In the present study, mtDNA haplogroups D and F were found to be beneficial for individuals' resistance to lung cancer, whereas haplogroups G and M7 were associated with an increased risk for lung cancer in a Han Chinese population from southwestern China. In addition, cigarette smoking was a risk factor for the 822 bp mtDNA deletion and mtDNA haplogroups D was susceptible to damage from external ROS caused by cigarette smoking. The findings provided evidences from the point of mtDNA that the gene-environment interaction does exist in the individual susceptibility to lung cancer. To our knowledge, this is the first study to report the association of lung cancer risk with the mtDNA variations and of the cigarette smoking with the 822 bp mtDNA deletion that begins between 15587–15591 nps and ends between 16408–16412 nps.
The 822 bp mtDNA deletion was accidentally found to be present in many samples when the primers mtDNA-1 and mtDNA-2 were used to amplify the mtDNA fragment for subsequent sequencing of HVS I in the study. In principle, by using the nested-PCR protocol, it is possible to concentrate deleted molecules at the first PCR step and detect single molecules. Thus, all samples (cases and controls) were then re-amplified using the more sensitive nested-PCR method to detect extremely low levels of the mtDNA deletion. To investigate the role of the mtDNA deletion in the study population, the frequencies of mtDNA deletion were compared between light cigarette smoking subjects and heavy cigarette smoking subjects of combined cases and controls. The comparison revealed that the mtDNA deletion was significantly more frequent in heavy smoking subjects compared with light smoking subjects. Multivariate logistic regression analysis revealed that cigarette smoking was a risk factor for the mtDNA deletion. Many of the substances in cigarette smoke are chemicals that may create ROS within human body to introduce oxidative stress which was thought to be involved in lung carcinogenesis 
and mtDNA mutations 
. Recently, it has been reported that the increased mtDNA copy number was associated with future development of lung cancer among heavy smokers for compensation for damage due to limited DNA repair capacity of mitochondrial 
. Thus, the prior findings that heavy cigarette smokers would have higher internal doses of ROS 
and increased mtDNA copy number 
than lighter cigarette smokers supported our findings that heavy smokers would have increased frequencies of the mtDNA deletion compared with light cigarette smokers.
MtDNA deletions are generally believed to be the results of oxyradical-induced DNA damage, but the mechanism of deletion is poorly understood. Most mtDNA deletions are predominantly (~85%) flanked by short direct repeats 
. Presently, there are two proposals about the formation of mtDNA deletions. One proposal is that mtDNA deletion could be generated through a slipped-stand replication mechanism 
. But the proposal was challenged by the recent modification of the strand-displacement model which argues that large regions of single-stranded DNA do not exist, rather, the lagging-strand template is largely protected by RNA 
. Another proposal is that mtDNA deletions could be generated during repair of damaged DNA caused by increased oxidative stress from whatever causes 
. The later proposal was supported by many evidences from E. coli
, mice to human cells 
. The 822 bp mtDNA deletion detected in the study is also flanked by 5 bp short direct repeats (CTCCG
) (). Our findings provided indirect evidence to support the later proposal that the mtDNA deletions could be created during repair of damaged DNA generated by cigarette smoking. Understanding the mechanism involved in generation and subsequent clonal expansion is worthwhile to investigate further.
Compared with controls, the mtDNA deletion was found to be enriched in cases. In order to investigate whether the mtDNA deletion was associated with some mtDNA haplogroups, the frequencies of the mtDNA deletion in major mtDNA haplogroups of combined cases and controls were analyzed and the data revealed that the deletion occurred significantly more frequently in mtDNA haplogroups D and multivariate logistic regression analyses revealed that haplogroup D might be a risk factor for the mtDNA deletion. Since the cigarette smoking was a risk factor for the mtDNA deletion and no female cigarette smoking subjects were gathered in the study, the male subjects pooled from cases and controls were stratified by cigarette smoking into male cigarette smoking subjects and male non-cigarette smoking subjects to analyze further the association of mtDNA deletion with mtDNA haplogroups. The analysis of the frequencies of the mtDNA deletion in major mtDNA haplogroups among male cigarette smoking subjects also revealed that haplogroup D might be risk factors for the mtDNA deletion. No similar significant difference was found among male non-cigarette smoking subjects. However, mtDNA haplogroup G was found to be a risk factor for the deletion among male non-cigarette smoking subjects. Interestingly, the deletion was enriched in female non-cigarette smoking subjects compared with male non-cigarette smoking subjects of combined cases and control. One reason we hypothesized to explain this difference is that cooking oil fumes could be a greater risk factor for the mtDNA deletion for females who cook far more often than men in southwestern China.
Several mtDNA haplogroups were found to play roles in human longevity, carcinogenesis, Leber's hereditary optic neuropathy (LHON), and other metabolic and degenerative diseases. MtDNA haplogroup D is one of these mtDNA haplogroups. Haplogroup D is defined by the specific variation C5178A in mitochondrial NADH dehydrogenase subunit 2 (ND2). Prior studies showed that the protective effect of a Leu→Met substitution at amino acid 237 (L237M) of ND2 (C5178A) against oxidative damage to mitochondria not only contributes to human longevity 
, but also provides strong anti-atherosclerotic effects in diabetic patients and protects against myocardial infarction 
. Thus, the protective effect of haplogroup D against oxidative damage might also decrease the risk of lung cancer. The frequency of haplogroup J, which was found to be correlated with lower efficiency of the electron transport chain (ETC), diminished ATP, diminished ROS production, and accumulation in elderly people, was increased in patients with LHON and multiple sclerosis due to its limited power to compensate the mitochondrial energetic deficiency 
. Similarly, haplogroup D could account for the increased frequency of the C5178A variant in elderly people, which is caused by decreased oxidative damage 
. However, once the external ROS created by cigarette smoking increases, the frequency of the mtDNA deletion increases in cigarette-smoking subjects with haplogroup D. One reason we hypothesized to explain the phenomena that the mtDNA haplogroup D was found to be protective in lung cancer while the mtDNA deletion was enriched in male cigarette-smoking subjects with mtDNA haplogroup D of combined cases and controls is that individuals with haplogroup D might be susceptible to damage of external ROS caused by cigarette smoking. The methionine residues have been proposed to constitute an important anti-oxidant defense mechanism 
. According to the predicted model of human ND2 molecular 
, the Leu→Met substitution at amino acid 237 (L237M) of ND2 (C5178A) is exposed at the surface of complex I and may play important roles in protective effect against oxidative damage to mitochondria as an efficient oxidant scavenger 
. Localization of the methionine residue (Leu→Met) on the surface of the complex I might also be a target for damage of increased ROS whatever caused. The damaged residue or surface structure of the complex I might cause or exacerbate the deletion of mtDNA. The other reason we supposed to explain the phenomena is that the lung cancer risk and the mtDNA deletion were not influenced solely by the mtDNA variations. The nuclear background in which the mtDNA haplogroups are classified and the mtDNA deletion was found may also contribute to the individuals' susceptibility to lung cancer risk and the mtDNA deletion. Investigating the generation of endogenous ROS and the protective effect against oxidative damage to mitochondria under different conditions of different mtDNA haplogroups with the same nuclear background, or investigating the different nuclear background with the same mtDNA haplogroups, may help us, at least in part, to explore the fundamental mechanisms.
Haplogroup F was also identified less often in lung cancer cases as haplogroup D, but no significant difference was found in the frequencies of the mtDNA deletion among male cigarette-smoking subjects with haplogroup F. Haplogroup F has been divided into sub-haplogroups F1–4 in East Asia 
. We speculate that haplogroup F might confer decreased lung cancer risk through some antioxidant defense mechanism. It is worthwhile to investigate further the role of haplogroup F in lung cancer resistance.
Both haplogroups M7 and G were found to be risk factors for lung cancer in this study. Haplogroup M7 was defined by a specific variation at np T9824C 
and Haplogroup G was classified with the presence of the combined RFLP-4830 Hae
II site/+4831 Hha
I site 
. Previous studies have demonstrated that M7b1'2, a sub-haplogroup of M7, was found to increase the penetrance of LHON 
and haplogroup M7 was a risk factor for mild acute mountain sickness (AMS) 
. In the present study, haplogroups G and M7 were found to be present in controls at frequencies of 2.78% and 5.95%, respectively, but were present in cases of lung cancer at frequencies of 7.92% and 12.67%, respectively. The findings that no significant difference was observed in the frequencies of the mtDNA deletion in male cigarette smoking subjects with haplogroup M7 or G indicated that the two haplogroups might better tolerate the damage of external ROS caused by cigarette smoking due to their relatively higher endogenous ROS production, which was supported by the subsequent finding that the frequencies of the mtDNA deletion increased in male non-cigarette smoking subjects with haplogroup G.
Though we conducted a population-based case-control study, there were several limitations. First, due to limited cases, we did not analyze the distribution of the mtDNA haplogroups or the mtDNA deletion in each type of lung cancer to investigate whether the risk of a certain type of lung cancer was associated with one or more mtDNA haplogroups or with the mtDNA deletion. Second, because the distribution of mtDNA haplogroups varies spatially in China, large-scale studies are required to clarify the association of mtDNA variation with lung cancer risk in other populations.
In summary, we conducted a case-control study to investigate the role of mtDNA variations in individual risk of lung cancer in a Han Chinese population from southwestern China. Our findings suggested that mtDNA haplogroups G and M7 might be risk factors for lung cancer, whereas haplogroups D and F might decrease the risk of lung cancer in the study population. In addition, the study revealed that mtDNA haplogroup D might be relatively more susceptible to DNA damage from the external ROS caused by cumulative cigarette smoking. And the 822 bp mtDNA deletion, which was positively associated with cigarette smoking, would be a potential biomarker for the exogenous and endogenous exposures that are associated with subsequent cigarette-smoking-related diseases.