mtDNA Methylation Research Articles

DNA methylation at hepatitis B virus integrants and flanking host mitochondrially encoded cytochrome C oxidase III.

It is widely accepted that hepatitis B virus (HBV) integrants in the human genome are one of the key factors in liver carcinogenesis. Although it is difficult to observe pre/post-HBV infection genomic-level changes in the same clinical sample pairs, they can be observed using artificially infected HBV cell lines such as HepG2.2.15. A detailed HBV integration analysis comparing HepG2.2.15 with HepG2 cells, especially their mitochondrial (mt) DNA, was conducted using next-generation sequencing (NGS)-based integration analysis. Following target DNA enrichment for elements of the HBV genome, NGS was used to identify HBV integration sites in the mtDNA and DNA methylation was analyzed using semi-quantitative pyrosequencing at the boundaries of the integrated region. The results revealed the HBV integration site in the mtDNA of HepG2.215, most notably the insertion of the HBV preCore, X gene fragment in exon 1 of mitochondrially encoded cytochrome C oxidase III (MT-CO3; ChrM 9652), along with a ‘CACCA’ microhomology sequence. Both boundaries of the integrated region were concordant and highly methylated (HBV side, 92.3%; MT-CO3 side, 95.5%) relative to those observed in nonintegrated HepG2 (4.3%), HepG2.2.15 (3.0%) and PLC/PRF/5 (4.0%) cells. In conclusion, HBV integration sites were successfully identified in the MT-CO3 gene along with a ‘CACCA’ microhomology sequence using NGS-based analysis and mitochondrial heteroplasmy was identified. The present study also revealed that the HBV/MT-CO3-integrated boundary DNA was hypermethylated at both the HBV and MT-CO3 sides.

Association between diesel exhaust exposure and mitochondrial DNA methylation.

Diesel exhaust is an established human carcinogen, however the mechanisms by which it leads to cancer development are not fully understood. Mitochondrial dysfunction is an established contributor to carcinogenesis. Recent studies have improved our understanding of the role played by epigenetic modifications in the mitochondrial genome on tumorigenesis. In this study, we aim to evaluate the association between diesel engine exhaust (DEE) exposure with mitochondrial DNA (mtDNA) methylation levels in workers exposed to DEE. The study population consisted of 53 male workers employed at a diesel engine manufacturing facility in Northern China who were routinely exposed to diesel exhaust in their occupational setting, as well as 55 unexposed male control workers from other unrelated factories in the same geographic area. Exposure to DEE, elemental carbon, organic carbon, and particulate matter (PM2.5) were assessed. mtDNA methylation for CpG sites (CpGs) from seven mitochondrial genes (D-Loop, MT-RNR1, MT-CO2, MT-CO3, MT-ATP6, MT-ATP8, MT-ND5) was measured in blood samples. Linear regression models were used to estimate the associations between DEE, elemental carbon, organic carbon and PM2.5 exposures with mtDNA methylation levels, adjusting for potential confounders. DEE exposure was associated with decreased MT-ATP6 (difference = -35.6%, P-value = 0.019) and MT-ATP8 methylation (difference = -30%, P-value = 0.029) compared to unexposed controls. Exposures to elemental carbon, organic carbon, and PM2.5 were also significantly and inversely associated with methylation in MT-ATP6 and MT-ATP8 genes (all P-values < 0.05). Our findings suggest that DEE exposure perturbs mtDNA methylation, which may be of importance for tumorigenesis.

Association between diesel exhaust exposure and mitochondrial DNA methylation

Background: Diesel exhaust is an established human carcinogen, however the mechanisms by which it leads to cancer development are not fully understood. Mitochondrial dysfunction is an established contributor to carcinogenesis, and recent studies have improved our understanding of the role played by epigenetic modifications in the mitochondrial genome on tumorigenesis. In light of these developments, we aim to evaluate the association between diesel engine exhaust (DEE) exposure with mitochondrial DNA (mtDNA) methylation levels in workers exposed to DEE. Methods: The study population consisted of 53 male workers employed at a diesel engine manufacturing facility in Northern China who were routinely exposed to diesel exhaust in their occupational setting, as well as 55 unexposed male control workers from other unrelated factories in the same geographic area. Exposure to DEE, elemental carbon as a DEE surrogate, organic carbon, and particulate matter (PM2.5) were assessed. mtDNA methylation for CpG sites (CpGs) from seven mitochondrial genes (D-Loop, MT-RNR1, MT-CO2, MT-CO3, MT-ATP6, MT-ATP8, MT-ND5) was measured in blood samples. Linear regression models were used to estimate the associations between DEE and mtDNA methylation levels, adjusting for potential confounders. The associations between elemental carbon, organic carbon, and PM2.5 exposures with mtDNA methylation levels were also assessed. Results: DEE exposure was associated with decreased MT-ATP6 (difference = -35.6%, p-value = 0.019) and MT-ATP8 methylation (difference = -30%, p-value = 0.029) compared to unexposed controls. Exposures to elemental carbon, organic carbon, and PM2.5 were also significantly and inversely associated with methylation in MT-ATP6 and MT-ATP8 genes (all p-values &amp;#x3c; 0.05). Conclusions: Our findings suggest that DEE exposure perturbs mtDNA methylation, which may be of importance for tumorigenesis. Keywords: Diesel exhaust, mitochondrial DNA methylation

Geometric Constraints Regulate Energy Metabolism and Cellular Contractility in Vascular Smooth Muscle Cells by Coordinating Mitochondrial DNA Methylation.

Vascular smooth muscle cells (SMCs) can adapt to changes in cellular geometric cues; however, the underlying mechanisms remain elusive. Using 2D micropatterned substrates to engineer cell geometry, it is found that in comparison with an elongated geometry, a square-shaped geometry causes the nuclear-to-cytoplasmic redistribution of DNA methyltransferase 1 (DNMT1), hypermethylation of mitochondrial DNA (mtDNA), repression of mtDNA gene transcription, and impairment of mitochondrial function. Using irregularly arranged versus circumferentially aligned vascular grafts to control cell geometry in 3D growth, it is demonstrated that cell geometry, mtDNA methylation, and vessel contractility are closely related. DNMT1 redistribution is found to be dependent on the phosphoinositide 3-kinase and protein kinase B (AKT) signaling pathways. Cell elongation activates cytosolic phospholipase A2, a nuclear mechanosensor that, when inhibited, hinders AKT phosphorylation, DNMT1 nuclear accumulation, and energy production. The findings of this study provide insights into the effects of cell geometry on SMC function and its potential implications in the optimization of vascular grafts.

Targeted Mitochondrial Epigenetics: A New Direction in Alzheimer's Disease Treatment.

Mitochondrial epigenetic alterations are closely related to Alzheimer’s disease (AD), which is described in this review. Reports of the alteration of mitochondrial DNA (mtDNA) methylation in AD demonstrate that the disruption of the dynamic balance of mtDNA methylation and demethylation leads to damage to the mitochondrial electron transport chain and the obstruction of mitochondrial biogenesis, which is the most studied mitochondrial epigenetic change. Mitochondrial noncoding RNA modifications and the post-translational modification of mitochondrial nucleoproteins have been observed in neurodegenerative diseases and related diseases that increase the risk of AD. Although there are still relatively few mitochondrial noncoding RNA modifications and mitochondrial nuclear protein post-translational modifications reported in AD, we have reason to believe that these mitochondrial epigenetic modifications also play an important role in the AD process. This review provides a new research direction for the AD mechanism, starting from mitochondrial epigenetics. Further, this review summarizes therapeutic approaches to targeted mitochondrial epigenetics, which is the first systematic summary of therapeutic approaches in the field, including folic acid supplementation, mitochondrial-targeting antioxidants, and targeted ubiquitin-specific proteases, providing a reference for therapeutic targets for AD.

Mini-review: Mitochondrial DNA methylation in type 2 diabetes and obesity.

Type 2 diabetes (T2D) and obesity are two of the most challenging public health problems of our time. Therefore, understanding the molecular mechanisms that contribute to these complex metabolic disorders is essential. An underlying pathophysiological condition of T2D and obesity is insulin resistance (IR), a reduced biological response to insulin in peripheral tissues such as the liver, adipose tissue, and skeletal muscle. Many factors contribute to IR, including lifestyle variables such as a high-fat diet and physical inactivity, genetics, and impaired mitochondrial function. It is well established that impaired mitochondria structure and function occur in insulin-resistant skeletal muscle volunteers with T2D or obesity. Therefore, it could be hypothesized that the mitochondrial abnormalities are due to epigenetic regulation of mitochondrial and nuclear-encoded genes that code for mitochondrial structure and function. In this review, we describe the normal function and structure of mitochondria and highlight some of the key studies that demonstrate mitochondrial abnormalities in skeletal muscle of volunteers with T2D and obesity. Additionally, we describe epigenetic modifications in the context of IR and mitochondrial abnormalities, emphasizing mitochondria DNA (mtDNA) methylation, an emerging area of research.

Toxicity of environmental pollutants for mitochondrialDNA alteration

<p indent="0mm">Mitochondria, as the main site of cellular aerobic respiration, are organelles that provide energy for cells. The mitochondrial genomes are independent of the nuclear genome, known as mitochondrial DNA (mtDNA). mtDNA encodes 37 genes, including 13 respiratory chain-related polypeptides, 22 tRNAs and 2 rRNA genes. Mitochondrial abnormalities can directly reduce cellular ATP synthesis and thus generate insufficient cellular energy. The regulation of mtDNA copy number and epigenetics is crucial for the basic functions of mitochondria. After entering the cells of organisms, environmental pollutants elevate reactive oxygen species (ROS), causing oxidative stress in the organism and resulting in abnormal metabolism and various diseases. Compared with nuclear DNA, mtDNA is more susceptible to oxidative stress because of its proximity to the site of oxidative phosphorylation (OXPHOS) and lack of histone protection and sufficient DNA damage repair capacity. Exposure to various environmental pollutants can induce excessive accumulation of ROS, which leads to changes in mtDNA copy number and epigenetic modifications of mtDNA. An increasing number of studies focus on abnormal mtDNA as a possible biomarker for the exposure and toxicity of environmental pollutants. In this article, we briefly introduce the physiological functions and regulatory mechanisms of mitochondria and mtDNA copy number. Environment pollutant exposures often cause mitochondrial damage, which may alter mtDNA copy number, leading to mitochondrial abnormalities and impairing cell function. Studies have found that mitochondrial dysfunction is related to the occurrence and development of various diseases (e.g., cancer, diabetes, cardiovascular disease, neurodegenerative diseases, etc.). Noteworthily, decreased mtDNA copy number in germ cells blocks embryonic development. Next, along with the associated proteins found in recent studies, possible epigenetic modifications present on mtDNA (e.g., 5-methylcytosine, 5-hydroxymethylcytosine and N6-methyladenine) are also summarized. A study identified mtDNMT1 in the mitochondrial matrix, which was suggested to be a methyltransferase for mtDNA 5mC. Since 5hmC modification on mtDNA was first reported in 2011, the results of studies on mtDNA 5hmC have been conflicting. However, some research groups found that Tet1 and Tet2 may be involved in the formation of mtDNA 5hmC. A higher level of 6mA modification than nuclear DNA was suggested to be detected in mtDNA, and the METTL4 protein is a potential mtDNA 6mA methyltransferase. Nonetheless, the study on mtDNA methylation is of intensive interest. We reviewed the effects of multiple common environmental pollutants (e.g., PM, black carbon, nicotine, heavy metal particles, etc.) on mitochondrial DNA from two aspects: (1) Environmental pollutants exposure causes mtDNA copy number changes, which may increase disease risk; (2) environmental pollutant exposure alters methylation levels of certain genes in mtDNA. In the first aspect, exposure to different pollutants, or even the same pollutant, resulted in different changes in mtDNA copy number. It suggests that the same environmental pollutants may also affect mtDNA copy number through different mechanisms and pathways. In the second aspect, changes in the methylation levels of D-loop and gene regions on mtDNA caused by pollutant exposure can affect mitochondrial physiological function by affecting mitochondrial DNA replication and mitochondrial-encoded protein expression. We prospect and discuss how to perform further study on the effect of environmental pollutants on mtDNA and its molecular mechanism. The following two aspects should be improved in future pollutants-mtDNA studies: (1) Develop more convenient methods for the extraction of mtDNA from less than million cells to a high purity. This will facilitate the detection and sequencing of mtDNA for diverse purposes; (2) to achieve accurate identification of mtDNA methylation sites in small number of cells and eliminate the interference of mtDNA heterogeneity, the mtDNA methylation sequencing method should be further innovated.

Mitochondrial genome undergoes de novo DNA methylation that protects mtDNA against oxidative damage during the peri-implantation window.

Mitochondrial remodeling during the peri-implantation stage is the hallmark event essential for normal embryogenesis. Among the changes, enhanced oxidative phosphorylation is critical for supporting high energy demands of postimplantation embryos, but increases mitochondrial oxidative stress, which in turn threatens mitochondrial DNA (mtDNA) stability. However, how mitochondria protect their own histone-lacking mtDNA, during this stage remains unclear. Concurrently, the mitochondrial genome gain DNA methylation by this stage. Its spatiotemporal coincidence with enhanced mitochondrial stress led us to ask if mtDNA methylation has a role in maintaining mitochondrial genome stability. Herein, we report that mitochondrial genome undergoes de novo mtDNA methylation that can protect mtDNA against enhanced oxidative damage during the peri-implantation window. Mitochondrial genome gains extensive mtDNA methylation during transition from blastocysts to postimplantation embryos, thus establishing relatively hypermethylated mtDNA from hypomethylated state in blastocysts. Mechanistic study revealed that DNA methyltransferase 3A (DNMT3A) and DNMT3B enter mitochondria during this process and bind to mtDNA, via their unique mitochondrial targeting sequences. Importantly, loss- and gain-of-function analyses indicated that DNMT3A and DNMT3B are responsible for catalyzing de novo mtDNA methylation, in a synergistic manner. Finally, we proved, invivo and invitro, that increased mtDNA methylation functions to protect mitochondrial genome against mtDNA damage induced by increased mitochondrial oxidative stress. Together, we reveal mtDNA methylation dynamics and its underlying mechanism during the critical developmental window. We also provide the functional link between mitochondrial epigenetic remodeling and metabolic changes, which reveals a role for nuclear-mitochondrial crosstalk in establishing mitoepigenetics and maintaining mitochondrial homeostasis.

Aberrant mitochondrial DNA methylation and declined pulmonary function in a population with polycyclic aromatic hydrocarbon composition in particulate matter

Air pollution exposure has been found to be associated with epigenetic modification of the mitochondrial genome, which could subsequently induce adverse health outcomes. However, very limited studies exist regarding the association between fine particulate matter (PM2.5) exposure and pulmonary function at the molecular level of mitochondrial epigenetic changes. This study aimed to investigate the association of platelet mitochondrial DNA (mtDNA) methylation with occupational PM2.5 exposure and pulmonary function. First, 768 participants were occupationally exposed to polycyclic aromatic hydrocarbon (PAH)-enriched PM2.5 in a coke-oven plant in East China. The levels of PM2.5, PAH components bound to PM2.5, and urinary PAH metabolites in the workplace environment were measured as an internal dose, respectively. mtDNA methylation was measured by bisulfite pyrosequencing of two genes of ATP synthase (MT-ATP6 and MT-ATP8). Mediation analysis was conducted to evaluate the role of mtDNA methylation in pulmonary alteration induced by PAH. A decreasing trend of platelet mtDNA methylation was observed with increase in PM2.5 exposure across all participants. As an important PAH metabolite in urine, 1-hydroxypyrene (1-OHP) was significantly negatively associated with FEV1/FVC (Forced Expiratory Volume in 1s/Forced Vital Capacity) ratio. The participants with high serum folate levels (≥10 nmol/L) showed positive association between MT-ATP6 methylation and FEV1/FVC ratio. Mediation analysis suggested that MT-ATP6 methylation mediated the significant association of urinary 1-OHP with FEV1/FVC. Our findings suggested the methylation of platelet mitochondrial gene MT-ATP6 and FEV1/FVC to be negatively associated with PM exposure. Platelet mtDNA methylation acted as an intermediary between PAH exposure and lung function decline. The mitochondrial epigenetic regulation in platelets, in response to PM exposure, might be involved in subsequent progress of abnormal pulmonary function.

Association between mitochondrial DNA methylation and internal exposure to polycyclic aromatic hydrocarbons (PAHs), nitrated-PAHs (NPAHs) and oxygenated-PAHs (OPAHs) in young adults from Tianjin, China

Polycyclic aromatic hydrocarbons (PAHs), nitrated-PAHs (NPAHs) and oxygenated-PAHs (OPAHs) are environmental pollutants with adverse effects on human health. The correlation between the concentrations of PAHs, NPAHs and OPAHs in human plasma and the methylation level of mitochondrial DNA (mtDNA) was investigated using data from 110 plasma samples collected in Tianjin, China. The median concentrations of PAHs, NPAHs and OPAHs were 16.0 (IQR: 14.4–20.7) ng/mL, 82.2 (IQR: 63.1–97.6) ng/mL and 49.6 (IQR: 28.6–53.8) ng/mL, and the mean proportions were 13.4%, 56.5% and 30.1%, respectively. Bisulfite-PCR pyrosequencing was used to measure the methylation level of MT-CO1 and tRNA-Leu. The methylation levels of two mitochondrial genes (MT-CO1, tRNA-Leu) including four CpG sites (MT-CO1-P1, MT-CO1-P2, tRNA-Leu-P1 and tRNA-Leu-P2) were 0.67% ± 1.38%, 13.54% ± 2.59%, 7.23% ± 5.35% and 1.64% ± 2.94%, respectively. To the best of our knowledge, this is the first time that significant correlations were found between PAHs and their derivatives exposure and mtDNA methylation levels.

Mitochondrial DNA in Visceral Adipose Tissue in Severe Obesity: From Copy Number to D-Loop Methylation.

Peripheral alterations of mitochondrial DNA copy number (mtDNAcn) in obesity and associated co-morbidities have been previously shown. Furthermore, the possibility that methylation could occur in the mtDNA (in particular in the displacement loop, D-Loop) and regulate its functions has been raised. However, limited data about mtDNA methylation in adipose tissue are currently available. Since a strict crosstalk between the nucleus and mitochondria exists, especially in terms of the one-carbon cycle (that supports methylation reactions in the cell), we investigated methylation in selected areas of the mitochondrial and nuclear DNA and their expression in visceral adipose tissue (VAT) samples of patients with severe obesity. VAT biopsies were collected from surgery patients to isolate DNA and RNA. Gene expression and mtDNAcn were assessed through qPCR. DNA methylation in both nuclear and mitochondrial areas were determined through bisulfite pyrosequencing. Methylation levels of the mtDNA were only marginally associated with the obesity degree (higher D-Loop methylation in severe obesity) and were not correlated with mtDNAcn. A significant correlation between D-Loop methylation and LINE-1 methylation was observed in VAT samples, and this was independent from the obesity degree. A progressive reduction of mtDNAcn and increase in NRF1 expression levels were measured in VAT in severe obesity. NRF1 expression was directly correlated with PPARG and MTHFR expression levels, while mtDNAcn was associated to TFAM expression. The correlation between mtDNAcn and TFAM expression was affected by the obesity status. This evidence supports the hypothesis that mtDNA alterations occur in obesity and a complex dynamic correlation between mitochondrial and nuclear DNA methylation exists, highlighting the need for further investigations.

Integrated mitoepigenetic signalling mechanisms associated with airborne particulate matter exposure: A cross-sectional pilot study

Airborne particulate matter (PM) is known to be associated with various chronic human ailments, however, precise underlying molecular mechanisms remains elusive. Based on the place of residence and amount of PM exposure, the study groups were divided into high-risk group and low-risk group. Different parameters related to oxidative DNA damage and repair, integrated stress response, mito-nuclear crosstalk, mitochondrial biogenesis, functioning and epigenetic machinery were assessed. The results of our pilot study showed that in comparison to the low-risk group, the levels of mitochondrial stress and oxidative DNA damage were significantly higher in the high-risk group. In addition, the upregulated expression levels of OMA1, DELE1, and HRI genes in the high-risk group confirmed the onset of an integrated stress response. These disturbances in the mitochondrial genome were further found to be associated with the vital processes of mitochondrial biogenesis. The high-risk group reported significant alterations in the mtDNA copy number and expression of mitochondrial fission and fusion genes. Importantly, we also observed higher expression levels of mitochondrial transcription factor TFAM, a key regulator of mtDNA replication and transcription in the high-risk group. Moreover, these changes were accompanied by the disturbances in the expression of TET genes and levels of mtDNA methylation. This impaired mtDNA methylation profile further correlated with the disturbed activity of electron chain complexes and expression of the mitochondria function genes in the high-risk group. Altogether, our observations provide vital molecular insights of the integrated mitoepigenetic signalling network in the airborne PM exposed individuals, hitherto unreported.

Ultra-deep whole genome bisulfite sequencing reveals a single methylation hotspot in human brain mitochondrial DNA

ABSTRACT While DNA methylation is established as a major regulator of gene expression in the nucleus, the existence of mitochondrial DNA (mtDNA) methylation remains controversial. Here, we characterized the mtDNA methylation landscape in the prefrontal cortex of neurological healthy individuals (n=26) and patients with Parkinson’s disease (n=27), using a combination of whole-genome bisulphite sequencing (WGBS) and bisulphite-independent methods. Accurate mtDNA mapping from WGBS data required alignment to an mtDNA reference only, to avoid misalignment to nuclear mitochondrial pseudogenes. Once correctly aligned, WGBS data provided ultra-deep mtDNA coverage (16,723 ± 7,711) and revealed overall very low levels of cytosine methylation. The highest methylation levels (5.49 ± 0.97%) were found on CpG position m.545, located in the heavy-strand promoter 1 region. The m.545 methylation was validated using a combination of methylation-sensitive DNA digestion and quantitative PCR analysis. We detected no association between mtDNA methylation profile and Parkinson’s disease. Interestingly, m.545 methylation correlated with the levels of mtDNA transcripts, suggesting a putative role in regulating mtDNA gene expression. In addition, we propose a robust framework for methylation analysis of mtDNA from WGBS data, which is less prone to false-positive findings due to misalignment of nuclear mitochondrial pseudogene sequences.

The Mitochondrial Epigenome: An Unexplored Avenue to Explain Unexplained Myopathies?

Mutations in either mitochondrial DNA (mtDNA) or nuclear genes that encode mitochondrial proteins may lead to dysfunctional mitochondria, giving rise to mitochondrial diseases. Some mitochondrial myopathies, however, present without a known underlying cause. Interestingly, methylation of mtDNA has been associated with various clinical pathologies. The present study set out to assess whether mtDNA methylation could explain impaired mitochondrial function in patients diagnosed with myopathy without known underlying genetic mutations. Enhanced mtDNA methylation was indicated by pyrosequencing for muscle biopsies of 14 myopathy patients compared to four healthy controls, at selected cytosines in the Cytochrome B (CYTB) gene, but not within the displacement loop (D-loop) region. The mtDNA methylation patterns of the four healthy muscle biopsies were highly consistent and showed intriguing tissue-specific differences at particular cytosines with control skin fibroblasts cultured in vitro. Within individual myopathy patients, the overall mtDNA methylation pattern correlated well between muscle and skin fibroblasts. Despite this correlation, a pilot analysis of four myopathy and five healthy fibroblast samples did not reveal a disease-associated difference in mtDNA methylation. We did, however, detect increased expression of solute carrier family 25A26 (SLC25A26), encoding the importer of S-adenosylmethionine, together with enhanced mtDNA copy numbers in myopathy fibroblasts compared to healthy controls. To confirm that pyrosequencing indeed reflected DNA methylation and not bisulfite accessibility, mass spectrometry was employed. Although no myopathy-related differences in total amount of methylated cytosines were detected at this stage, a significant contribution of contaminating nuclear DNA (nDNA) was revealed, and steps to improve enrichment for mtDNA are reported. In conclusion, in this explorative study we show that analyzing the mitochondrial genome beyond its sequence opens novel avenues to identify potential molecular biomarkers assisting in the diagnosis of unexplained myopathies.

Placental mtDNA copy number and methylation in association with macrosomia in healthy pregnancy.

Fetal growth and development depend on metabolic energy from placental mitochondria. However, the impact of placental mitochondria on the occurrence of macrosomia remains unclear. We aimed to explore the association between macrosomia without gestational diabetes mellitus (non-GDM) and changes in placental mitochondrial DNA (mtDNA) copy number and methylation. Fifty-four newborns with macrosomia and 54 normal birthweight controls were enrolled in this study. Placental mtDNA copy number and mRNA expression of nuclear genes related to mitochondrial replication or ATP synthesis-related genes were measured by real-time quantitative polymerase chain reaction (qPCR). Methylation levels of the non-coding regulatory region D-loop and ATP synthesis-related genes were detected by targeted bisulfite sequencing. Newborns with macrosomia had lower placental mtDNA copy number and higher methylation rates of the CpG15 site in the D-loop region (D-CpG15) and CpG6 site in the cytochrome C oxidase III (COX3) gene (COX3-CpG6) than normal birth weight newborns. After adjusting for potential covariates (gestational age, prepregnancy BMI, and infant sex), decreased placental mtDNA copy number (adjusted odds ratio [aOR]=2.09, 95% confidence interval [CI] 1.03-4.25), elevated methylation rate of D-CpG15 (aOR=2.06, 95% CI 1.03-4.09) and COX3-CpG6 (aOR=2.13, 95% CI 1.08-4.20) remained significantly associated with a higher risk of macrosomia. Reduced mtDNA copy number and increased methylation levels of specific loci at mtDNA would increase the risk of macrosomia. However, the detailed molecular mechanism needs further identification.

Mitochondrial DNA and Epigenetics: Investigating Interactions with the One-Carbon Metabolism in Obesity.

Mitochondrial DNA copy number (mtDNAcn) has been proposed for use as a surrogate biomarker of mitochondrial health, and evidence suggests that mtDNA might be methylated. Intermediates of the one-carbon cycle (1CC), which is duplicated in the cytoplasm and mitochondria, have a major role in modulating the impact of diet on the epigenome. Moreover, epigenetic pathways and the redox system are linked by the metabolism of glutathione (GSH). In a cohort of 101 normal-weight and 97 overweight/obese subjects, we evaluated mtDNAcn and methylation levels in both mitochondrial and nuclear areas to test the association of these marks with body weight, metabolic profile, and availability of 1CC intermediates associated with diet. Body composition was associated with 1CC intermediate availability. Reduced levels of GSH were measured in the overweight/obese group (p = 1.3∗10−5). A high BMI was associated with lower LINE-1 (p = 0.004) and nominally lower methylenetetrahydrofolate reductase (MTHFR) gene methylation (p = 0.047). mtDNAcn was lower in overweight/obese subjects (p = 0.004) and independently correlated with MTHFR methylation levels (p = 0.005) but not to LINE-1 methylation levels (p = 0.086). DNA methylation has been detected in the light strand but not in the heavy strand of the mtDNA. Although mtDNA methylation in the light strand did not differ between overweight/obese and normal-weight subjects, it was nominally correlated with homocysteine levels (p = 0.035) and MTHFR methylation (p = 0.033). This evidence suggests that increased body weight might perturb mitochondrial-nuclear homeostasis affecting the availability of nutrients acting as intermediates of the one-carbon cycle.

Peripheral mitochondrial DNA, telomere length and DNA methylation as predictors of live birth inin vitrofertilization cycles.

To evaluate whether telomere length (TL), mitochondrial-DNA (mt-DNA) or epigenetic age estimators based on DNA methylation (DNAm) pattern could be considered reliable predictors of in-vitro-fertilization (IVF) success in terms of live birth rate. Prospective cohort study. Infertility Unit of the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. 181 women aged 37-39 years who underwent IVF at a single-centre between January 2017 and December 2018. On the day of recruitment, blood samples were collected, and genomic DNA was isolated from white blood cells. TL, mt-DNA and DNAm assessment was performed using quantitative real-time polymerase chain reaction (qPCR). Biological age (DNAm age) was computed as the algorithm based on methylation pattern of five genes. Epigenetic age acceleration was estimated from the residuals of the linear model of epigenetic age regressed on chronological age. Long Interspersed Nuclear Elements (LINE)-1 methylation pattern was used as a surrogate for global DNA methylation. This study investigated whether peripheral TL, mt-DNA and DNAm could predict live birth in IVF cycles. TL, mt-DNA and LINE-1 methylation were not associated with IVF success. Conversely, DNAm age resulted significantly lower in women who had a live birth compared to women who did not (36.1 ± 4.2 and 37.3 ± 3.3 years, respectively, p = 0.04). For DNAm age, odds ratio (OR) for live birth per year of age was 0.90 (95%CI: 0.82-0.99, p = 0.036) after adjusting for FSH and antral follicle count (AFC) and 0.90 (95%CI: 0.82-0.99, p = 0.028) after adjusting also for number of oocytes retrieved. A significant association also emerged for epigenetic age acceleration after adjustments (OR = 0.91, 95%CI: 0.83-1.00, p = 0.048). DNAm age is associated with IVF success but the magnitude of this association is insufficient to claim a clinical use. However, our findings are promising and warrant further investigation. Assessment of biological age using different epigenetic clocks or focusing on different tissues may reveal new predictors of IVF success.

The exposure levels and health risk assessment of antibiotics in urine and its association with platelet mitochondrial DNA methylation in adults from Tianjin, China: A preliminary study

There has been extensive research on antibiotics exposure in adults by biomonitoring, but the biological mechanisms and potential risks to human health remain limited. In this study, 102 adults aged 26–44 years in Tianjin were studied and 23 common antibiotics in urine were analyzed by Liquid chromatography-mass spectrometry (LC-MS). All antibiotics were detected in urine, with an overall detection frequency of 40.4% (the detection frequencies of phenothiazines, quinolones, sulfonamides, tetracyclines, and chloramphenicol were 77%, 54%, 24%, 28%, and 49%, respectively.). Ofloxacin and enrofloxacin had the highest detection frequencies (85% and 81%), with median concentrations of 0.26 (IQR: 0.05–1.36) and 0.09 (IQR: 0.03–0.14) ng/mL, respectively. Based on health risk assessment, the predicted estimated daily exposures (EDEs) ranged from 0 μg/kg/day to 13.98 μg/kg/day. The hazard quotient (HQ) values of all the antibiotics except ofloxacin and ciprofloxacin were bellow one, which are considered safe. For all blood samples, the mitochondrial DNA (mtDNA) methylation levels in the MT-ATP6 (ranging between 3.86% and 34.18%) were slightly higher than MT-ATP8 and MT-ND5 (ranging between 0.57% and 9.32%, 1.08% and 19.62%, respectively). Furthermore, mtDNA methylation from MT-ATP6, MT-ATP8 and MT-ND5 were measured by bisulfite-PCR pyrosequencing. The association (P < 0.05) was found between mtDNA methylation level (MT-ATP8 and MT-ND5) and individual antibiotics including chlorpromazine, ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, sulfaquinoxaline, sulfachloropyridazine, chloramphenicol, and thiamphenicol, indicating that persistent exposure to low-dose multiple antibiotics may affect the mtDNA methylation level and in turn pose health risks.

Low guanine content and biased nucleotide distribution in vertebrate mtDNA can cause overestimation of non-CpG methylation.

ABSTRACTMitochondrial DNA (mtDNA) methylation in vertebrates has been hotly debated for over 40 years. Most contrasting results have been reported following bisulfite sequencing (BS-seq) analyses. We addressed whether BS-seq experimental and analysis conditions influenced the estimation of the levels of methylation in specific mtDNA sequences. We found false positive non-CpG methylation in the CHH context (fpCHH) using unmethylated Sus scrofa mtDNA BS-seq data. fpCHH methylation was detected on the top/plus strand of mtDNA within low guanine content regions. These top/plus strand sequences of fpCHH regions would become extremely AT-rich sequences after BS-conversion, whilst bottom/minus strand sequences remained almost unchanged. These unique sequences caused BS-seq aligners to falsely assign the origin of each strand in fpCHH regions, resulting in false methylation calls. fpCHH methylation detection was enhanced by short sequence reads, short library inserts, skewed top/bottom read ratios and non-directional read mapping modes. We confirmed no detectable CHH methylation in fpCHH regions by BS-amplicon sequencing. The fpCHH peaks were located in the D-loop, ATP6, ND2, ND4L, ND5 and ND6 regions and identified in our S. scrofa ovary and oocyte data and human BS-seq data sets. We conclude that non-CpG methylation could potentially be overestimated in specific sequence regions by BS-seq analysis.

Mitochondrial DNA methylation drift and postoperative delirium in mice.

Mitochondrial dysfunction is linked to the etiopathogenesis of postoperative delirium (POD), which severely affects the prognosis of elderly patients undergoing surgery. The methylation of mitochondrial DNA (mtDNA), a new and incompletely described phenomenon that regulates the structure and function of mitochondria, is associated with ageing. However, the relationship between mtDNA methylation and POD has not been established. To explore the potential roles of mitochondrial epigenetic regulation in POD. A randomised animal study. Eighty-eight 6-month-old and one hundred seventy-six 18-month-old male C57BL/6N mice. POD was induced by abdominal surgery under 1.4% isoflurane for 2 h. Behavioural tests were performed at 24 h before surgery and at 6, 9 and 24 h after surgery. 5-methylcytosine (5-mC) at five CpG sites of the displacement loop (D-loop) and at 60 CpG sites of coding gene loci in the mitochondrial genome after surgery of the hippocampus, prefrontal cortex, amygdala and anterior cingulate cortex in 6 and 18-month-old mice were detected using bisulfite pyrosequencing. Mitochondrial structure, mitochondrial gene expression and mtDNA copy number were also examined using Electron microscopy and real time PCR to find the association with mtDNA methylation. The mtDNA methylation drift manifested as a decrease in the methylation levels at the D-loop and an increase or decrease in the methylation levels at several coding gene loci, ultimately resulting in reduced mtDNA copy numbers, altered mitochondrial gene expression and damaged mitochondrial structures in the hippocampus and prefrontal cortex after surgery. The activation of Silent information regulator-1 (SIRT1) ameliorated anaesthesia-induced and surgery-induced mitochondrial dysfunction and delirium-like behaviours by regulating mtDNA methyltransferase-mediated mtDNA methylation. These data support the existence of epigenetic mtDNA regulation in POD; however, further studies are required to explore the specific mechanisms. No 20181204 Drum tower hospital.