The three major findings of this study were as follows: 1) Using the classic twin study design, we demonstrated that genetic factors were statistically significantly associated with variation in mtDNA content in peripheral blood lymphocytes. 2) Using a case–control study, we found that low mtDNA content was associated with a statistically significantly increased renal cell carcinoma risk. These results indicate that mtDNA content may be useful to identify subsets of individuals at increased risk of renal cell carcinoma. 3) Results of both case–control and twin studies indicated that mtDNA content is associated with smoking.
Although a number of protein factors encoded by nuclear genes have been shown to be involved in the replication, transcription, and maintenance of mtDNA, the extent to which mtDNA content is influenced by genetic factors has not been clearly established. Curran et al. (34
) in a family-based study among Mexican Americans showed that mtDNA content has a genetic component. However, such a study design cannot distinguish between shared environmental and/or genetic factors (24
). A twin study is the most powerful tool for direct assessment of a genetic component (35
), and we have now demonstrated high heritability of mtDNA content with such a classic twin study design.
The genetic loci and biologic mechanisms that regulate mtDNA content remain to be elucidated. Curran et al. (34
) found evidence that two genomic regions (10q11 and a small region on mtDNA) may harbor genes influencing variations in mtDNA content. Furthermore, they identified a total of 829 genes that had a statistically significant correlation with mtDNA content by analyzing genome-wide quantitative transcriptional profiles. In addition, a p53-binding sequence has been identified in mtDNA, indicating that p53 might be involved in the regulation of mtDNA replication (36
). Other factors such as Ras and p66shc have also been reported to contribute to the regulation of mtDNA content (37
). Further studies are warranted to identify genes that are responsible for mtDNA content regulation.
Although we estimated that the strong association between renal cell carcinoma and mtDNA content evident in twins was due solely to genetic factors, some of the additive genetic variance could reflect stronger intrauterine association among monozygotic twins than among dizygotic twins. This possibility would have to be evaluated in a different design such as a half-sib study with different mothers.
In the case–control study of renal cell carcinoma, we found that lower mtDNA content was associated with a 1.56-fold increased risk of renal cell carcinoma. To the best of our knowledge, this is the first molecular epidemiological study to evaluate mtDNA content in lymphocytes as a susceptibility biomarker for cancer. Reduced mtDNA content (or mtDNA depletion) has been previously detected in tumor tissue samples and has been associated with tumor progression. For example, Yamada et al. (38
) reported that mtDNA content was statistically significantly decreased in hepatocellular carcinoma as compared with the corresponding noncancerous liver tissues and that patients with low mtDNA content had shorter 5-year survival rates than those with higher mtDNA content. Wu et al. (17
) found that mtDNA depletion existed in gastric cancer and was associated with poor differentiation of cancer cells. In addition, Meierhofer et al. (23
) reported a decrease in mtDNA content in 34 (91%) of 37 renal cell carcinomas compared with control kidney tissue. Selvanayagam et al. (39
) likewise noted a decrease of mtDNA content and mRNA coding for NADH dehydrogenase subunit 3 in eight (61%) of 13 renal cell carcinomas compared with adjacent normal tissue. This phenomenon was also observed in five of six renal carcinoma cell lines (ie, SKRC29, SKRC42, CAK11, CAK12, SW 839, and ACHN) (39
). Thus, mtDNA depletion may be an important phenotype associated with neoplastic transformation of renal cells.
The mechanism underlying the role of mtDNA depletion in tumorigenesis remains under investigation. Warburg (40
) initiated research on the alteration of mitochondrial respiratory function in cancer more than half a century ago. He hypothesized that “injury” to the respiratory machinery was a critical event in carcinogenesis. Subsequently, a number of reports showed that mutations and depletion of mtDNA were common events in various types of malignancies (19
). Chandel et al. (43
) demonstrated that mtDNA depletion made cells resistant to apoptosis. Murine C2C12 skeletal myoblasts with depleted mtDNA exhibit an invasive phenotype and the overexpression of several tumor-specific markers (34
). Biswas et al. (44
) reported that depletion of mtDNA activated nuclear factor-kappa B (NFκB) and/or Rel factors through inactivation of NFκB inhibitor β. It has been demonstrated that mtDNA depletion alters mitochondrial gene expression and causes deficiency in oxidative phosphorylation and enhanced production of reactive oxidative species in aerobic metabolism, resulting in disruption of cellular functions (42
). However, further studies are warranted to fully elucidate the functional mechanism of mtDNA depletion.
Our case–control data indicated a non–statistically significantly higher percentage of smokers among control subjects than among case patients and a non–statistically significantly higher number of pack-years of smoking among control subjects. Although tobacco smoking is considered to be a risk factor for renal cell carcinoma, the data are still inconsistent. For example, a recent meta-analysis (45
) with 24 studies examining smoking and renal cell carcinoma observed 12 non–statistically significant associations. Likewise, a systematic review (46)
of 11 studies examining risk factors for renal cell cancer found an association between smoking and renal cell carcinoma among only seven of the studies. More studies on smoking intensity, duration, and duration since quitting are warranted to further elucidate the association between smoking and renal cell carcinoma.
In the current study, stratified analyses indicated that mtDNA profile may be modified by sex or smoking status. Specifically, women had statistically significantly higher mtDNA content than men among both case patients and control subjects. In the twin study, there was a similar trend (1.15 copies, 95% CI = 1.11 to 1.21 copies, vs 1.20 copies, 95% CI = 1.17 to 1.24 copies), although statistical significance was not reached (P
= .169). The biologic plausibility of this association is not clear. In addition, our results also showed that ever smokers had higher mtDNA content than never smokers among both case patients with renal cell carcinoma and healthy control subjects, as well as in twin study participants. Consistent with our findings, a previous study (47
) reported increased mtDNA content in response to cigarette smoking. This increase was thought to be a compensatory mechanism for oxidative damage to mtDNA and respiratory chain components caused by smoking.
Our study had several limitations. Although our data indicated that mtDNA content was associated with a statistically significant increased risk for renal cell carcinoma, the cause–effect relationship between mtDNA content and cancer is subject to the scrutiny of reverse causation. This is a limitation inherent in case–control study design. Future prospective epidemiological studies are needed to investigate the hypothesis that mtDNA depletion predisposes individuals to increased cancer risk. Another limitation of the current case–control study is the moderate sample size, which limits the statistical power to detect interactions between mtDNA content and environmental risk factors in renal cell carcinoma etiology. Future studies with larger sample size are warranted to further investigate the complex interplay between mtDNA content and other risk factors. Finally, our analysis was restricted to white subjects; data from other ethnic groups would be valuable for comparison.
In summary, our study provides evidence that decreased mtDNA content is statistically significantly associated with risk of renal cell carcinoma. Our study also demonstrates a strong genetic component for mtDNA content that warrants additional research into the utility of mtDNA content as a genetic marker for cancer susceptibility. These results also indicate that phenotypic screening for mtDNA content might be a useful screening test for renal cell carcinoma.