GWAS and ExWAS of blood Mitochondrial DNA copy number identifies 71 loci and highlights a potential causal role in dementia

Background: Mitochondrial DNA copy number (mtDNA-CN) is an accessible blood-based measurement believed to capture underlying mitochondrial function. The specific biological processes underpinning its regulation, and whether those processes are causative for disease, is an area of active investigation.Methods: We developed a novel method for array-based mtDNA-CN estimation suitable for biobank-scale studies, called 'AutoMitoC'. We applied AutoMitoC to 395,781 UKBiobank study participants and performed genome and exome-wide association studies, identifying novel common and rare genetic determinants. Finally, we performed two-sample Mendelian Randomization to assess whether genetically low mtDNA-CN influenced select mitochondrial phenotypes.Results: Overall, genetic analyses identified 71 loci for mtDNA-CN, which implicated several genes involved in rare mtDNA depletion disorders, dNTP metabolism, and the mitochondrial central dogma. Rare variant analysis identified SAMHD1 mutation carriers as having higher mtDNA-CN (beta=0.23 SDs; 95% CI, 0.18- 0.29; P=2.6x10-19), a potential therapeutic target for patients with mtDNA depletion disorders, but at increased risk of breast cancer (OR=1.91; 95% CI, 1.52-2.40; P=2.7x10-8). Finally, Mendelian randomization analyses suggest a causal effect of low mtDNA-CN on dementia risk (OR=1.94 per 1 SD decrease in mtDNA-CN; 95% CI, 1.55-2.32; P=7.5x10-4).Conclusions: Altogether, our genetic findings indicate that mtDNA-CN is a complex biomarker reflecting specific mitochondrial processes related to mtDNA regulation, and that these processes are causally related to human diseases.Funding: No funds supported this specific investigation. Awards and positions supporting authors include: Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarships Doctoral Award (MC, PM); CIHR Post-Doctoral Fellowship Award (RM); Wellcome Trust Grant number: 099313/B/12/A; Crasnow Travel Scholarship; Bongani Mayosi UCT-PHRI Scholarship 2019/2020 (TM); Wellcome Trust Health Research Board Irish Clinical Academic Training (ICAT) Programme Grant Number: 203930/B/16/Z (CJ); European Research Council COSIP Grant Number: 640580 (MO); E.J. Moran Campbell Internal Career Research Award (MP); CISCO Professorship in Integrated Health Systems and Canada Research Chair in Genetic and Molecular Epidemiology (GP).

Mitochondrial DNA Copy Number in Cleavage Stage Human Embryos—Impact on Infertility Outcome

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between mitochondrial DNA (mtDNA) content of cleavage-stage preimplantation embryos and their developmental potential. A total of 69 couples underwent IVF treatment (averaged women age: 36.5, SD 4.9) and produced a total of 314 embryos. A single blastomere was biopsied from each embryo at the cleavage stage (day-3 post-fertilization) subjected to low-pass next generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number amount was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 6.3 ± 7.5 versus 7.1 ± 5.8, p < 0.004; U Mann–Whitney test), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (6.6 ± 4.8 vs. 8.5 ± 13.6, p 0.09), sex (6.6 ± 4.1 vs. 6.2 ± 6.8, p 0.16), maternal age (6.9 ± 7.8 vs. 6.7 ± 4.5, p 0.14) or its ability to implant (7.4 ± 6.6 vs. 5.1 ± 4.6, p 0.18). The mtDNA content cannot serve as a useful biomarker at this point in development. However, further studies investigating both quantitative and qualitative aspects of mtDNA are still required to fully evaluate the relationship between mitochondrial DNA and human reproduction.

Decreased mitochondrial D-loop region methylation mediates an increase in mitochondrial DNA copy number in CADASIL

Background Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a typical neurodegenerative disease associated with mitochondrial dysfunction. Methylation of the D-loop region and mitochondrial DNA copy number (mtDNAcn) play a critical role in the maintenance of mitochondrial function. However, the association between D-loop region methylation, mtDNAcn and CADASIL remains unclear. Methods Overall, 162 individuals were recruited, including 66 CADASIL patients and 96 age- and sex-matched controls. After extracting genomic DNA from the peripheral white blood cells, levels of D-loop methylation and mtDNAcn were assessed using MethylTarget sequencing and real-time PCR, respectively. Results We observed increased mtDNAcn and decreased D-loop methylation levels in CADASIL patients compared to the control group, regardless of gender stratification. Besides, we found a negative correlation between D-loop methylation levels and mtDNAcn. Mediation effect analysis shows that the proportion of the association between mtDNAcn and CADASIL that is mediated by D-loop methylation is 11.6% (95% CI 5.6, 22.6). After gender stratification, the proportions of such associations that are mediated by D-loop methylation in males and females were 7.2% (95% CI 2.4, 19.8) and 22.0% (95% CI 7.4, 50.1), respectively. Conclusion Decreased methylation of the D-loop region mediates increased mtDNAcn in CADASIL, which may be caused by a compensatory mechanism of mitochondrial dysfunction in patients with CADASIL.

Secoisolariciresinol Diglucoside Improves Ovarian Reserve in Aging Mouse by Inhibiting Oxidative Stress

Ovarian reserve is a key factor in the reproductive function of the ovaries. Ovarian aging is characterized by a gradual decline in the quantity and quality of follicles. The underlying mechanism of ovarian aging is complex and age-related oxidative stress is considered one of the most likely factors. Secoisolariciresinol diglucoside (SDG) has been shown to have good scavenging ability against reactive oxygen species (ROS) which slowly accumulates in ovarian tissues. However, it is unknown whether SDG had beneficial effects on aging ovaries. In this study, we used 37-week-old female C57BL/6J mouse as a natural reproductive aging model to evaluate the role of SDG in ovarian aging. SDG (7 and 70 mg/kg) intragastric administration was performed in the mice daily. After 8 weeks, the effects of SDG on aging ovaries were evaluated by counting the number of follicles and the expression of follicle-stimulating hormone receptors (FSHR) in the ovary. The mechanism of SDG on the aging ovaries was further explored through ovarian metabolomics. It was found that SDG can effectively increase the number of growing follicles and increase the expression of the FSHR protein. The metabolomics results showed that the ovaries in the SDG intervention group achieved better uptake and transport of nutrients, including amino acids and glucose that are necessary for the development of oocytes. At the same time, the ovaries of the SDG intervention group showed that the drug reduced ROS generation. Additionally, we found that ovarian telomere length and ovarian mitochondrial DNA copy number that are highly susceptible to ROS damage and are also related to aging. The results showed that SDG can significantly increase mitochondrial DNA copy number and slow down the process of telomere shortening. These data indicate that SDG improves ovarian reserve by inhibiting oxidative stress.

Mitochondrial DNA copy number as a prognostic marker is age-dependent in adult glioblastoma

Background Glioblastoma (GBM) is the most common and aggressive form of glioma. GBM frequently displays chromosome (chr) 7 gain, chr 10 loss and/or EGFR amplification (chr7+/chr10-/EGFRamp). Overall survival (OS) is 15 months after treatment. In young adults, IDH1/2 mutations are associated with longer survival. In children, histone H3 mutations portend a dismal prognosis. Novel reliable prognostic markers are needed in GBM. We assessed the prognostic value of mitochondrial DNA (mtDNA) copy number in adult GBM. Methods mtDNA copy number was assessed using real-time quantitative PCR in 232 primary GBM. Methylation of POLG and TFAM genes, involved in mtDNA replication, was assessed by bisulfite-pyrosequencing in 44 and 51 cases, respectively. Results Median age at diagnosis was 56.6 years-old and median OS, 13.3 months. 153/232 GBM (66 %) displayed chr7+/chr10-/EGFRamp, 23 (9.9 %) IDH1/2 mutation, 3 (1.3 %) H3 mutation and 53 (22.8 %) no key genetic alterations. GBM were divided into two groups, “Low” (n = 116) and “High” (n = 116), according to the median mtDNA/nuclear DNA ratio (237.7). There was no significant difference in OS between the two groups. By dividing the whole cohort according to the median age at diagnosis, OS was longer in the “High” vs “Low” subgroup (27.3 vs 15 months, p = 0.0203) in young adult GBM (n = 117) and longer in the “Low” vs “High” subgroup (14.5 vs 10.2 months, p = 0.0116) in older adult GBM (n = 115). POLG was highly methylated, whereas TFAM remained unmethylated. Conclusion mtDNA copy number may be a novel prognostic biomarker in GBM, its impact depending on age.

Associations of mitochondrial DNA copy number with incident risks of gastrointestinal cancers: a prospective case-cohort study

Background Epidemiological investigations implied that inter-individual variations of mitochondrial DNA copy number (mtDNAcn) could trigger predisposition to multiple cancers, but evidence regarding gastrointestinal cancers (GICs) was still uncertain. Methods We conducted a case-cohort study within the prospective Dongfeng-Tongji cohort, including incident cases of colorectal cancer (CRC, n=278), gastric cancer (GC, n=138), and esophageal cancer (EC, n=72) as well as a random subcohort (n=1173), who were followed up from baseline to the end of 2018. Baseline blood mtDNAcn was determined with quantitative PCR assay, and associations of mtDNAcn with the GICs risks were estimated by using weighted Cox proportional hazards models. Results Significant U-shaped associations were observed between mtDNAcn and risks of CRC, GC, EC, and total GICs. Compared to subjects within the 2nd quartile (Q2) mtDNAcn subgroup, those within the 1st (Q1), 3rd (Q3) and 4th (Q4) quartile subgroups showed increased risks of CRC [HR(95%CI)=2.27(1.47-3.52), 1.65(1.04-2.62), and 2.81(1.85-4.28), respectively] and total GICs [HR(95%CI)=1.84(1.30-2.60), 1.47(1.03-2.10), and 2.51(1.82-3.47), respectively], and those within Q4 subgroup present elevated GC and EC risks [HR(95%CI)=2.16(1.31-3.54) and 2.38(1.13-5.02), respectively]. Similar associations of mtDNAcn with CRC and total GICs risks remained in stratified analyzes by age, gender, and smoking status. Notably, there were joint effects of age and smoking status with mtDNAcn on CRC and total GICs risks. Conclusions This prospectively case-cohort study showed U-shaped associations between mtDNAcn and incident risks of GICs, but further researches are needed to confirm these results and uncover underlying biological mechanisms.

Oxidative Stress Biomarkers and Mitochondrial DNA Copy Number Associated with APOE4 Allele and Cholinesterase Inhibitor Therapy in Patients with Alzheimer’s Disease

Studies of the oxidative/anti-oxidative status in patients with Alzheimer’s disease (AD) carrying different alleles of the apolipoprotein E (APOE) gene are currently inconclusive; meanwhile, data regarding mitochondrial DNA copy number (mtCN) remain limited. We herein determined the thiobarbituric acid reactive substances (TBARS), thiols, and mtCN in blood samples of 600 AD patients and 601 controls. A significantly higher oxidative TBARS (1.64 μmol/L), lower antioxidative thiols (1.60 μmol/L), and lower mtCN (2.34 log Delta Ct) were found in the AD cohort as compared to the non-AD cohort (1.54 μmol/L, 1.71 μmol/L, 2.46 log Delta Ct). We further identified the ε4 alleles (APOE4) and separated subjects into three groups according to the number of APOE4. A significant trend was noted in the TBARS levels of both AD and non-AD cohorts, highest in the homozygous two alleles (1.86 and 1.80 μmol/L), followed by heterozygous one allele (1.70 and 1.74 μmol/L), and lowest in the no APOE4 allele (1.56 and 1.48 μmol/L). Similar trends of lower thiols and mtCN were also found in the AD cohort. In our study of the influence of cholinesterase inhibitor therapy, we found significantly reduced TBARS levels, and elevated mtCN in AD patients receiving rivastigmine and galantamine therapy. Our study demonstrates associations between the APOE4 allele and oxidative stress biomarkers and mtCN. Using cholinesterase inhibitor therapy may benefit AD patients through attenuation of oxidative stress and manipulation of the mtCN.

Mitochondrial DNA Copy Number as a Marker and Mediator of Stroke Prognosis: Observational and Mendelian Randomization Analyses

Background:Low buffy coat mitochondrial DNA copy number (mtDNA-CN) is associated with incident risk of stroke and post-stroke mortality; however, its prognostic utility has not been extensively explored.Objective:To investigate whether low buffy coat mtDNA-CN is a marker and causal determinant of post-stroke outcomes using epidemiological and genetic studies.Methods:First, we performed association testing between baseline buffy coat mtDNA-CN measurements and 1-month post-stroke outcomes in 3498 acute, first stroke cases from 25 countries from the international, multicenter case-control study, “Importance of Conventional and Emerging Risk Factors of Stroke in Different Regions and Ethnic Groups of the World” (INTERSTROKE). Then, we performed two-sample Mendelian Randomization analyses to evaluate potential causative effects of low mtDNA-CN on 3-month modified Rankin Scale (mRS). Genetic variants associated with mtDNA-CN levels were derived from the UKBiobank study (N=383476), and corresponding effects on 3-month mRS were ascertained from the Genetics of Ischemic Stroke funCtional Outcome study (GISCOME; N=6021).Results:A 1-standard deviation (SD) lower mtDNA-CN at baseline was associated with stroke severity (baseline mRS; OR=1.27; 95% CI, 1.19-1.36; P=4.7x10-12). Independent of baseline stroke severity, lower mtDNA-CN was associated with increased odds of greater 1-month disability (ordinal mRS; OR=1.16; 95% CI, 1.08-1.24; P=4.4x10-5), poor functional outcome status (mRS 3-6 vs. 0-2; OR=1.21; 95% CI, 1.08-1.34; P=6.9x10-4), and mortality (OR=1.35; 95% CI, 1.14-1.59; P=3.9x10-4). Subgroup analyses demonstrated consistent effects across stroke type, sex, age, country income level, and education level. In addition, mtDNA-CN significantly improved reclassification of poor functional outcome status (Net Reclassification Index (NRI)=0.16; 95% CI, 0.08-0.23; P=3.6x10-5) and mortality (NRI=0.31; 95% CI, 0.19-0.43; P=1.7x10-7) beyond known prognosticators. Using independent datasets, Mendelian Randomization revealed that a 1 SD decrease in genetically determined mtDNA-CN was associated with increased odds of greater 3-month disability quantified by ordinal mRS (OR=2.35; 95% CI, 1.13-4.90; P=0.02) and poor functional outcome status (OR=2.68; 95% CI, 1.05-6.86; P=0.04).Conclusions:Buffy coat mtDNA-CN is a novel and robust marker of post-stroke prognosis that may also be a causal determinant of post-stroke outcomes.Classification of Evidence:This study provides class II evidence that low buffy coat mtDNA-CN (>1-standard deviation) was associated with worse baseline severity and 1-month outcomes in patients with ischemic or hemorrhagic stroke.

Concurrent Measurement of Mitochondrial DNA Copy Number and ATP Concentration in Single Bovine Oocytes

To sustain energy-demanding developmental processes, oocytes must accumulate adequate stores of metabolic substrates and mitochondrial numbers prior to the initiation of maturation. In the past, researchers have utilized pooled samples to study oocyte metabolism, and studies that related multiple metabolic outcomes in single oocytes, such as ATP concentration and mitochondrial DNA copy number, were not possible. Such scenarios decreased sensitivity to intraoocyte metabolic relationships and made it difficult to obtain adequate sample numbers during studies with limited oocyte availability. Therefore, we developed and validated procedures to measure both mitochondrial DNA (mtDNA) copy number and ATP quantity in single oocytes. Validation of our procedures revealed that we could successfully divide oocyte lysates into quarters and measure consistent results from each of the aliquots for both ATP and mtDNA copy number. Coefficient of variation between the values retrieved for mtDNA copy number and ATP quantity quadruplicates were 4.72 ± 0.98 and 1.61 ± 1.19, respectively. We then utilized our methodology to concurrently measure mtDNA copy number and ATP quantity in germinal vesicle (GV) and metaphase two (MII) stage oocytes. Our methods revealed a significant increase in ATP levels (GV = 628.02 ± 199.53 pg, MII = 1326.24 ± 199.86 pg, p < 0.001) and mtDNA copy number (GV = 490,799.4 ± 544,745.9 copies, MII = 1,087,126.9 ± 902,202.8 copies, p = 0.035) in MII compared to GV stage oocytes. This finding is consistent with published literature and provides further validation of the accuracy of our methods. The ability to produce consistent readings and expected results from aliquots of the lysate from a single oocyte reveals the sensitivity and feasibility of using this method.

Telomere Shortening linked to Disability and Mitochondrial DNA Copy Number in Patients with Relapsing-Remitting Multiple Sclerosis

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease that affects the nervous system. Peripheral blood leukocyte telomere length (LTL) and mitochondrial DNA copy number (mtDNA-CN) are potential biomarkers of disability and neurological damage. The present work evaluated LTL and mtDNA-CN in 75 relapsing-remittent MS (RRMS) patients 50 of whom had an Expanded Disability Status Scale (EDSS) 0 to 3 (mild-moderate disability), and 25 had an EDSS of 3.5 to 7 (severe disability). Absolute LTL and absolute mtDNA-CN were measured via real-time polymerase chain reaction (qPCR). The LTL and mtDNA-CN were significantly lower in RRMS severe disability than in RRMS mild-moderate disability (3.924 &plusmn; 0.124 vs 2.854 &plusmn; 0.092, p&lt;00001; 75.14 &plusmn; 1.77 vs 68.06 &plusmn; 1.608, p&lt;0.00001, respectively). The LTL and mtDNA-CN showed a linear correlation in RRMS with mild-moderate disability (r=0.2986, p=0.0351). In addition, in a binary logistic regression model the LTL can predict severe disability (AUC=0.697, p=0.0031, cutoff &le; 3.0875 Kb, sensitivity= 73.1%, specificity=62.5%), the prediction is improved by including age to the model (AUC=0.765, &lt;0.0001, sensitivity=78.26%, specificity=81.25%). Aging is closely linked to the development of disability in RRMS and can be evaluated through LTL and mtDNA-CN absolute quantification.