation rate in33loci of gynecologic cancer tissues was 0~9.68%, the loci with highest detection rate in autosomal STR were Penta E,D5S818, FGA, and DXS10146in X-STR. No STR mutation was found inAMEL、Penta D and HPRTB, the mutation rate of the other loci was higherthan DNA average mutation rate. It influenced the correct genotype of allele.There was no significant difference and correlation on STR mutation rate oftwo genetic markers.The χ2tests showed that there were no significant difference ofautosomal and X chromosomal STR mutation in gynecologic cancer tissues ofdifferent tumor types, tissue types and degree of differentiation. However,because no STR mutation was found in gynecologic benign tumor, there wassignificant difference of STR mutation between benign tumor and cancer onlyin autosomal and the whole33STRs. There was no significant difference inX-STR. The rank tests showed that no significant difference of STR mutationin autosome and X chromosome of different clinical stage.2STR mutation in breast cancer tissue4types of STR mutation were also found in breast cancer, the detectionrate of each mutation types was different. The results of statistical analysisshowed that there was no significant difference in individual mutation andlocus mutation of three mutation types in autosomal and the whole33STRs.But it’s significantil different in X-STR. The detection rate of additional allelewas highest and the LOH was lowest.The range of STR mutation rate on locus level was0~4.76%in breastcancer. There were less loci with STR mutation than that in gynecologiccancer. The results showed that there was no significant difference andcorrelation between two genetic markers.The χ2tests showed that the mutation rate of autosomal STR in breastcancer with different degree of differentiation was significantly different, butno different in X chromosomal and the whole33STRs. The rank tests showedthe difference of STR mutation in breast cancer with three clinical stageswasn’t significant.3The difference of STR mutation in gynecologic and breast cancer The gynecologic cancer and breast cancer were impacted by the samehormones, but the STR mutation rate was different. Observing STR mutationrate on locus level of gynecologic cancer and breast cancer, we found the lociwith STR mutation in gynecologic cancer was significantly more than that inbreast cancer and the STR mutation rate of same locus in gynecologic canceris higher than that in breast cancer. The mutation occurred in the autosomalSTR loci in gynecologic cancer, but it was more common in X-STR in breastcancer.4The result of microdissectionThe results showed that the genotype of stromal cells and normal tissuewas consistent, but there were differences between tumor cells and tumortissue. There were3mutation types in tumor cells including: Anew、LOH andpLOH, and the number of LOH detection in tumor cells was more than that incorresponding tumor tissue.Conclusion:1In autosomal STR and X-STR, the STR mutation rate of locus andindividual in gynecologic cancer is significantly higher than that in breastcancer, so the value of judicial practice of breast cancer is greater than that ofgynecologic cancer.2In gynecologic cancer, STR mutation was found in all loci except forAMEL, Penta D and HPRTB. It suggests that the33forensic commonly usedSTR loci in gynecological cancer tissue are poorly stable and not suitable forindividual identification and paternity test in the tumor tissue.3The study finds16autosomal STRs and4X-STRs without STRmutation. It can be used as alternative loci in individual identification of breastcancer and guide in further study in a large sample.4The autosomal STR mutation in breast cancer tissues is correlated withdegree of tumor differentiation. Poorly differentiated tumor tissues with higherSTR mutation rate suggest that we should be cautious in the actual caseencountering such tumor tissue.5Genotype of stromal cells separated from tumor tissue accurately by microdissection can represent the genotype of tumor individual. It’s aneffective way to solve the source identification of tumor tissue, especially fortumor tissue with clear boundary of tumor cells and stromal cells.