Study Of Genetic Susceptibility Of Thyroid Papillary Cancer Based On The STR Multiplex PCR

ObjectiveUsing microdissection and STR multiplex application technology to explore the polymorphism of STR loci in the fresh tissues of papillary thyroid cancer(PTC)and to find the region of susceptibility gene in papillary thyroid cancer.Studying the types and the rule of STR mutation in PTC cells to found the loci associated with the occurrence and development of PTC and better understand the mechanism of PTC occurrence and development.To provide reference for the PTC molecular diagnosis and the targeted drugs discovery.Method1.Samples collection: 68 papillary thyroid cancer specimens and their corresponding normal tissue(adjacent tissue)samples with clear pathological diagnosis were investigated.262 healthy people peripheral blood were collected as control group.The patient’s sex,age,tissue type,and whether metastasis were recorded.2.Microdissection: Using laser capture microdissection to separate papillary thyroid cancer tumor cells from normal stromal cells of adjacent normal tissues.3.DNA extraction: DNA was extracted from papillary thyroid cancer tumor cells and normal stromal cells of adjacent normal tissues using Chelex-100 and QIAamp DNA FFPE Kit.Genomes DNA in blood were extracted with Chelex-100 and BloodGene Mini Kit.Then DNA was quantified with Bio Spec-nano.4.STR genotype: DNA was amplified using Ampf?STRòIdentifilerò PCR Amplification Kit,GoldeneyeTM DNA ID System 20 A amplification system and HuaxiaTM Platinum amplification system including 23 loci.Electrophoresis of PCR products were carried out on ABI 3500 Genetic Analyzer.To obtain STR genotype of tumor cells,normal stromal cells and healthy people peripheral blood,the data was analyzed by Gene Mapperò ID-X 1.4 software.Record the locus and type of STR mutation.5.Verification of STR mutations: Mutation was conformed by repeat detect and different kits amplfilication.6.Statistical analysis: The PowerStatsV12 software was utilized for calculating the allelic frequencies.The STR mutation rate of mutation types,different chromosomal and loci was analyzed by counting method.The c2 tests were used to analyze the difference of STR mutation in different sex,age,tissue type,and whether metastasis.All these data was analyzed by SPSS v17.0.ResultsThe frequency of D19S433-12 in the experimental group was significantly higher than that in the control group(P<0.05),while the frequency of D16S539-10 was obviously lower(P<0.05).No statistically significant difference between theoretical frequency and actual frequency of homozygote was found(P>0.05).Four kinds of STR mutation types were found in papillary thyroid cancer tissues including additional alleles(Aadd),new alleles(Anew),complete loss of heterozygosity(LOH)and partial loss of heterozygosity(pLOH).The first three mutation types result in genotype change(STRGA).The same results were obtained by repeat tests.The results of statistical analysis showed that the mutation rate of three mutation types that make the genotype change was different.Chromosomal 2 and chromosomal 5 show higher STR mutation.The highest mutation rate in 23 loci is D2S1338.CSF1 PO,Penta E,D6S1043 have the highest STRGA detection rate(0.0294).No STR mutation was found in VWA,D16S539,and Penta D.The c2 tests showed that there was significance difference of STR mutation in papillary thyroid cancer of different sex(P<0.01),different age group(P<0.0001),but no significance difference of tumor size and mutation or not(P>0.05).Conclusion1.There may be predisposing genes of papillary thyroid cancer near the locus of D19S433-12,while there may be suppressor gene of papillary thyroid cancer near the locus of D16S539-10,and homozygote frequency increased or not has nothing to do with PTC.2.Papillary thyroid cancer may associated with mutation in CSF1 PO,Penta E and D6S1043 STR loci.Also have relations with mutation on No.2,No.5 chromosome.3.Female and age between 40-59 people more likely to get papillary thyroid cancer and easy to cause genotype alter.4.STR mutation has no obvious relation with tumor size and metastasis.These four kinds of STR mutation types may be an early molecular event happened in PTC,and throughout the whole development process of PTC,has nothing to do with the prognosis of PTC.5.We didn’t find STR mutation in VWA,D16S539,Penta D.These loci can be used in individual identification of PTC.