Simultaneous assessment of mitochondrial DNA copy number and nuclear epigenetic age towards predictive models of development and aging.

OBJECTIVE: Mitochondrial dysfunction and nuclear epigenetic alterations, two hallmarks of aging, are associated with aberrant development and complex disease risk. Here, we report a method for the simultaneous assessment of mitochondrial DNA copy number (mtDNA-CN) and DNA methylation age (DNAm age) from the same DNA extraction using quantitative polymerase chain reaction (qPCR) and array data, respectively. RESULT: We present methods for the concurrent estimation of mtDNA-CN and DNAm age from the same DNA samples. This includes qPCR to estimate mtDNA-CN, representing the number of circular mitochondrial genomes in a cell, and DNA methylation microarray data to estimate the epigenetic age of an individual. Further, we provide a method for the combination of these metrics into a shared metric termed 'mtEpiAge'. This approach provides a valuable tool for exploring the interplay between mitochondrial dysfunction and nuclear epigenetic alterations, and their associations with disease and aging.

Mitochondrial DNA copy number reduction via in vitro TFAM knockout remodels the nuclear epigenome and transcriptome.

Mitochondrial DNA copy number (mtDNA-CN) is associated with several age-related chronic diseases and is a predictor of all-cause mortality. Here, we examine site-specific differential nuclear DNA (nDNA) methylation and differential gene expression resulting from in vitro reduction of mtDNA-CN to uncover shared genes and biological pathways mediating the effect of mtDNA-CN on disease. Epigenome and transcriptome profiles were generated for three independent human embryonic kidney (HEK293T) cell lines harbouring a mitochondrial transcription factor A (TFAM) heterozygous knockout generated via CRISPR-Cas9, and matched control lines. We identified 4,242 differentially methylated sites, 228 differentially methylated regions, and 179 differentially expressed genes associated with mtDNA-CN. Integrated analysis uncovered 381 Gene-CpG pairs. GABAA receptor genes and related pathways, the neuroactive ligand receptor interaction pathway, ABCD1/2 gene activity, and cell signalling processes were overrepresented, providing insight into the underlying biological mechanisms facilitating these associations. We also report evidence implicating chromatin state regulatory mechanisms as modulators of mtDNA-CN effect on gene expression. We demonstrate that mitochondrial DNA variation signals to the nuclear DNA epigenome and transcriptome and may lead to nuclear remodelling relevant to development, aging, and complex disease.

Longitudinal change in mitochondrial heteroplasmy exhibits positive selection for deleterious variants.

A common feature of human aging is the acquisition of somatic mutations, and mitochondria are particularly prone to mutation due to their inefficient DNA repair and close proximity to reactive oxygen species, leading to a state of mitochondrial DNA heteroplasmy1,2. Cross-sectional studies have demonstrated that detection of heteroplasmy increases with participant age3, a phenomenon that has been attributed to genetic drift4-7. In this first large-scale longitudinal study, we measured heteroplasmy in two prospective cohorts (combined n=1405) at two timepoints (mean time between visits, 8.6 years), demonstrating that deleterious heteroplasmies were more likely to increase in variant allele fraction (VAF). We further demonstrated that increase in VAF was associated with increased risk of overall mortality. These results challenge the claim that somatic mtDNA mutations arise mainly due to genetic drift, instead demonstrating positive selection for predicted deleterious mutations at the cellular level, despite an negative impact on overall mortality.

Mitochondrial DNA copy number is associated with incident chronic kidney disease and proteinuria in the AIDS linked to the intravenous experience cohort.

We evaluated the prospective association of mitochondrial DNA copy number (mtDNA CN) with markers of kidney function among a cohort of persons who inject drugs (PWID). This is a Prospective cohort study nested in the AIDS linked to the intravenous experience cohort (community-based cohort of PWID in Baltimore, MD). mtDNA CN was measured at two time-points 5 years apart using a real-time polymerase chain reaction. Kidney function (estimated glomerular filtration rate [eGFR], serum creatinine, urine protein) was measured annually. We used linear mixed effects models to evaluate kidney function trajectories (N = 946) and Cox regression models to assess hazard of incident CKD (eGFR < 60 at two consecutive visits, N = 739) and proteinuria (urine protein:creatinine ratio > 200, N = 573) by level of mtDNA CN (Low [lowest quartile], vs high [other three quartiles]. Models were adjusted for demographic and behavioral characteristics, HIV and/or HCV infection, and comorbidity burden. Low mtDNA CN was independently associated with higher hazard of incident CKD (aHR: 2.33, 95% CI 1.42, 3.80) and proteinuria (aHR: 1.42, 95% CI 1.04, 1.96). Participants with low mtDNA CN had greater declines in eGFR and greater increases in serum creatinine over time. Low mtDNA CN is associated with more rapid kidney function decline and risk of incident CKD and proteinuria.

Mitochondrial DNA copy number, metabolic syndrome, and insulin sensitivity: Insights from the Sugar, Hypertension, and Physical Exercise studies.

Mitochondrial DNA copy number (mtDNA-CN) measured in blood has been associated with many aging-related diseases, with higher mtDNA-CN typically associated with lower disease risk. Exercise training is an excellent preventative tool against aging-related disorders and has been shown to increase mitochondrial function in muscle. Using the Sugar, Hypertension, and Physical Exercise cohorts (N = 105), we evaluated the effect of 6-months of exercise intervention on mtDNA-CN measured in blood. Although there was no significant relationship between exercise intervention and mtDNA-CN change (P = 0.29), there was a nominally significant association between mtDNA-CN and metabolic syndrome (P = 0.04), which has been seen in previous literature. We also identified a nominally significant association between higher mtDNA-CN and higher insulin sensitivity (P = 0.02).

Evaluation of mitochondrial DNA copy number estimation techniques.

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 can influence mortality and cardiovascular disease via methylation of nuclear DNA CpGs.

BACKGROUND: Mitochondrial DNA copy number (mtDNA-CN) has been associated with a variety of aging-related diseases, including all-cause mortality. However, the mechanism by which mtDNA-CN influences disease is not currently understood. One such mechanism may be through regulation of nuclear gene expression via the modification of nuclear DNA (nDNA) methylation. METHODS: To investigate this hypothesis, we assessed the relationship between mtDNA-CN and nDNA methylation in 2507 African American (AA) and European American (EA) participants from the Atherosclerosis Risk in Communities (ARIC) study. To validate our findings, we assayed an additional 2528 participants from the Cardiovascular Health Study (CHS) (N = 533) and Framingham Heart Study (FHS) (N = 1995). We further assessed the effect of experimental modification of mtDNA-CN through knockout of TFAM, a regulator of mtDNA replication, via CRISPR-Cas9. RESULTS: Thirty-four independent CpGs were associated with mtDNA-CN at genome-wide significance (P < 5 × 10- 8). Meta-analysis across all cohorts identified six mtDNA-CN-associated CpGs at genome-wide significance (P < 5 × 10- 8). Additionally, over half of these CpGs were associated with phenotypes known to be associated with mtDNA-CN, including coronary heart disease, cardiovascular disease, and mortality. Experimental modification of mtDNA-CN demonstrated that modulation of mtDNA-CN results in changes in nDNA methylation and gene expression of specific CpGs and nearby transcripts. Strikingly, the "neuroactive ligand receptor interaction" KEGG pathway was found to be highly overrepresented in the ARIC cohort (P = 5.24 × 10- 12), as well as the TFAM knockout methylation (P = 4.41 × 10- 4) and expression (P = 4.30 × 10- 4) studies. CONCLUSIONS: These results demonstrate that changes in mtDNA-CN influence nDNA methylation at specific loci and result in differential expression of specific genes that may impact human health and disease via altered cell signaling.

Effect of Sex and Underlying Disease on the Genetic Association of QT Interval and Sudden Cardiac Death.

Background Sudden cardiac death (SCD) accounts for ≈300 000 deaths annually in the United States. Men have a higher risk of SCD and are more likely to have underlying coronary artery disease, while women are more likely to have arrhythmic events in the setting of inherited or acquired QT prolongation. Moreover, there is evidence of sex differences in the genetics of QT interval duration. Using sex- and coronary artery disease-stratified analyses, we assess differences in genetic association between longer QT interval and SCD risk. Methods and Results We examined 2282 SCD subjects and 3561 Finnish controls. The SCD subjects were stratified by underlying disease (ischemic versus nonischemic) and by sex. We used logistic regression to test for association between the top QT interval-associated single-nucleotide polymorphism, rs12143842 (in the NOS1AP locus), and SCD risk. We also performed Mendelian randomization to test for causal association of QT interval in the various subgroups. No statistically significant differences were observed between the sexes for associations with rs12143842, despite the odds ratio being higher in females across all subgroup analyses. Consistent with our hypothesis, female non-ischemics had the highest odds ratio point estimate for association between rs12143842 and SCD risk and male ischemics the lowest odds ratio point estimate (P=0.036 for difference). Similar trends were observed for the Mendelian randomization analysis. Conclusions While individual subgroup comparisons did not achieve traditional criteria for statistical significance, this study is consistent with the hypothesis that the causal association of longer QT interval on SCD risk is stronger in women and nonischemic individuals.

In Situ Enzymatic Conversion of Nannochloropsis oceanica IMET1 Biomass into Fatty Acid Methyl Esters.

Conventionally, production of methyl ester fuels from microalgae occurs through an energy-intensive two-step chemical extraction and transesterification process. To improve the energy efficiency, we performed in situ enzymatic conversion of whole algae biomass from an oleaginous heterokont microalga Nannochloropsis oceanica IMET1 with the immobilized lipase from Candida antarctica. The fatty acid methyl ester yield reached 107.7% for dry Nannochloropsis biomass at biomass to t-butanol to methanol weight ratio of 1:2:0.5 and a reaction time of 12 h at 25 °C, representing the first report of efficient whole algae biomass conversion into fatty acid methyl esters at room temperature. Different forms of algal biomass including wet Nannochloropsis biomass were tested. The maximum yield of wet biomass was 81.5%. Enzyme activity remained higher than 95% after 55 days of treatment (equal to 110 cycles of reaction) under the conditions optimized for dry algae biomass conversion. The low reaction temperature, high enzyme stability, and high yield from this study indicate in situ enzymatic conversion of dry algae biomass may potentially be used as an energy-efficient method for algal methyl ester fuel production while allowing co-product recovery.