A Novel Next-Generation Sequencing–Based Approach for Concurrent Detection of Mitochondrial DNA Copy Number and Mutation

Abstract

Numerous studies have identified essential contributions of altered mitochondrial DNA (mtDNA) copy number and mutations in many common disorders, including cancer. To date, capture-based next-generation sequencing (NGS) has been widely applied to detect mtDNA mutations, although it lacks the ability to assess mtDNA copy number. The current strategy for quantifying mtDNA copy number relies mainly on real-time quantitative PCR, which is limited in degraded samples. A novel capture-based NGS approach was developed using both mtDNA and nuclear DNA probes to capture target fragments, enabling simultaneous detection of mtDNA mutations and copy number in different sample types. First, the impact of selecting reference genes on mtDNA copy number calculation was evaluated, and finally, 3 nuclear DNA fragments of 4000 bp were selected as an internal reference for detection. Then, the effective application of this approach was verified in DNA samples of formalin-fixed, paraffin-embedded specimens and body fluids, indicating the widespread applicability. This approach showed more accurate and stable results in detecting mtDNA copy number compared with real-time quantitative PCR in degraded DNA samples. Moreover, data indicated this approach had good reproducibility in detecting both mtDNA copy number and mutations among three sample types. Altogether, a versatile and cost-effective capture-based NGS approach has been developed for concurrent detection of mtDNA copy number and mutations, which has numerous applications in research and diagnosis. Numerous studies have identified essential contributions of altered mitochondrial DNA (mtDNA) copy number and mutations in many common disorders, including cancer. To date, capture-based next-generation sequencing (NGS) has been widely applied to detect mtDNA mutations, although it lacks the ability to assess mtDNA copy number. The current strategy for quantifying mtDNA copy number relies mainly on real-time quantitative PCR, which is limited in degraded samples. A novel capture-based NGS approach was developed using both mtDNA and nuclear DNA probes to capture target fragments, enabling simultaneous detection of mtDNA mutations and copy number in different sample types. First, the impact of selecting reference genes on mtDNA copy number calculation was evaluated, and finally, 3 nuclear DNA fragments of 4000 bp were selected as an internal reference for detection. Then, the effective application of this approach was verified in DNA samples of formalin-fixed, paraffin-embedded specimens and body fluids, indicating the widespread applicability. This approach showed more accurate and stable results in detecting mtDNA copy number compared with real-time quantitative PCR in degraded DNA samples. Moreover, data indicated this approach had good reproducibility in detecting both mtDNA copy number and mutations among three sample types. Altogether, a versatile and cost-effective capture-based NGS approach has been developed for concurrent detection of mtDNA copy number and mutations, which has numerous applications in research and diagnosis. As the key organelle in eukaryotic cells, mitochondria functions vitally in regulating oxidative phosphorylation, central carbon metabolism, and the biosynthesis of intermediates for cell growth.1Murphy M.P. Hartley R.C. Mitochondria as a therapeutic target for common pathologies.Nat Rev Drug Discov. 2018; 17: 865-886Crossref PubMed Scopus (191) Google Scholar Mitochondria contain their own genome, a maternally inherited double-strand circular DNA molecule.2McBride H.M. Neuspiel M. Wasiak S. Mitochondria: more than just a powerhouse.Curr Biol. 2006; 16: R551-R560Abstract Full Text Full Text PDF PubMed Scopus (1192) Google Scholar The human mitochondrial DNA (mtDNA) contains 16,569 bp, which encode 13 polypeptides of the respiratory chain, 22 transfer RNAs, and two ribosomal RNAs.3Taanman J.W. The mitochondrial genome: structure, transcription, translation and replication.Biochim Biophys Acta. 1999; 1410: 103-123Crossref PubMed Scopus (904) Google Scholar Compared with two copies of nuclear DNA (nDNA), the copy number of mtDNA per mitochondrion ranges from 2 to 10, with 103 to 104 copies per cell depending on the different cell types.4Legros F. Malka F. Frachon P. Lombes A. Rojo M. Organization and dynamics of human mitochondrial DNA.J Cell Sci. 2004; 117: 2653-2662Crossref PubMed Scopus (264) Google Scholar Numerous studies have reported significant associations between an abnormal change of mtDNA copy number and different diseases, including many types of cancers.1Murphy M.P. Hartley R.C. Mitochondria as a therapeutic target for common pathologies.Nat Rev Drug Discov. 2018; 17: 865-886Crossref PubMed Scopus (191) Google Scholar Because of a lack of protective histones and an inefficient DNA repair system, mtDNA is more susceptible to replication error or damage by endogenous reactive oxygen species.5Tuppen H.A. Blakely E.L. Turnbull D.M. Taylor R.W. Mitochondrial DNA mutations and human disease.Biochim Biophys Acta. 2010; 1797: 113-128Crossref PubMed Scopus (387) Google Scholar,6Yuan Y. Ju Y.S. Kim Y. Li J. Wang Y. Yoon C.J. Yang Y. Martincorena I. Creighton C.J. Weinstein J.N. Xu Y. Han L. Kim H.L. Nakagawa H. Park K. Campbell P.J. Liang H. PCAWG ConsortiumComprehensive molecular characterization of mitochondrial genomes in human cancers.Nat Genet. 2020; 52: 342-352Crossref PubMed Scopus (47) Google Scholar Consequently, the mutation rate in mtDNA is approximately 10 times higher than in nDNA. Because numerous mutated and normal mtDNA co-exist in the same cell, this phenomenon of intracellular mtDNA mixture is defined as heteroplasmy.7Li M. Schonberg A. Schaefer M. Schroeder R. Nasidze I. Stoneking M. Detecting heteroplasmy from high-throughput sequencing of complete human mitochondrial DNA genomes.Am J Hum Genet. 2010; 87: 237-249Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar A series of studies reported that heteroplasmies throughout the mitochondrial genome are common in normal and pathologic conditions.8Porporato P.E. Filigheddu N. Pedro J.M.B. Kroemer G. Galluzzi L. Mitochondrial metabolism and cancer.Cell Res. 2018; 28: 265-280Crossref PubMed Scopus (306) Google Scholar, 9Orrenius S. Gogvadze V. Zhivotovsky B. Mitochondrial oxidative stress: implications for cell death.Annu Rev Pharmacol Toxicol. 2007; 47: 143-183Crossref PubMed Scopus (921) Google Scholar, 10Giorgi C. Danese A. Missiroli S. Patergnani S. Pinton P. Calcium dynamics as a machine for decoding signals.Trends Cell Biol. 2018; 28: 258-273Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Furthermore, the frequency of heteroplasmic variants has been shown to vary considerably among different tissues, even in the same individual.11Stewart J.B. Chinnery P.F. The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease.Nat Rev Genet. 2015; 16: 530-542Crossref PubMed Scopus (355) Google Scholar Recently, mitochondrial dysfunction induced by the heteroplasmic mtDNA mutations was found extensively to play essential roles in tumor initiation and progression.12Polyak K. Li Y. Zhu H. Lengauer C. Willson J.K. Markowitz S.D. Trush M.A. Kinzler K.W. Vogelstein B. Somatic mutations of the mitochondrial genome in human colorectal tumours.Nat Genet. 1998; 20: 291-293Crossref PubMed Scopus (705) Google Scholar,13Larman T.C. DePalma S.R. Hadjipanayis A.G. Protopopov A. Zhang J. Gabriel S.B. Chin L. Seidman C.E. Kucherlapati R. Seidman J.G. Cancer Genome Atlas Research NetworkSpectrum of somatic mitochondrial mutations in five cancers.Proc Natl Acad Sci U S A. 2012; 109: 14087-14091Crossref PubMed Scopus (140) Google Scholar Moreover, the detection of mtDNA copy number and mutations has been reported to have potential application as a promising clinical biomarker in multiple diseases, especially in cancers.14Xing J. Chen M. Wood C.G. Lin J. Spitz M.R. Ma J. Amos C.I. Shields P.G. Benowitz N.L. Gu J. de Andrade M. Swan G.E. Wu X. Mitochondrial DNA content: its genetic heritability and association with renal cell carcinoma.J Natl Cancer Inst. 2008; 100: 1104-1112Crossref PubMed Scopus (182) Google Scholar In the past, mtDNA content has only been estimated by Southern blot hybridization, which requires large amounts of samples and laborious work. Recently, real-time quantitative PCR (qPCR) was used extensively in the measurement of relative mtDNA copy number by measuring the ratio between mtDNA and nDNA.15Gonzalez-Hunt C.P. Rooney J.P. Ryde I.T. Anbalagan C. Joglekar R. Meyer J.N. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage.Curr Protoc Toxicol. 2016; 67: 20.11.21-20.11.25Crossref Scopus (44) Google Scholar However, the use of external calibrators varies among laboratories, which is essential for analytical performance in qPCR and makes data comparison impractical.16Regier N. Frey B. Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar.BMC Mol Biol. 2010; 11: 57Crossref PubMed Scopus (52) Google Scholar In addition, the application of qPCR is limited considerably by the low quality of detected DNA samples,17Wai K.T. Gunn P. Barash M. Development of the MitoQ assay as a real-time quantification of mitochondrial DNA in degraded samples.Int J Legal Med. 2019; 133: 411-417Crossref PubMed Scopus (1) Google Scholar frequently resulting in failure or inaccuracy of detection. Sanger sequencing is used widely to detect mtDNA mutations, has a low cost, and is easy to use. However, this method is not sensitive enough to identify mutations with a low heteroplasmy level (approximately <15%).18Irwin J.A. Saunier J.L. Niederstatter H. Strouss K.M. Sturk K.A. Diegoli T.M. Brandstatter A. Parson W. Parsons T.J. Investigation of heteroplasmy in the human mitochondrial DNA control region: a synthesis of observations from more than 5000 global population samples.J Mol Evol. 2009; 68: 516-527Crossref PubMed Scopus (115) Google Scholar Recently, next-generation sequencing (NGS), a massively parallel sequencing technique, has been used increasingly in sensitive and high-throughput detection of mtDNA mutations.19Yin C. Li D.Y. Guo X. Cao H.Y. Chen Y.B. Zhou F. Ge N.J. Liu Y. Guo S.S. Zhao Z. Yang H.S. Xing J.L. NGS-based profiling reveals a critical contributing role of somatic D-loop mtDNA mutations in HBV-related hepatocarcinogenesis.Ann Oncol. 2019; 30: 953-962Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar Usually, to reduce sequencing cost, mtDNA needs to be enriched from genomic DNA by PCR-based or capture-based methods.20Mamanova L. Coffey A.J. Scott C.E. Kozarewa I. Turner E.H. Kumar A. Howard E. Shendure J. Turner D.J. Target-enrichment strategies for next-generation sequencing.Nat Methods. 2010; 7: 111-118Crossref PubMed Scopus (766) Google Scholar Several studies also have shown that mtDNA mutations could be identified from exome sequencing data.21Zhang P. Lehmann B.D. Samuels D.C. Zhao S. Zhao Y.Y. Shyr Y. Guo Y. Estimating relative mitochondrial DNA copy number using high throughput sequencing data.Genomics. 2017; 109: 457-462Crossref PubMed Scopus (9) Google Scholar However, because of the relatively low coverage of mtDNA in these data sets, most of the mtDNA mutations with a low heteroplasmic level cannot be detected effectively.11Stewart J.B. Chinnery P.F. The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease.Nat Rev Genet. 2015; 16: 530-542Crossref PubMed Scopus (355) Google Scholar In addition, both capture-based and PCR-based mtDNA sequencing could detect only mtDNA mutations, but could not estimate the mtDNA copy number. With an increasing number of studies focusing on the mitochondrial dysfunction in diseases, more requirements are needed for the detection of mtDNA copy number alteration and mtDNA mutation. To date, only whole-genome sequencing data including mtDNA and nuclear DNA be used to the mutation and copy number of mtDNA on a series of W. I. J. S. S. H. M. M. K. Turner M.R. C. J. C. S. S. J.W. Chinnery P.F. Mitochondrial DNA mutations and relative copy number in and control human 2017; PubMed Scopus Google Scholar The of in S. of of next-generation sequencing Rev Genet. 2016; 17: PubMed Scopus Google Scholar are more for and quantitative detection of The developed a capture-based NGS approach that the concurrent detection of both mutations and copy number of The application of this method in different sample types was its This novel method a for clinical of mtDNA detection. 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NGS-based profiling reveals a critical contributing role of somatic D-loop mtDNA mutations in HBV-related hepatocarcinogenesis.Ann Oncol. 2019; 30: 953-962Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar genomic DNA for was applied to the the was used to the sequencing using Then, was on the for sequencing of 10 cell 10 and 10 using with 2 to the The was to mtDNA and in reference and the PCR and the selected reference genes are shown in PCR was as Y. Guo S. C. Guo X. Liu M. Z. Zhao Z. Y. Xing J. 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The of mtDNA copy number detected by the current approach and qPCR were compared in three different sample types The data showed that good of mtDNA copy number detected by two was only in DNA samples from cell but not in and DNA samples and that this approach is with a the current that qPCR be used in DNA samples with low the of this capture-based novel approach was among different types of specimens by the of two were good of mtDNA copy number between two in DNA samples from cell and and results were for the number and and heteroplasmy level and of mtDNA mutations in three sample types and data good reproducibility for mtDNA copy number and mutation detection. In addition, two sequencing used most widely for NGS and were applied to the of this novel approach for detection of mtDNA from the current showed the of mtDNA mutation and heteroplasmy between two the high of this capture-based approach The developed a capture-based NGS approach for concurrent detection of mtDNA copy number and mutations, which mtDNA profiling in different sample the and of detection. This novel approach has two is that the of both mtDNA copy number and mutation be by only which the functions of both qPCR and is the widespread of this by which effective detection of mtDNA be in DNA samples with low are used widely to mtDNA copy number However, that accurate quantification have been E. N. N. A. A. N. S. M. K. H. A. S. K. E. Somatic mitochondrial mutation using sequencing of the mitochondrial genome reveals tumor in and cell 2020; PubMed Scopus (5) Google Scholar The of relative quantification results in data comparison are among different laboratories, even among different in the same The application of is especially with DNA including DNA from samples and body Recently, increasing has the of nuclear the of approximately bp to S. A. J. J. V. D.C. DNA in with 2019; PubMed Scopus Google K. P. J. Ma H. DNA analysis in of Res. 2019; PubMed Scopus Google Scholar In the of the of mtDNA was using the and the (approximately of mtDNA compared with was found in both and indicating the of mtDNA K. S. Y. X. L. X. X. and J. these also that qPCR be used in DNA samples with low be used only to mtDNA copy number and not to mtDNA mutation To date, mtDNA mutations were detected mainly by sequencing including Sanger sequencing and J.B. Chinnery P.F. The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease.Nat Rev Genet. 2015; 16: 530-542Crossref PubMed Scopus (355) Google C.P. Rooney J.P. Ryde I.T. Anbalagan C. Joglekar R. Meyer J.N. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage.Curr Protoc Toxicol. 2016; 67: 20.11.21-20.11.25Crossref Scopus (44) Google Scholar including capture-based and NGS are used for sensitive and high-throughput detection of mtDNA mutations by mtDNA from genomic DNA L. Coffey A.J. Scott C.E. Kozarewa I. Turner E.H. Kumar A. Howard E. Shendure J. Turner D.J. Target-enrichment strategies for next-generation sequencing.Nat Methods. 2010; 7: 111-118Crossref PubMed Scopus (766) Google Scholar Because an sequencing strategy has been reported to coverage and more PCR Y. Guo S. C. Guo X. Liu M. Z. Zhao Z. Y. Xing J. PCR-based coverage and mutation detection in mitochondrial DNA Mol 2020; Full Text Full Text PDF PubMed Scopus Google Scholar was a to capture-based sequencing to detect mtDNA mutations found in C. Li D.Y. Guo X. Cao H.Y. Chen Y.B. Zhou F. Ge N.J. Liu Y. Guo S.S. Zhao Z. Yang H.S. Xing J.L. NGS-based profiling reveals a critical contributing role of somatic D-loop mtDNA mutations in HBV-related hepatocarcinogenesis.Ann Oncol. 2019; 30: 953-962Abstract Full Text Full Text PDF PubMed Scopus (5) Google C. Liu Y. Guo X. Li W. Yang J. Zhou F. W. Y. Yang H. Xing J. effective strategy to in mtDNA next-generation sequencing of multiple samples.J Mol 2019; Full Text Full Text PDF PubMed Scopus (5) Google X. Guo X. Li X. C. Chen C. W. Z. Zhang J. Li B. Yang H. Xing J. sequencing reveals and of somatic mtDNA mutations in and colorectal J 2018; PubMed Scopus Google Scholar However, both target sequencing only the mtDNA mutations, not the copy number. was in an approach that could the simultaneous detection of the and of detection. In this capture-based NGS nuclear DNA fragments were selected for and used as internal for mtDNA copy number calculation on capture sequencing data of As the results by mtDNA probes and probes the same system, it is to detect the mtDNA copy number and mutations in a and This approach was and the and reproducibility in detecting mtDNA copy number and mutation was on measurement of the mtDNA copy number by has been widely as a and mtDNA copy number Y. Ju Y.S. Kim Y. Li J. Wang Y. Yoon C.J. Yang Y. Martincorena I. Creighton C.J. Weinstein J.N. Xu Y. Han L. Kim H.L. Nakagawa H. Park K. Campbell P.J. Liang H. PCAWG ConsortiumComprehensive molecular characterization of mitochondrial genomes in human cancers.Nat Genet. 2020; 52: 342-352Crossref PubMed Scopus (47) Google Scholar, Zhang Y. J. A. Taylor J. E. N. E. of mitochondrial DNA copy number with 2017; PubMed Scopus Google Scholar, R. C. Yang S. C. J. J. M. E. N. Taylor K. J. E. of mitochondrial DNA copy number 2020; PubMed Scopus Google Scholar because of the of However, for the capture-based approach the in a of mtDNA copy number, to the different capture of the probes mtDNA and fragments, to the ratio of mtDNA to reference in the current this method be used for a relative mtDNA copy number but not the In addition, this capture-based NGS approach also had a performance in DNA its compared with the this capture-based novel approach has a in cost, the and of detection. A estimate of the three approaches is in it is more for this approach to be used in a pathologic diagnosis. application of this novel approach tumor sequencing is copy number and mutation profiling of mtDNA in the of genetic and Yang and for and

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