Association Of Mitochondrial DNA Variations With Lung Cancer Risk In A Han Chinese Population From Southwestern China

BackgroundLung cancer has replaced liver cancer to become the leading cause of cancer-relateddeaths in China, accounting for22.7%of all cancer deaths. The rates of morbidity andmortality continue to rise rapidly and the lung cancer patients will reach one million in2025if no effective control measures were taken. Cigarette smoking and asbestos are thetwo major causes of lung cancer, however, not all of those who have been exposed to therisk factors will develop lung cancer, suggesting that other causes, including geneticsusceptibility, might contribute to the individual lung cancer risk and that gene-environmentinteractions may exist.Until now, many studies have been focused on the genetic variants of nuclear genomicDNA encoding genes to investigate the gene–environment interaction of genetic variants ofgenes with lung cancer risk. However, very few studies have investigated the role ofmitochondrial DNA (mtDNA) variations in individual susceptibility to lung cancer.Through the Krebs cycle and oxidative phosphorylation (OXPHOS), mitochondria produceboth ATP to help cells survive and approximately85%of intracellular reactive oxygenspecies (ROS), which can promote cellular differentiation or induce apoptosis. HumanmtDNA is a circular molecule of approximately16.5kb, encoding22transfer RNAs(tRNAs),2ribosomal RNAs (rRNAs) and13respiratory chain subunits that are essentialfor the respiratory functions of the mitochondria. Prior studies have demonstrated that somemtDNA haplogroups are associated with human susceptibility to metabolic anddegenerative diseases, and influence longevity and carcinogenesis in conditions wheremitochondrial ROS production is thought to play a role. In addition, the common and“non-pathological” mtDNA variations that define mtDNA haplogroups have been found todetermine differences in OXPHOS performance and ROS production in mice and humans. It has been established that mtDNA is particularly susceptible to oxidative damage andmutation due to the high rate of ROS production and limited DNA-repair capacity inmitochondria. Cigarette smoking is one exposure that induces oxidative stress by creatingROS within the human body. Chronic oxidative stress may induce mtDNA damage, leadingto point mutations, insertions or deletions. The accumulation of oxidative damage and theresulting sequence variations in mtDNA may ultimately lead to abnormal OXPHOS inaffected cells, which may play a role in the occurrence of mitochondrial-related diseases.Recently, the increased mtDNA copy number for compensation for damage has been foundto be positively associated with subsequent lung cancer risk among heavy smokers.Given that the common and “non-pathological” mtDNA variations that define anmtDNA haplogroup contribute to differences in OXPHOS performance and intracellularROS production and that the level of ROS present is an important determinant of cancerrisk, we hypothesize that the mtDNA variations defining some mtDNA haplogroups mayplay a role in susceptibility to lung cancer. To test this hypothesis, a case-control study wasconducted to investigate the role of mtDNA variation in lung cancer risk in a Han Chinesepopulation from southwestern China. In addition, the gene-environment interactionsbetween the mtDNA variations and cumulative cigarette smoking were analyzed.Materials and MethodsStudy group:Patients (n=442) with primary lung cancer diagnosed from September2007to December2008were recruited from the Institute of Human Respiratory Disease ofXinqiao Hospital. All patients were newly diagnosed, histologically confirmed andpreviously untreated.504age and sex-matched control samples were collected fromindividuals at the Center of Physical Examination of Xinqiao Hospital between November2007and December2008. The exclusion criterion for the control group was any history ofcancer. All of the subjects were unrelated at least within three generations. After explainingthe purpose and procedures of the study, all participants signed a written informed consentform and completed a detailed questionnaire regarding their smoking habits. Blood sampleswere drawn into Na-EDTA tubes from all subjects and stored at-70oC for genomic DNAextraction. The study was approved by the Ethical Committee of Xinqiao Hospital, ThirdMilitary Medical University.MtDNA haplogrouping:Genomic DNA was extracted from whole blood using the QIAamp DNA Blood Mini Kit according to the manufacturer’s instructions (QIAGEN,Maryland, USA). The entire mtDNA sample was amplified into22overlapping PCRfragments and then digested with14restriction endonucleases. A negative control wasincluded in each PCR-restriction fragment length polymorphism (PCR-RFLP) analysis formtDNA haplogrouping to avoid artificial contamination caused by potential samplecrossover. PCR-RFLP analysis was supplemented by sequencing the hypervariable segmentI (HVS-I, from positions16,024to16,383, relative to the revised Cambridge ReferenceSequence of mtDNA, rCRS).Detection of an822bp deletion in mtDNA: The amplification of the fragment ofmtDNA822bp deletion was performed by using PCR with a pair of specific primersdesigned according to the gene sequence issued by Genbank. The PCR products werevisualized using electrophoresis on1.5%agarose gels stained with ethidium bromide. Fivebands containing the deletion selected randomly were cut from agarose gels and DNA waspurified using a DNA Gel Extraction Kit (GENERAY, Shanghai, China). Then the purifiedDNA was cloned into the pMD18-T vector according to the manufacturer’s instructions(TaKaRa, Dalian, China). Twenty clones were randomly selected for subsequent sequencing.The locations of the deletion(s) boundaries were determined by alignment of the PCRproduct sequence with the rCRS of mtDNA. In order to detect extremely low levels of themtDNA deletion, a nested-PCR method was applied. The PCR products were visualizedusing electrophoresis on2.0%agarose gels stained with ethidium bromide.ResultsMtDNA haplogroupingMultivariate logistic regression analyses with adjustments for age, gender, andsmoking revealed that, on the basis of a p value of <0.05, haplogroups D and F wereassociated with individuals’ lung cancer resistance (OR=0.465,95%CI=0.329–0.656, p<0.001; and OR=0.622,95%CI=0.425–0.909, p=0.014, respectively), whilehaplogroups G and M7might be risk factors for lung cancer (OR=3.924,95%CI=1.757–6.689, p <0.001; and OR=2.037,95%CI=1.253–3.312, p=0.004, respectively).Detection of the small deletion in mtDNAPrimers mtDNA-1and mtDNA-2were primarily used to detect the deletion of mtDNA.In the wide type mtDNA, the primers only yielded a product with1129bp. In subjects with the mtDNA deletion, the primers amplified products with1129bp and an aberrant productabout300bp. DNA fragments purified from five randomly selected bands containing thedeletion were cloned into pMD18-T vector and sequenced. Sequencing data revealed thatthe deleted portion of mtDNA fragment was approximately822bp, starting between15587–15591nucleotide positions (nps) and ending between16408–16412nps. Becauseboth ends of the deleted region have the5bp same nucleotides (CTCCG,5bp short directrepeats), the exact start and end points of the deletion could not be determined. In order todetect extremely low levels of the mtDNA deletion, a nested-PCR method was applied forall of the cases and controls. In the wide type mtDNA, the primers mtDNA-1and mtDNA-3only yielded a product with956bp. In subjects with the mtDNA deletion, the primersamplified products with956bp and an aberrant product with134bp.Distribution of the822bp mtDNA deletion among cases and controlsPearson’s chi-square test or Fisher’s exact test revealed that the deletion occurredsignificantly more frequently in heavy smoking subjects (p <0.001). Compared withcontrols, the mtDNA deletion was significantly enriched in cases of lung cancer (p <0.001).Multivariate logistic regression analyses with adjustment for age, gender, and smokinghabits revealed that the risk of lung cancer cases for the mtDNA deletion was3.8timeshigher than that of controls (OR=3.776,95%CI=2.662-5.355, adjusted p <0.001).Multivariate logistic regression analyses adjusted for age revealed that the risk of femalenon-smoking subjects for the mtDNA deletion was5.8times higher than that of malenon-smoking subjects (OR=5.814,95%CI=3.279-10.309, adjusted p <0.001).Distribution of the822bp mtDNA deletion in major mtDNA haplogroups ofcombined cases and controlsPearson’s chi-square test or Fisher’s exact test revealed that the deletion occurredsignificantly more frequently in subjects with mtDNA haplogroups D (p <0.001).Multivariate logistic regression analyses adjusted for age, gender and smoking habitsrevealed that haplogroup D might be a risk factor for the822bp mtDNA deletion (OR=1.906,95%CI=1.359-2.738, adjusted p <0.001). Haplogroup D was found to be a riskfactor for the mtDNA deletion among male cigarette smoking subjects (OR=2.752,95%CI=1.699-4.456, adjusted p <0.001). The deletion was enriched in subjects with mtDNAhaplogroups G among male non-cigarette smoking subjects (p=0.036). Conclution:(1)The haplogroups D and F were related to individual lung cancerresistance;(2)The hplogroups G and M7might be risk factors for lung cancer;(3)Cigarettesmoking was a risk factor for the822bp mtDNA deletion;(4)The haplogroup D might besusceptible to DNA damage from external ROS caused by heavy cigarette smoking.