Relationship Between Sequence Polymorphisms In The D-loop Region Of Mitochondrial DNA And The Risk And Outcome Of Non-hodgkin Lymphoma

Objective： The mitochondrial D-loop in a variety of tumor cells hasbecome a research hotspot， but the related research between mtDNAD-loop and malignant lymphoma is relatively less. This experiment tostudy the single nucleotide Polymorphisms （SNPs） in mitochondrialD-loop region and explore the correlation between SNPs and the risk andoutcome of non-Hodgkin lymphoma.Methods：1Blood samples were collected from133NHL Patients who receivedtreatment in the resPiratory department at the Fourth Hospital of HebeiUnivercity from January2001to January2004. All of the Patients belong toHan nationality of Hebei area. Blood samples were also collected from159healthy controls.All Patients clinical data is relatively compeletly.2The mitochondria DNA of non-hodgkin lymphoma Patients and healthycontrols were extracted using the GENMED Whole Blood MitochondrialDNA extraction kit，and stored them at-80℃for later use.3Using PCR to amplificate the mitochondrial DNA D-loop region，theforword Primer5’-CCCCATGCTTACAAGCAAGT-3’（nucleotides16190-16209） and reverse Primers5’-GCTTTGAGGAGGTAAGCTAC-3’（nucleotides602-583），were used for amplification of a982-bp Product fromthe mtDNA D-loop region. PCR was Performed according to the ProtocolPCR Master Mix Kit and Purified Prior to sequencing. the Products were thenseparated on the ABIPRISM Genetic Analyzer3100. Polymorphisms wereconfirmed by repeated analyses from both strands. Subsequently tested thesingle nucleotide Polymorphisms of mitochondrial DNA D-loop region.4The Patients were followed up by telephone for10years，and then the 10year survival rate was calculated and its ralationship with clinical featureswas analyzed. AC₀00021database was referred to in order to find SNPsfrom the982bP mitochondrial D-loop region，and the relationship betweendifferent SNPs and10year survival rate was analyzed.5Statistical analysis：the SNPs sites showing frequency was analyzed byχ2test; The survival curve was calculated using the Kaplan–Meier method，and compared with the log-rank test. Multivariate survival analysis wasPerformed using a COX Proportional Hazards model. All of the statisticalanalysis was done with the SPSS18.0software Package. A P value of <0.05was considered statistically significant.Results：1133NHL （including54cases of diffuse large B cell lymphoma，41cases of T cell lymphoma，and38other types of non-Hodgkin lymphoma）were enrolled in this study，a total of91Patients with10years follow-up data，including32DLBCL，33TCL and26other types of non-Hodgkin lymphoma.2SNPs were detected in140sites within the982-bp mitochondria D-loopregion from the blood samples of the healthy controls and NHL Patients.TheSNPs with a rare allele frequency>5%in either controls or NHL Patients wereused for cancer risk analysis， a total of26SNPs were selected.3When individual SNPs were analyzed in NHL，a statistically significantdecrease of SNP frequency for73A/G，263A/G�'�315C/Cinsert was observedin NHL Patients（P<0.05）， the difference was statistically significant.Additional SNPs of200G/A were significantly associated with an risk fordiffuse large B-cell lymphoma（DLBCL）（P<0.05）， the difference wasstatistically significant. The minor allele of nucleotides16362C/T and16362Del/A is associated with the risk of T-cell lymphoma（P<0.05），the differencewas statistically significant..4No statistical difference for SNPs frequency distribution referring toage （With60years old is bounded） and sex was observed （P>0.05）; Theclinical characteristics including Ann Arbor Stage and IPI score display theirPotential association with10-year survival rate （P <0.05）. 5According to the26SNPs which had been found tocalculate thesurvival curve， finding that The nucleotide site146C/T、309Cinsert/C、315Cinsert/C、199C/T、16304C/T were identified for their association with NHLoverall survival at statistically significant levels. We analysised therelationship among the SNPs、clinical characteristics and10year survival ratewith the Cox Proportional hazards model， finding that the16304C/T alleleswere identified as independent Predictors for NHL outcome. The length ofsurvival for Patients with the minor allele16304C genotype was significantlyless than that for Patients with the frequent allele16304T （relative risk，0.513；95%CI，0.266–0.989；P=0.046） at the16304site.Conclusions：1Mitochondrial DNA D-loop region was a highly Polymorphic region.2The minor alleles of nucleotides73A/G，263A/G，315C/C insert wereassociated with a decreased risk. The people who carry the minor allele of73G、263G、315Cinsert were an increased risk for NHL.3SNPs of200A/G were significantly associated with the diffuse largeB-cell lymphoma （DLBCL）， the minor allele of200G was an increased riskfor this illniss.4The minor allele of nucleotides16362C/T and249Del/A werespecifically associated with the decreased risk of T-cell lymphoma，the minorallele of16362T and249A were an increased risk for this illniss.5The nucleotide site146C/T、309Cinsert/C、315Cinsert/C、199C/T、16304C/T and Ann Arbor stage，IPI were identified for their association withNHL overall survival. The16304alleles were identified as independentPredictors for NHL outcome.