The Relevance Of Single Nucleotide Polymorphisms At D-loop Region Of Mitochondrial DNA And Prognosis Of Epithelial Ovarian Cancer Patients

Objective: Mitochondrial DNA (mtDNA) is the nique genetic materialexcept for the nuclear genes. Mitochondria known as “power plant” is animportant organelle in eukaryotes, and is the center of energy metabolism incells. MtDNA is believed to be more vulnerable to damage than nuclear DNA(nDNA) due to exposure to a high oxygen environment directly, lacking ofprotective histones, and limited capacity for DNA repair. The mutations ofmtDNA are easy to happen and accumulate, leading to the tumorigenesis. Sothe mutations of mtDNA are popular in cancer tissues.The human mtDNA is a16569bp, closed circular, double-strandedmolecule and it contains coding region and non-coding region. The codingregion encodes37genes, which composed by13protein genes, two ribosomeRNAs and22tRNA. D-loop region is the only non-coding region of mtDNAand contains3hypervariable regions. The D-loop plays an importantregulatory role in mtDNA transcription and replication because it contains theimportant structures of replication origin of the heavy strand and transcriptionpromoters of the light and heavy strand, so D-loop is known as the controlregion of mtDNA. The recent studies have reported that D-loop regionmutations were associated with cancer risk and prognosis of many tumors, butthere are few study on the relationship between single nucleotidepolymorphisms (SNPs) at D-loop region of mtDNA and prognosis ofepithelial ovarian cancer (EOC). In this study, we analyze the relevance ofSNPs at D-loop region of mitochondrial DNA and prognosis of EOC patients,and access the relationship between clinical characteristics and prognosis ofEOC patients, which contribute to providing theoretical basis for a reasonabletreatment plan and prognosis evaluation of EOC patients. Methods:1Sample collection and patients following-up:1This study included89patients who received operative treatment in the Second Hospital of HebeiMedical University between2005and2009. The participants werehistologically confirmed primary epithelial ovarian cancer and Han nationality.The patients did not have neoadjuvant chemotherapy or hormonic therapybefore surgical treatment. Formalin-fixed, paraffin-embedded tissues forexperiment were collected after operation. The patients’ clinical characteristics(gender, age, clinical stage, et al) were accurately recorded. We followed upfor3-years survival status of post-operation patients by outpatient clinic,phones and letters. The follow-up deadline was December2012.2mtDNA extraction, amplification and sequencing: The mtDNA wasextracted with DNA purification kit from formalin-fixed, paraffin-embeddedtissues. The target gene of qualified DNA samples was then amplified by PCR.The products were confirmed by agarose gel electrophoresis and purified priorto sequencing. Cycle sequencing was carried out with the Big Dye TerminatorCycle Sequencing Ready Reaction Kit (Applied Biosystems) and the productswere then separated on the ABI Genetic Analyzer3730XL (AppliedBiosystem). Polymorphisms were confirmed by repeating the analysis on bothstrands.3Statistical analysis: Statistical analysis was performed using the IBMSPSS19.0software package (IBM Company, New York, USA). Survivalanalysis was performed by the Kaplan-,Meier and compared,by the log-,ranktest in each model. Factors related to the prognosis were analyzed by Coxregression model. A P-value of <0.05was considered statistically significance.Results:1The univariate survival analysis showed that the SNP sites ofnucleotides309C/T,324C/G and446C/A were identified for prediction ofpost-operational survival. The3-year survival rate of309C genotype and Tgenotype were69.88%and33.33%. Patients of C genotype has longersurvival time than T genotype patients (P=0.021). The3-year survival rate of 324C genotype and G genotype were69.88%and33.33%. The length ofsurvival of patients with C genotype was significantly longer than that ofpatients with G genotype (P=0.004). The3-year survival rate of446Cgenotype,and A genotype were69.05%and40%. The prognosis of patientswith T genotype was superior to that of patients with C genotype (P=0.044).2The univariate survival analysis showed that clinical characteristics,such as types of pathological tissue, chemotherapy, were not associated withthe post-operative survival rate of EOC cancer patients. Age, FIGO stage andtumor residual size were statistically significant predictors of the length ofpost-operational survival.3The factors which have significant difference in univariate analysiswere then analyzed by Cox regression model. It showed that the alleles309and324, FIGO.stage were as independent predictors for EOC outcome. Thedeath risk of patients with T genotype was4.063times of C genotype at the309allele. The death risk of patients with G genotype was5.636times of Cgenotype at the324allele. The mortality risk of patients with stage Ⅲ-Ⅳgroup was2.929times of stage Ⅰ-Ⅱ group.4The SNP of D-loop and age-at-onset of EOC: The SNP sites ofnucleotide248,524and16304alleles were identified for their associationwith age-at-onset by the log-rank test. The age-at-onset of patients with theminor allele248G was lower than that of patients with248A. The same wasseen for the rare allele16304T genotype when compared with matched. allele16304C at the16304site. But the age-at-onset.of patients with the minorallele524C was higher than that of patients with frequent524deletion.Conclusions:1The SNPs of309C/T,324C/G of mtDNA were independent factors forprognosis of EOC patients. The length of survival of patients with T genotypewas significantly longer than that of patients with C genotype.2Age, FIGO stage and tumor residual size were statistically significantpredictors of the length of EOC post-operational survival. And FIGO stage isindependent predictors of EOC outcome. Patients of stage Ⅰ-Ⅱ group have longer survival time than stage Ⅲ-Ⅳgroup patients.3Single nucleotide polymorphisms in the D-loop could be used as thepredictive markers for age-at-onset in EOC patients. Accordingly, the analysisof genetic polymorphisms may help identify EOC patient subgroups with highrisk of early onset.