Identification Of Sequence Polymorphisms In The D-loop Region Of Mitochondrial DNA As Risk Biomarkers For Liposarcoma

Objective : Single nucleotide polymorphisms（SNPs） in the Displacement- loop（D-loop） region of mitochondrial DNA have been reported to be associated with cancer risk in various types of cancer. The mitochondrial D-loop in a variety of tumor cells has become a research hotspot, but the related research between mt DNA D-loop and liposarcoma is relatively less. To assess the frequency of D-loop SNPs and establish correlations with liposarcoma risk,we sequenced the D-loop of 82 liposarcoma patients and analyzed their use as predictive biomarkers for liposarcoma risk in this experiment.Methods:1 Blood samples were collected from 82 liposarcoma Patients who received treatment in the Fourth Hospital of Hebei University from January 2002 to January 2012. Blood samples were also collected from 100 healthy controls. We collected the Patients’ clinical data to make it completely.2 The mitochondria DNA of liposarcoma Patients and healthy controls were extracted using the GENMED Whole Blood Mitochondrial DNA extraction kit,and stored them at- 20 for later use. ℃3 Using PCR to amplificate the mitochondrial DNA D-loop region, the forword Primer 5’-CCCCATGCTTACAAGCAAGT-3’（nucleotides 16190-16209） and reverse Primers 5’-GCTTTGAGGAGGTAAGCTAC- 3’（nucleotides 602-583）,were used for amplification of a 982-bp Product from the mt DNA D-loop region. PCR was Performed according to the Protocol PCR Master Mix Kit and Purified Prior to sequencing. the Products were then separated on the ABIPRISM Genetic Analyzer 3100. Polymorphisms were confirmed by repeated analyses from both strands. Subsequently tested the single nucleotide Polymorphisms of mitochondrial DNA D-loop region.4 The Patients were followed up by telephone for 10 years,and collected the patients’ clinical manifestation and feature at the same time, AC₀00021 database was referred to in order to find SNPs from the 982 b P mitochondrial D-loop region,and the relationship between different SNPs and 10 year survival rate was analyzed.5 Statistical analysis:the SNPs sites showing frequency was analyzed by χ2 test.All of the statistical analysis was done with the SPSS 18.0 software Package. A P value of <0.05 was considered statistically significant.Results:1 82 liposarcoma were enrolled in this study,SNPs were detected in 140 sites within the 982-bp mitochondria D-loop region from the blood samples of the healthy controls and liposarcoma Patients.The SNPs with a rare allele frequency >5% in either controls or NHL Patients were used for cancer risk analysis, a total of 19 SNPs were selected.2 When individual SNPs were analyzed in liposarcoma,a statistically significant decrease of SNP frequency for73A/G, 315C/Cinsert, 523-524 delect, 16290C/T, 16319G/A, 16356T/C was observed in liposarcoma Patients（P<0.05）, the difference was tatistically significant. The minor alleles of nucleotides 73 G,523–524del, 16290 T, 16319 A, 16356 C were associated with an increased risk for liposarcoma patients, whereas the insertion of C at the site 315（located within the D310） were associated with a decreased risk for liposarcoma patients.Conclusions:1 Mitochondrial DNA D-loop region was a highly Polymorphic region.2 The minor alleles of nucleotides 73 G,523–524del, 16290 T, 16319 A, 16356 C were associated with a increased risk. The people who carry the minor allele of 73 G,523–524del, 16290 T, 16319 A, 16356 C were an increased risk for liposarcoma.3 The minor alleles of nucleotides 315C/C insert were associated with a decreased risk. The minor allele of 315 C insert was an decreased risk for this illness.