Mitochondrial DNA copy number (mtDNA-CN), a measure of the number of mitochondrial genomes per cell, is a minimally invasive proxy measure for mitochondrial function and has been associated with several aging-related diseases. Although quantitative real-time PCR (qPCR) is the current gold standard method for measuring mtDNA-CN, mtDNA-CN can also be measured from genotyping microarray probe intensities and DNA sequencing read counts. To conduct a comprehensive examination on the performance of these methods, we use known mtDNA-CN correlates (age, sex, white blood cell count, Duffy locus genotype, incident cardiovascular disease) to evaluate mtDNA-CN calculated from qPCR, two microarray platforms, as well as whole genome (WGS) and whole exome sequence (WES) data across 1,085 participants from the Atherosclerosis Risk in Communities (ARIC) study and 3,489 participants from the Multi-Ethnic Study of Atherosclerosis (MESA). We observe mtDNA-CN derived from WGS data is significantly more associated with known correlates compared to all other methods (p < 0.001). Additionally, mtDNA-CN measured from WGS is on average more significantly associated with traits by 5.6 orders of magnitude and has effect size estimates 5.8 times more extreme than the current gold standard of qPCR. We further investigated the role of DNA extraction method on mtDNA-CN estimate reproducibility and found mtDNA-CN estimated from cell lysate is significantly less variable than traditional phenol-chloroform-isoamyl alcohol (p = 5.44x10^-4) and silica-based column selection (p = 2.82x10^-7). In conclusion, we recommend the field moves towards more accurate methods for mtDNA-CN, as well as re-analyze trait associations as more WGS data becomes available from larger initiatives such as TOPMed.

Mitochondrial DNA copy number (mtDNA-CN) is a proxy for mitochondrial function and is associated with aging-related diseases. However, it is unclear how mtDNA-CN measured in blood can reflect diseases that primarily manifest in other tissues. Using the Genotype-Tissue Expression Project, we interrogated relationships between mtDNA-CN measured in whole blood and gene expression from whole blood and 47 additional tissues in 419 individuals. mtDNA-CN was significantly associated with expression of 700 genes in whole blood, including nuclear genes required for mtDNA replication. Significant enrichment was observed for splicing and ubiquitin-mediated proteolysis pathways, as well as target genes for the mitochondrial transcription factor NRF1. In nonblood tissues, there were more significantly associated genes than expected in 30 tissues, suggesting that global gene expression in those tissues is correlated with blood-derived mtDNA-CN. Neurodegenerative disease pathways were significantly associated in multiple tissues, and in an independent data set, the UK Biobank, we observed that higher mtDNA-CN was significantly associated with lower rates of both prevalent (OR = 0.89, CI = 0.83; 0.96) and incident neurodegenerative disease (HR = 0.95, 95% CI = 0.91;0.98). The observation that mtDNA-CN measured in blood is associated with gene expression in other tissues suggests that blood-derived mtDNA-CN can reflect metabolic health across multiple tissues. Identification of key pathways including splicing, RNA binding, and catalysis reinforces the importance of mitochondria in maintaining cellular homeostasis. Finally, validation of the role of mtDNA CN in neurodegenerative disease in a large independent cohort study solidifies the link between blood-derived mtDNA-CN, altered gene expression in multiple tissues, and aging-related disease.

Sudden cardiac death (SCD) is a major public health burden. Mitochondrial dysfunction has been implicated in a wide range of cardiovascular diseases including cardiomyopathy, heart failure, and arrhythmias, but it is unknown if it also contributes to SCD risk. We sought to examine the prospective association between mtDNA copy number (mtDNA-CN), a surrogate marker of mitochondrial function, and SCD risk. We measured baseline mtDNA-CN in 11 093 participants from the Atherosclerosis Risk in Communities (ARIC) study. mtDNA copy number was calculated from probe intensities of mitochondrial single nucleotide polymorphisms (SNP) on the Affymetrix Genome-Wide Human SNP Array 6.0. Sudden cardiac death was defined as a sudden pulseless condition presumed due to a ventricular tachyarrhythmia in a previously stable individual without evidence of a non-cardiac cause of cardiac arrest. Sudden cardiac death cases were reviewed and adjudicated by an expert committee. During a median follow-up of 20.4 years, we observed 361 SCD cases. After adjusting for age, race, sex, and centre, the hazard ratio for SCD comparing the 1st to the 5th quintiles of mtDNA-CN was 2.24 (95% confidence interval 1.58–3.19; P-trend <0.001). When further adjusting for traditional cardiovascular disease risk factors, prevalent coronary heart disease, heart rate, QT interval, and QRS duration, the association remained statistically significant. Spline regression models showed that the association was approximately linear over the range of mtDNA-CN values. No apparent interaction by race or by sex was detected. In this community-based prospective study, mtDNA-CN in peripheral blood was inversely associated with the risk of SCD.

Aim: Prenatal alcohol exposure (PAE) can cause fetal alcohol spectrum disorders (FASD). Previously, we assessed PAE in brain tissue from mouse models, however whether these changes are present in humans remains unknown. Materials & methods: In this report, we show some identical changes in DNA methylation in the buccal swabs of six children with FASD using the 450K array. Results: The changes occur in genes related to protocadherins, glutamatergic synapses, and hippo signaling. The results were found to be similar in another heterogeneous replication group of six FASD children. Conclusion: The replicated results suggest that children born with FASD have unique DNA methylation defects that can be influenced by sex and medication exposure. Ultimately, with future clinical development, assessment of DNA methylation from buccal swabs can provide a novel strategy for the diagnosis of FASD.

Background: The mechanism of action of olanzapine in treating schizophrenia is not clear. This research reports the effects of a therapeutic equivalent treatment of olanzapine on DNA methylation in a rat model in vivo. Genome-wide DNA methylation was assessed using a MeDIP-chip analysis. All methylated DNA immunoprecipitation (MeDIP), sample labelling, hybridization and processing were performed by Arraystar Inc (Rockville, MD, USA). The identified gene promoters showing significant alterations to DNA methylation were then subjected to Ingenuity Pathway Analysis (Ingenuity System Inc, CA, USA). Results: The results show that olanzapine causes an increase in methylation in 1,140, 1,294 and 1,313 genes and a decrease in methylation in 633, 565 and 532 genes in the hippocampus, cerebellum and liver, respectively. Most genes affected are tissue specific. Only 41 affected genes (approximately 3%) showed an increase and no gene showed a decrease in methylation in all three tissues. Further, the two brain regions shared 123 affected genes (approximately 10%). The affected genes are enriched in pathways affecting dopamine signalling, molecular transport, nervous system development and functions in the hippocampus; ephrin receptor signalling and synaptic long-term potentiation in the cerebellum; and tissue morphology, cellular assembly and organization in the liver. Also, the affected genes included those previously implicated in psychosis. Conclusions: The known functions of affected genes suggest that the observed epigenetic changes may underlie the amelioration of symptoms as well as accounting for certain adverse effects including the metabolic syndrome. The results give insights into the mechanism of action of olanzapine, therapeutic effects and the side effects of antipsychotics.

Despite their singular origin, monozygotic twin pairs often display discordance for complex disorders including schizophrenia. It is a common (1%) and often familial disease with a discordance rate of ~50% in monozygotic twins. This high discordance is often explained by the role of yet unknown environmental, random, and epigenetic factors. The involvement of DNA methylation in this disease appears logical, but remains to be established. We have used blood DNA from two pairs of monozygotic twins discordant for schizophrenia and their parents in order to assess genome-wide methylation using a NimbleGen Methylation Promoter Microarray. The genome-wide results show that differentially methylated regions (DMRs) exist between members representing discordant monozygotic twins. Some DMRs are shared with parent(s) and others appear to be de novo. We found twenty-seven genes affected by DMR changes that were shared in the affected member of two discordant monozygotic pairs from unrelated families. Interestingly, the genes affected by pair specific DMRs share specific networks. Specifically, this study has identified two networks; “cell death and survival” and a “cellular movement and immune cell trafficking”. These two networks and the genes affected have been previously implicated in the aetiology of schizophrenia. The results are compatible with the suggestion that DNA methylation may contribute to the discordance of monozygotic twins for schizophrenia. Also, this may be accomplished by the direct effect of gene specific methylation changes on specific biological networks rather than individual genes. It supports the extensive genetic, epigenetic and phenotypic heterogeneity implicated in schizophrenia.