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      Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection

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          Abstract

          The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5’-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI 95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI 95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration.

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          CpG methylation patterns of human mitochondrial DNA

          The epigenetic modification of mitochondrial DNA (mtDNA) is still in controversy. To clarify this point, we applied the gold standard method for DNA methylation, bisulfite pyrosequencing, to examine human mtDNA methylation status. Before bisulfite conversion, BamHI was used to digest DNA to open the loop of mtDNA. The results demonstrated that the linear mtDNA had significantly higher bisulfite conversion efficiency compared with circular mtDNA. Furthermore, the methylation values obtained from linear mtDNA were significantly lower than that of circular mtDNA, which was verified by SEQUENOM MassARRAY. The above impacts of circular structure were also observed in lung DNA samples but not in saliva DNA samples. Mitochondrial genome methylation of blood samples and saliva samples from 14 unrelated individuals was detected. The detected regions covered 83 CpG sites across mtDNA including D-loop, 12 S rRNA, 16 S rRNA, ND1, COXI, ND3, ND4, ND5, CYTB. We found that the average methylation levels of nine regions were all less than 2% for both sample types. In conclusion, our findings firstly show that the circular structure of mtDNA affects bisulfite conversion efficiency, which leads to overestimation of mtDNA methylation values. CpG methylation in human mtDNA is a very rare event at most DNA regions.
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            Regionally specific and genome-wide analyses conclusively demonstrate the absence of CpG methylation in human mitochondrial DNA.

            Although CpG methylation clearly distributes genome-wide in vertebrate nuclear DNA, the state of methylation in the vertebrate mitochondrial genome has been unclear. Several recent reports using immunoprecipitation, mass spectrometry, and enzyme-linked immunosorbent assay methods concluded that human mitochondrial DNA (mtDNA) has much more than the 2 to 5% CpG methylation previously estimated. However, these methods do not provide information as to the sites or frequency of methylation at each CpG site. Here, we have used the more definitive bisulfite genomic sequencing method to examine CpG methylation in HCT116 human cells and primary human cells to independently answer these two questions. We found no evidence of CpG methylation at a biologically significant level in these regions of the human mitochondrial genome. Furthermore, unbiased next-generation sequencing of sodium bisulfite treated total DNA from HCT116 cells and analysis of genome-wide sodium bisulfite sequencing data sets from several other DNA sources confirmed this absence of CpG methylation in mtDNA. Based on our findings using regionally specific and genome-wide approaches with multiple human cell sources, we can definitively conclude that CpG methylation is absent in mtDNA. It is highly unlikely that CpG methylation plays any role in direct control of mitochondrial function.
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              Placental mitochondrial methylation and exposure to airborne particulate matter in the early life environment: An ENVIRONAGE birth cohort study

              Most research to date has focused on epigenetic modifications in the nuclear genome, with little attention devoted to mitochondrial DNA (mtDNA). Placental mtDNA content has been shown to respond to environmental exposures that induce oxidative stress, including airborne particulate matter (PM). Damaged or non-functioning mitochondria are specifically degraded through mitophagy, exemplified by lower mtDNA content, and could be primed by epigenetic modifications in the mtDNA. We studied placental mtDNA methylation in the context of the early life exposome. We investigated placental tissue from 381 mother-newborn pairs that were enrolled in the ENVIRONAGE birth cohort. We determined mtDNA methylation by bisulfite-pyrosequencing in 2 regions, i.e., the D-loop control region and 12S rRNA (MT-RNR1), and measured mtDNA content by qPCR. PM2.5 exposure was calculated for each participant's home address using a dispersion model. An interquartile range (IQR) increment in PM2.5 exposure over the entire pregnancy was positively associated with mtDNA methylation (MT-RNR1: +0.91%, P = 0.01 and D-loop: +0.21%, P = 0.05) and inversely associated with mtDNA content (relative change of −15.60%, P = 0.001) in placental tissue. mtDNA methylation was estimated to mediate 54% [P = 0.01 (MT-RNR1)] and 27% [P = 0.06 (D-loop)] of the inverse association between PM2.5 exposure and mtDNA content. This study provides new insight into the mechanisms of altered mitochondrial function in the early life environment. Epigenetic modifications in the mitochondrial genome, especially in the MT-RNR1 region, substantially mediate the association between PM2.5 exposure during gestation and placental mtDNA content, which could reflect signs of mitophagy and mitochondrial death.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draft
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                8 February 2018
                2018
                : 13
                : 2
                : e0192722
                Affiliations
                [1 ] Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, United States of America
                [2 ] EpigenDx, Inc., Hopkinton, MA, United States of America
                [3 ] Harvard Medical School, Boston, MA, United States of America
                University of Perugia, ITALY
                Author notes

                Competing Interests: The commercial affiliation of MP and LY with EpigenDx does not alter our adherence to PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0002-9434-7835
                Article
                PONE-D-17-31204
                10.1371/journal.pone.0192722
                5805350
                29420656
                9ea9f4cb-e2a4-4ca2-876a-e1812723b05d
                © 2018 Owa et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 28 August 2017
                : 25 January 2018
                Page count
                Figures: 6, Tables: 1, Pages: 19
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R01ES023316
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R21ES024861
                Award Recipient :
                This study was supported by NIEHS/NIH grants R01ES023316 and R21ES024861 to TS. The NIEHS/NIH had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. MP and LY are affiliated with EpigenDx, Inc., a for-profit company specialized in DNA bisulfite pyrosequencing and methylation analysis (MP, Scientific Director; LY, President). MP and LY played supportive roles in study design and preparation of the manuscript. MP was also involved in data collection and analysis. EpigenDx provided financial support to MP and LY in the form of salaries. Costs of research performed by and at EpigenDx were paid by TS. CO and TS are affiliated with Massachusetts General Hospital Center for Caner Research, a non-profit research organization. TS is also affiliated with Harvard Medical School (Associate Professor of Medicine). CO and TS are supported by the abovementioned NIEHS/NIH grants for salaries and research expenses. No financial support, in any forms, was provided from EpigenDx to CO or TS. CO was involved in data collection and analysis, and manuscript preparation. TS played primary roles in study design and preparation of the manuscript. TS was exclusively responsible for decisions of contents to be published without being influenced by EpigenDx.
                Categories
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                All deep sequencing data files are available from the NCBI Sequence Read Archive (SRA) database (accession numbers SRP126859 and SRP126863).

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