Study On The Alteration Of Autosomal And Y Chromosomal STR Loci In Nasopharyngeal Carcinoma

Objective:Some special cases are often encountered in forensic individual identification and paternity identification.Tumor tissue（usually formaldehyde-fixed paraffin embedded tissue）is the only reference sample.Some studies have shown that STR locis are mutated in tumor tissues,which makes the genotyping of tumor tissues inconsistent with that of normal tissues in the same individual.The purpose of the study is to investigate the variation types and regularity of alleles of autosomal and Y-chromosomal STR loci in nasopharyngeal carcinoma,and to lay a foundation for the application of special samples such as nasopharyngeal carcinoma in forensic practice.Methods:1.Samples collection:The cancer tissues and normal tissues of 79patients with nasopharyngeal carcinoma diagnosed by pathology were collected.Among them,12 cases of nasopharyngeal carcinoma tissues were pathological biopsy tissues and the control tissues were oral swabs of the same individual.Other 67 cancerous tissues were paraffin embedded tissues and blood from the same one was collected as control.The fixed time of paraffin embedded tissue was less than one week and the preservation time was not more than one year.Among the 79 patients with nasopharyngeal carcinoma,56 were males and 23 were females.The clinical data of patients with nasopharyngeal carcinoma were recorded（including sex,age,clinical stage,etc）.2.DNA extraction:Genomic DNA of pathological biopsy tissues was extracted by phenol chloroform method.Oral swabs and blood were extracted by Chelex-100,and paraffin embedded tissues were extracted by TIANamp FFPE DNA Kit.3.STR genotype:AGCU Expressmarker 22 kit was used to amplify the extracted DNA samples.AGCU Database Y24 kit was used to amplify DNA samples from the male patients.Capillary electrophoresis of PCR products was carried out by AB3500 genetic Analyzer and GeneMapper^?ID-X software was used for STR typing.The DNA genotyping of normal tissues and tumor tissues of the same individual was compared,and the STR mutation sites and types were recorded.4.Statistical analysis:The data was analyzed by SPSS 13.0 software.The count data were represented by the number of cases and the rate,and the comparison between groups was performed byχ² test.Results:1.Genotyping results of sample DNAIn this study,the sample DNA was extract by phenol chloroform method,Chelex-100 and TIANamp FFPE DNA Kit.Commercial kit was used to amplify the extracted DNA samples and complete STR genotyping was obtained from all STR locis.And the allelic peak height of STR locus was more than 150 RFU（Relative Fluorescence Unit）.2.Results of mutations in autosomal STR and Y chromosomal STR in NPCBy using AGCU Expressmarker 22 fluorescence detection kit,26 cases were found to be having the alterations of STR which were different from normal control tissues in 79 nasopharyngeal carcinoma tissues.The individual incidence of autosomal STR mutation in nasopharyngeal carcinoma was32.9%.4 types of allelic alteration in autosomal STR loci were observed in nasopharyngeal carcinoma,including additional alleles(A_add)5 cases,new alleles(A_new)1 case,complete lost of heterozygosity（LOH）5 cases,partial lost of heterozygosity（pLOH）25 cases.The first three STR mutations could cause the genotype change and result in mistake.A total of 68 times of alterations were observed in 22 common STR loci.The STR locus with the highest number of mutations was D3S1358（12times）.D7S820,D19S433 and Amel loci had no alterations.12 STR loci（D3S1358,D13S317,D16S539,TH01,CSF1PO,vWA,D21S11,D18S51,D6S1043,D8S1179,D12S391,FGA）had the alteration of STR which could cause STR_GA,7 STR loci（Penta E,D2S441,TPOX,D2S1338,Penta D,D10S1248,D5S818）had only partial lost of heterozygosity,which did not result in STR_GA.By using AGCU Database Y24 fluorescence detection kit,2 cases had been detected STR_GA in 56 cases of male nasopharyngeal carcinoma.The individual incidence of Y chromosomal STR_GA was 3.6%.2 times of STR_GAA were observed in DYS389Ⅱand DYS643 loci,and no alterations were detected in the other 22 Y-STR loci.3.Comparison of autosomal STR_GA and Y-STR_GA in male NPCBy using AGCU Expressmarker 22 fluorescence detection kit,7 cases were found to be having STR_GA in 56 cases of male nasopharyngeal carcinoma tissues,the individual incidence of autosomal STR_GA was 12.5%.13 times of STR_GA were observed in autosomal STR loci,at the locus level,the incidence of STR_GA was 1.055%in male nasopharyngeal carcinoma.Byχ² test,there was no significant difference in the individual incidence of STR_GA between autosomes and Y chromosomes in male patients with nasopharyngeal carcinoma（p>0.05）.At the locus level,the incidence of autosomal STR_GA and Y chromosomal STR_GA was significantly different（P<0.05）.4.Statistics of clinical data related to nasopharyngeal carcinomaByχ² test,it was found that there was no difference in the distribution of STR mutation in sex,age and clinical stage of nasopharyngeal carcinoma based on the analysis of the clinical data of NPC patients and the present experimental results.Conclusions:1.The alterations of STR loci in autosomal STR and Y chromosomal STR were both observed in nasopharyngeal carcinoma tissues.The mutations which could cause STR genotype alteration were detected in 12 autosomal STR and 2 Y-STR loci,indicated that the 14 common STR loci were less stable in nasopharyngeal carcinoma tissues.The conclusions should be carefully drawn when the genotyping of nasopharyngeal carcinoma tissues was inconsistent with that of normal tissues.2.In this study,3 autosomal STR locis and 22 Y-STR locis had no alterations in nasopharyngeal carcinoma tissues,which could be used as candidate locis for individual identification of nasopharyngeal carcinoma.We can increase the sample size in the future,and verify whether they can be incorporated into the stable STR loci,which provides the basis for individual identification and paternity identification of special samples such as tumor tissue.3.There was no significant difference in the individual incidence of autosomal STR_GA and Y-STR_GA in nasopharyngeal carcinoma（P>0.05）.At the locus level,the incidence of autosomal STR_GA was higher than that of Y chromosomal STR_GA,and the difference was statistically significant（P<0.05）,indicated that the stability of Y-STR locis in nasopharyngeal carcinoma was better than that of autosomal STR locis.4.In this study,there was no difference in the distribution of autosomal STR alterations in sex,age,clinical stage of nasopharyngeal carcinoma.Whether it was related to prognosis need further study.