Mutagenesis Advance Access originally published online on February 4, 2008
Mutagenesis 2008 23(2):137-142; doi:10.1093/mutage/gen002
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Tandem duplication/triplication correlated with poly-cytosine stretch variation in human mitochondrial DNA D-loop region
1Department and Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China 2Department of Radiation Oncology, Taichung Veterans General Hospital, Taiwan, Republic of China 3Department of Urology, Taipei Medical University—Affiliated Taipei Municipal Wan-Fang Hospital, Taiwan, Republic of China 4Department of Surgery, Taipei Veterans General Hospital and National Yang-Ming University, Taipei, Taiwan, Republic of China 5Department of Medical Research and Education, Taipei Veterans General Hospital, Taiwan, Republic of China
Somatic mutations in the mitochondrial DNA (mtDNA) displacement loop (D-loop) region have been frequently detected in various human cancers. In a previous study, we identified a polyplasmic 260-bp tandem duplication and triplication mutation in the mtDNA D-loop of one gastric cancer. In the present study, we adopted a more sensitive back-to-back polymerase chain reaction method to screen for this 260-bp tandem duplication/triplication in 197 cancers and their adjacent non-cancerous tissues. Nine samples of primary cancer (4.6%) were found to harbor the tandem duplication/triplication and these were made up of four out of 31 (12.9%) gastric cancers, two out of 45 (4.4%) breast cancers, two out of 56 (3.6%) hepatocellular cancers and one out of 32 (3.1%) colon cancers, but no tandem duplication/triplication was present in any of 33 lung cancers. We also found an expanded and polyplasmic poly-cytosine (poly-C) stretch around nucleotide position (np) 568 in eight of the 197 (4.1%) cancer patients. All the eight cancer samples carried the 260-bp tandem duplication/triplication. In addition, we detected the np 568 poly-C length variations in 11 of 234 (4.7%) peripheral blood samples of non-cancer population and the 260-bp tandem duplication in nine of the 11 cases with the np 568 poly-C length variations. These observations suggest that the occurrence of the tandem duplication/triplication in mtDNA D-loop is not specific for cancer tissues, but highly associated with the poly-C length variations around np 568.
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Introduction |
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Human mitochondrial DNA (mtDNA) is a 16.6-kb circular double-strand DNA molecule, which encodes 13 essential polypeptides for the respiratory chain and a set of RNAs (2 rRNAs and 22 tRNAs) for intramitochondrial translation (1
). Among these tightly packed genes, there is an 
1.1 kb of non-coding DNA region named the displacement loop (D-loop), which contains sequences that are important for the initiation of mitochondrial replication and transcription (2). The DNA within the mitochondrion lacks protective histones and is therefore at risk due to continuous exposure to reactive oxygen species generated during oxidative phosphorylation. This, together with a relatively poor DNA repair system within the mitochondrion results in mtDNA being considerably more vulnerable to oxidative damage than nuclear DNA. Oxidative damages and the resultant mutations in the mtDNA are able to persist and accumulate at a rate of 10-fold higher than in nuclear DNA (3,4).
Over the past few years, many types of somatic mtDNA mutations have been found in various human cancers and have included point mutation, deletions, insertions and altered mtDNA copy number (5–10). Some of these mutations in mitochondrial genomes result in missense mRNA transcription or functional alterations in the mitochondrial genome-encoded proteins (5,11,12). On the other hand, somatic mutations that occur in the D-loop region have been suggested to be associated with a decrease in mtDNA copy number (13–15). Increasing evidence seems to reveal a correlation between the presence of mtDNA mutations and malignant clinicopathological features or poor disease prognosis (16–19).
In a previous study, we identified a 260-bp tandem duplication/triplication mutation in the mtDNA D-loop region of cancerous tissues from one gastric cancer patient (18). The duplicated/triplicated region was 260-bp fragment that is flanked by two poly-cytosine (poly-C) stretches at nucleotide positions (nps) 303–309 and 568–573. Tandem duplications in the mtDNA regulatory region have been identified in patients with mitochondrial myopathy (20–22), in aged subjects (23,24) and in a particular Caucasian mtDNA haplogroup (25). However, it is still unclear whether or not the tandem duplication/triplication within the mtDNA occurred in different types of human cancers. In this study, we adopted a sensitive back-to-back polymerase chain reaction (PCR) method to determine the occurrence of the tandem duplication/triplication in 197 human cancerous tissues, which were made up of 31 gastric, 45 breast, 56 hepatocellular, 32 colon and 33 lung cancers. In addition to the detection of the duplicated/triplicated region, the presence of length variations in the poly-C area around np 568 in these cancers was also analyzed.
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DNA samples
The primary cancerous tissues included gastric cancer (31 cases), breast cancer (45 cases), liver cancer (56 cases), colon cancer (32 cases) and lung cancer (33 cases); each sample was associated with an adjacent non-cancerous tissue sample obtained at the same time. The mtDNA D-loop point mutations in the same set of samples have been reported previously (8
,13,14,18,19). In total, 197 cancer patients were sampled with their consent at Taipei Veterans General Hospital and Taichung Veterans General Hospital. In addition, peripheral blood cells from 234 non-cancer volunteers were collected with their consent. All the tissues were stored in liquid nitrogen immediately after surgical resection following protocols approved by the medical ethics committee for conducting human research at the two hospitals. Total DNA from the tissue and blood samples was extracted using the QIAamp DNA Mini kit (QIAGEN, Germany) according to the manufacturer's instructions and then stored at –30°C until use.
Detection of the 260-bp tandem duplication/triplication in mtDNA D-loop region
For the conventional PCR method, the mtDNA D-loop region was amplified using the primers L76 (5'-CAC GCG ATA GCA TTG CGA GAC GCT G-3') and H602 (5'-GCT TTG AGG AGG TAA GCT AC-3') as described previously (8,13,14,18,19). In this study, tandem duplication/triplication occurring in mtDNA D-loop region was detected by a back-to-back PCR method as described previously (20,23). The mtDNA-specific primer pairs used in this method were H335 (5'-TAA GTG CTG TGG CCA GAA GC-3') and L336 (5'-AAC ACA TCT GCC AAA CCC-3'). PCR was carried out for 40 cycles in a 50-µl reaction mixture containing 100 ng DNA, 200 µM of each deoxyribonucleotide triphosphate (dNTP), 40 pmol of each primer, 1.0 U of Taq DNA polymerase and 1x reaction buffer. The PCR cycles consisted of 15 s denaturation at 94°C, 15 s annealing at 58°C and 90 s primer extension at 72°C. The PCR products were then separated by electrophoresis on a 1.5% agarose gel and detected under UV transillumination after ethidium bromide staining.
DNA sequencing
The primer pair L336 and H2216 (5'-TGT TGA GCT TGA ACG CTT TC-3') was used to amplify the mtDNA fragment containing the poly-C tract at np 568. PCR was performed for 35 cycles in a 50-µl reaction mixture containing 100 ng DNA, 200 µM of each dNTP, 40 pmol of each primer, 2.5 U of PfuUltra high-fidelity DNA polymerase (Stratagene, La Jolla, CA) and 1x PfuUltra HF reaction buffer (Stratagene, La Jolla, CA). The PCR cycles consisted of 30 s denaturation at 94°C, 30 s annealing at 58°C and 2-min primer extension at 72°C. The PCR products were sequenced with an ABI Big Dye Terminator (version 3.1) cycle sequencing ready reaction kit and an ABI PRISM 3100 sequencer (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.
Semiquantitative PCR
The proportion of the mtDNA molecule harboring the tandem duplication/triplication was measured using a semiquantitative PCR method as described previously (23). Briefly, total DNA of each tissue sample was serially diluted by 2-fold with distilled water. The primers L3540 (5'-TCT CAC CAT CGC TCT TCT AC-3') and H3887 (5'-TTG GTC TCT GCT AGT GTG GA-3') were used to amplify a 348-bp DNA fragment from total mtDNA molecules, and the primers L336 and H335 were used for the amplification of a 260-bp PCR product from the mtDNA molecules that harbors the tandem duplication. PCR was performed for 35 cycles in a 50-µl reaction mixture containing 100 ng DNA, 200 µM of each dNTP, 40 pmol of each primer, 1 U of Taq DNA polymerase and 1x reaction buffer. The PCR cycles consisted of 15 s denaturation at 94°C, 15 s annealing at 58°C and 40 s primer extension at 72°C. Amplified DNA fragments were separated by electrophoresis on 1.5% agarose gels and were detected fluorographically under UV light transillumination after staining with ethidium bromide. The proportion of the duplicated mtDNA was determined by the ratio of the highest dilution fold that allowed the 260-bp PCR product to be visible on the gel to that which allowed the 236-bp PCR product to be visibly amplified from the total mtDNA under identical condition.
Correlation with clinical parameters
Fisher's exact test was used to compare mtDNA tandem duplication/triplication and clinicopathological parameters. The overall survival rates of patients with and without the mtDNA mutation were analyzed by Kaplan–Meier estimates and compared by the log-rank test.
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Results |
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The 260-bp tandem duplication/triplication in mtDNA D-loop region
In a previous study (18
), we used conventional PCR method to amplify the mtDNA D-loop region between nps 76 and 602 and identified the 260-bp tandem duplication and triplication mutation of the mtDNA D-loop sequence between two poly-C stretches at nps 303–309 and 568–573 in one gastric cancer (patient no. 1142). To screen for occurrence of the tandem duplication/triplication in various other human cancer tissues, we adopted a back-to-back PCR method that was originally designed by Brockington et al. (20). We first tested the effectiveness of the back-to-back primers L336 and H335 for detecting the mutations using various plasmid DNA molecules including wild-type mtDNA, the 260-bp tandem mtDNA D-loop duplication and the 520-bp mtDNA D-loop triplication, the latter two DNA fragments being obtained from the triplication of gastric cancer of patient no. 1142 (Figure 1A). One 260-bp PCR product was amplified from the plasmid DNA carrying the tandem duplication in D-loop, whereas no PCR product was obtained from the DNA-free control and the plasmid DNA carrying the wild-type D-loop sequence. Moreover, two DNA fragments (260 and 520 bp) were amplified from the DNA template carrying the D-loop tandem triplication in D-loop (Figure 1A). Using the conventional PCR method, we only detected the tandem duplication/triplication in the gastric cancer sample of patient no. 1142, but not in the cancers of patients no. 902 and no. 934 (Figure 1B). However, we clearly identified the tandem duplication/triplication in the three gastric cancers and their non-cancerous tissues (Figure 1C). These results indicate that the tandem duplication/triplication mutations of the mtDNA D-loop can be more sensitively detected using the back-to-back PCR method.
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After screening for the tandem duplication/triplication mutations in 197 cancerous tissues, we found that only nine cancer samples (4.6%) harbored the 260-bp tandem duplication/triplication mutations (Table I) and these consisted of four gastric cancers, two breast cancers, two hepatocellular carcinoma and one colon cancer (Figure 2). The PCR of the adjacent non-cancerous tissues from these nine patients were also obtained and showed the same patterns as the cancerous tissues. However, the 260-bp tandem duplication/triplication of the mtDNA was not found in any of the 33 lung cancers that were tested and their adjacent non-cancerous tissues were also negative. Table II summarizes the age, gender and the mtDNA D-loop mutations in the primary cancers of the patients who were detected to harbor the tandem duplication/triplication. In addition, no significant association was found between the tandem duplication/triplication mutations in gastric cancer and clinicopathologic features (Table III).
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On the other hand, we also screened for the tandem duplication/triplication mutations in 234 peripheral blood samples of non-cancer population and found that nine blood samples (3.8%) carry the 260-bp tandem duplication. These results revealed that the tandem duplication/triplication mutations could be detectable in the primary cancer and adjacent non-cancerous tissue of some cancer patients as well as peripheral blood cells of some non-cancer subjects.
Using a semiquantitative PCR method, we determined the proportion of the duplicated mtDNA from some samples that carried the tandem duplication/triplication mutations. We found that 1.6% of the duplicated mtDNA was present in the gastric cancer of patient no. 934, while 12.5% the duplicated mtDNA was detected in the adjacent non-cancerous tissue. Similarly, the proportion of the duplicated mtDNA in the gastric cancer of patient no. 1145 was 0.2%, while that in the adjacent non-cancerous tissue was 25%. The proportion of the mtDNA molecules with the tandem duplication/triplication mutations in the cancer tissue was lower than that in the adjacent non-cancerous tissue.
Poly-C stretch length variation at np 568 of the mtDNA D-loop region
It has been identified that the 260-bp tandem duplication/triplication found in gastric cancer patient no. 1142 was flanked by two poly-C stretches around nps 310 and 568, and there were variations in the number of cytosine within the np 568 poly-C stretch in the case (18). Using direct sequencing, we found the np 568 poly-C stretch length variations in eight out of the 197 cancer patients (4.1%). Moreover, we found that wild-type control contains only six Cs in the poly-C stretch at np 568 and shows homoplasmy; however, eight of the nine (88.9%) cancerous samples and their adjacent non-cancerous tissues with the 260-bp tandem duplication/triplication mutations showed np 568 poly-C length variations (Figure 3). In the eight cases, it was found that there was a longer (>6 Cs) poly-C stretch around np 568 (Figure 3). Moreover, a mixture of mtDNA molecules containing different numbers of cytosine (9–13 Cs) within the poly-C stretch made the sequence analysis unreadable in PCR fragments spanning this region (Figure 3). Among the 234 non-cancer subjects, we also found the np 568 poly-C length variations in 11 cases (4.7%). In addition, nine of the 11 (81.8%) cases were detected to carry the 260-bp tandem duplication. These results clearly identify that the occurrence of the 260-bp tandem duplication/triplication and the presence of the poly-C length variations at np 568 were highly associated.
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Discussion |
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The 260-bp tandem duplication mutation in mtDNA D-loop was first identified by Brockington et al. in 1993 (20
). They suggested that the presence of the 260-bp in the mtDNA regulatory region was highly associated with large-scale mtDNA deletions in patients with mitochondrial myopathy. Manfredi et al. (21) reported no association between the 260-bp duplication and mtDNA deletions, but suggested that the duplication may be pathogenic per se, if present at high levels. Similar mtDNA duplications have been suggested to be age related (23,24) and have also been detected in a specific Caucasian haplotype (25). In the present study, we identified the mtDNA tandem duplication and triplication as present in a range of different human cancers for the first time. Although it was not associated with patient's age, gander and clinicopathologic parameters (Tables II and III), the occurrence of the mtDNA tandem duplication/triplication was highly associated with the poly-C length variations at np 568.
In an earlier study, the mtDNA D-loop 260-bp tandem duplication/triplication mutations were detected in one gastric cancer patient (no. 1142) using a conventional PCR method and were further confirmed using cloning and direct nucleotide sequencing (18). Using conventional PCR, the tandem duplication/triplication mutations were shown to occur at relatively high levels (30% for the duplication and 26% for the triplication) in gastric cancer of one patient, but were not detectable in the same patient's non-cancerous tissues (Figure 1B, 18). In the present study, we adopted the back-to-back PCR method to detect the 260-bp tandem duplication/triplication in both cancerous and non-cancerous tissues of the same subject (Figure 1C). Our results indicated that the back-to-back PCR method is more sensitive than the conventional PCR method for detecting the tandem duplication/triplication mutations. In addition, we also identified the same tandem duplication/triplication in eight more cancerous samples (three gastric cancers, two breast cancers, two liver cancers and one colon cancer), as well as their adjacent non-cancerous tissues (Figure 1C), though we could not detect the tandem duplication/triplication in seven of the cancer samples using the conventional PCR method (Figure 1B, 18). Therefore, the results indicate that the tandem duplication/triplication mutations are at relatively low levels in the eight cancer samples and their adjacent non-cancerous tissues. Moreover, unlike the gastric cancer patient no. 1142 who carried higher proportion of the mutant mtDNA in his gastric cancer, the proportions of the mtDNA harboring the tandem duplication/triplication in two gastric cancers (no. 934 and no. 1145) were lower than that in their adjacent non-cancerous tissues. We also found that the tandem duplication was detectable in the peripheral blood cells of non-cancer subjects. These observations suggest that the presence of the tandem duplication/triplication is not specific for cancer tissue.
The 260-bp tandem duplication/triplication mutations are flanked by two poly-C stretches around nps 310 and 568. Although it is still unclear how the duplication/triplication is generated, slip mispairing is proposed to be the most likely mechanism (25,26). According to the slip-mispairing model (25,27), the two flanking poly-C sequences might play an important role in mtDNA rearrangements. An increase in the length of the longer poly-C stretches as flanking sequences could facilitate the generation of the 260-bp duplication/triplication. In addition, it is possible that long C-tracts in mtDNA are very unstable, while short ones are perfectly stable. The long C-tracts might be further created by duplicating and fusing existing C-stretch during the 260-bp duplication/triplication.
Although mutations in the poly-C stretch around np 310 has been reported as the most frequent position for variations in the mtDNA among several human cancers (28,29), we found that only four of the nine patients with the 260-bp duplication/triplication carry somatic alterations in the poly-C stretch around np 310 in their cancers and three of the four somatic alterations are 1-bp deletion (8,13,14,18,19). However, we found that poly-C stretches around np 568 in eight of nine patients (both cancerous and adjacent non-cancerous tissues) with the duplication/triplication were extensive and polyplasmic. In addition, the mtDNA tandem duplication was found in nine of 11 non-cancer subjects who have the np 568 poly-C length variations, but not in the non-cancer subjects who do not have the np 568 poly-C length variations. Therefore, our observations suggest that the occurrence of the 260-bp tandem duplication/triplication is highly associated with the presence of length variations in the poly-C stretch around np 568. It is worth noting that we did not detect an extended and polyplasmic abnormality of the np 568 poly-C stretch in patient no. 1145’s gastric cancer and his adjacent non-cancerous tissues. Thus, the results suggest that the extended and/or polyplasmic abnormality of the np 568 poly-C stretch is highly associated but not required for the occurrence of the 260-bp duplication/triplication.
Several regulatory elements controlling mtDNA replication and transcription are located in the duplicated/triplicated sequences. These elements include the heavy-/light-strand promoters, the binding sites for mitochondrial transcription factor A and the conserved sequence boxes (II and III). According to the observation from a patient with a slowly progressive myopathy (21), a high proportion of mtDNA molecules with the 260-bp tandem duplication in the skeletal muscle shows some degree of pathogenicity. However, the evidence obtained from a study of transmitochondrial cybrids containing homoplasmic mtDNA with the 260-bp tandem duplication (26) revealed that oxidative phosphorylation was not impaired. Moreover, mtDNA copy number and steady-state levels of heavy-strand and light-strand transcripts were not altered in the cybrid cells with the duplication; this suggests that this mtDNA tandem duplication is not pathogenic (26). In our present study, no significant association was found between the tandem duplication/triplication of mtDNA and clinicopathological features (Table III). Based on these results, the functional significance of the tandem duplication/triplication mutations in cancer progression requires further investigation.
In summary, we have identified 260-bp tandem duplication/triplication mutations in the mtDNA D-loop region in 4.6% of a range of different human cancers, and their adjacent non-cancerous tissues, as well as in 3.8% of peripheral blood cells of non-cancer subjects. Moreover, the occurrence of these tandem duplication/triplications is highly associated with the presence of the poly-C stretch length variation at np 568. Our results suggest that the occurrence of the tandem duplication/triplication mutations in the mtDNA D-loop region is not specific for cancer tissue, but the mutations occur frequently in the subjects with the poly-C stretch length variation at np 568.
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Funding |
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The Taichung Veterans General Hospital (TCVGH-957105D, TCVGH-967106D); the Ministry of Education, Aim for the Top University Plan; the National Science Council, Taiwan, Republic of China (NSC 94-2320-B-010-063, NSC 95-2314-B-010-020-MY2).
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Acknowledgments |
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We thank Ms Shu-Hui Li for excellent technical assistance.
Conflict of interest statement: None declared.
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* To whom correspondence should be addressed. Tel: +886 2 28267327; Fax: +886 2 28264372; Email: hclee2{at}ym.edu.tw
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Received on August 23, 2007; revised on December 19, 2007; accepted on December 30, 2007.
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