Mitochondrial DNA variation across 56,434 individuals in gnomAD

Databases of allele frequency are extremely helpful for evaluating clinical variants of unknown significance; however, until now, genetic databases such as the Genome Aggregation Database (gnomAD) have ignored the mitochondrial genome (mtDNA). Here we present a pipeline to call mtDNA variants that addresses three technical challenges: (i) detecting homoplasmic and heteroplasmic variants, present respectively in all or a fraction of mtDNA molecules, (ii) circular mtDNA genome, and (iii) misalignment of nuclear sequences of mitochondrial origin (NUMTs). We observed that mtDNA copy number per cell varied across gnomAD cohorts and influenced the fraction of NUMT-derived false-positive variant calls, which can account for the majority of putative heteroplasmies. To avoid false positives, we excluded samples prone to NUMT misalignment (few mtDNA copies per cell), cell line artifacts (many mtDNA copies per cell), or with contamination and we reported variants with heteroplasmy greater than 10%. We applied this pipeline to 56,434 whole genome sequences in the gnomAD v3.1 database that includes individuals of European (58%), African (25%), Latino (10%), and Asian (5%) ancestry. Our gnomAD v3.1 release contains population frequencies for 10,850 unique mtDNA variants at more than half of all mtDNA bases. Importantly, we report frequencies within each nuclear ancestral population and mitochondrial haplogroup. Homoplasmic variants account for most variant calls (98%) and unique variants (85%). We observed that 1/250 individuals carry a pathogenic mtDNA variant with heteroplasmy above 10%. These mitochondrial population allele frequencies are publicly available at gnomad.broadinstitute.org and will aid in diagnostic interpretation and research studies.

Acute Exposure to Bisphenol A Causes Oxidative Stress Induction with Mitochondrial Origin in Saccharomyces cerevisiae Cells

Bisphenol A (BPA) is a major component of the most commonly used plastic products, such as disposable plastics, Tetra Paks, cans, sport protective equipment, or medical devices. Due to the accumulation of excessive amounts of plastic waste and the subsequent release of BPA into the environment, BPA is classified as a pollutant that is undesirable in the environment. To date, the most interesting finding is the ability of BPA to act as an endocrine disrupting compound due to its binding to estrogen receptors (ERs), and adverse physiological effects on living organisms may result from this action. Since evidence of the potential pro-oxidizing effects of BPA has accumulated over the last years, herein, we focus on the detection of oxidative stress and its origin following BPA exposure using pulsed-field gel electrophoresis, flow cytometry, fluorescent microscopy, and Western blot analysis. Saccharomyces cerevisiae cells served as a model system, as these cells lack ERs allowing us to dissect the ER-dependent and -independent effects of BPA. Our data show that high concentrations of BPA affect cell survival and cause increased intracellular oxidation in yeast, which is primarily generated in the mitochondrion. However, an acute BPA exposure does not lead to significant oxidative damage to DNA or proteins.

Circulating extracellular DNA is in association with continuous metabolic syndrome score in healthy adolescents

Obesity is associated with chronic low-grade inflammation that eventually leads to metabolic complications. Extracellular DNA (ecDNA) is a damage-associated molecular pattern. Extracellular mitochondrial DNA can activate innate immunity. We hypothesized that ecDNA, especially of mitochondrial origin could be associated with components of the metabolic syndrome in young healthy probands. In a cross-sectional study healthy adolescents (n=1249) provided blood samples. Anthropometric data, blood pressure and blood counts were assessed. In addition, biochemical analysis of sera or plasma was conducted including the quantification of advanced oxidation protein products (AOPP) as a marker of oxidative stress induced by neutrophil or monocyte activation. Plasma ecDNA was isolated and measured using fluorometry. Nuclear and mitochondrial DNA were quantified using real time PCR. Males had higher total plasma ecDNA (15 (11-21) vs 11 (8-17) ng/ml; median (IQR)), nuclear (1760 (956-3273) vs 1153 (600-2292) GE/ml) and mitochondrial DNA (37181 (14836-90896) vs 30089 (12587-72286) GE/ml). EcDNA correlated positively with the continuous metabolic syndrome score (r= 0.158 for males and r= 0.134 for females). Stronger correlations were found between ecDNA of mitochondrial origin and AOPP (r= 0.202 and 0.186 for males and females respectively). Multivariate regression analysis revealed associations of nuclear DNA with leukocyte and erythrocyte counts. The results of this study on healthy adolescents show that circulating ecDNA is associated with the risk of metabolic syndrome, not with obesity per se. The association between mitochondrial ecDNA and AOPP requires further attention as it supports a potential role of mitochondria-induced sterile inflammation in the pathogenesis of the metabolic syndrome.

Cytosolic dsDNA of mitochondrial origin induces cytotoxicity and neurodegeneration in cellular and zebrafish models of Parkinson’s disease

Mitochondrial dysfunction and lysosomal dysfunction have been implicated in Parkinson’s disease (PD), but the links between these dysfunctions in PD pathogenesis are still largely unknown. Here we report that cytosolic dsDNA of mitochondrial origin escaping from lysosomal degradation was shown to induce cytotoxicity in cultured cells and PD phenotypes in vivo. The depletion of PINK1, GBA and/or ATP13A2 causes increases in cytosolic dsDNA of mitochondrial origin and induces type I interferon (IFN) responses and cell death in cultured cell lines. These phenotypes are rescued by the overexpression of DNase II, a lysosomal DNase that degrades discarded mitochondrial DNA, or the depletion of IFI16, which acts as a sensor for cytosolic dsDNA of mitochondrial origin. Reducing the abundance of cytosolic dsDNA by overexpressing human DNase II ameliorates movement disorders and dopaminergic cell loss in gba mutant PD model zebrafish. Furthermore, IFI16 and cytosolic dsDNA puncta of mitochondrial origin accumulate in the brain of patients with PD. These results support a common causative role for the cytosolic leakage of mitochondrial DNA in PD pathogenesis.

Deoxyribonuclease activity negative correlates with extracellular DNA in uncomplicated singleton pregnancies in the third trimester

Objectives It is not clear, which factors affect extracellular DNA (ecDNA) concentrations in healthy women with singleton uncomplicated pregnancies, although deoxyribonucleases (DNases) are hypothesized to be responsible for the cleavage of plasma ecDNA. The aim of this study was to analyze potential determinants of total ecDNA including plasma DNase activity. Methods Plasma samples were collected from 48 healthy women with singleton uncomplicated pregnancies in the third trimester (gestation week 37). DNA was isolated and quantified using fluorometry and real time PCR. DNase activity was assessed using the single radial enzyme-diffusion method. Results Neither ecDNA, nor DNase activity were affected by maternal age or BMI. DNase activity negatively correlated with total plasma ecDNA (r=−0.40, p=0.007). Similar associations were found for ecDNA of nuclear and mitochondrial origin, but not with fetal DNA quantified using Y-targeted PCR in male fetus-bearing pregnancies. Conclusions The role of plasma ecDNA of fetal and maternal origin is studied in the pathogenesis of pregnancy-complications. The results indicate that plasma DNase activity could negatively regulate ecDNA concentrations and should, thus, be analyzed in preeclampsia, preterm birth and other ecDNA-related pregnancy complications.

High efficiency and clinical relevance of exome sequencing in the daily practice of neurogenetics

ObjectiveTo assess the efficiency and relevance of clinical exome sequencing (cES) as a first-tier or second-tier test for the diagnosis of progressive neurological disorders in the daily practice of Neurology and Genetic Departments.MethodsSixty-seven probands with various progressive neurological disorders (cerebellar ataxias, neuromuscular disorders, spastic paraplegias, movement disorders and individuals with complex phenotypes labelled ‘other’) were recruited over a 4-year period regardless of their age, gender, familial history and clinical framework. Individuals could have had prior genetic tests as long as it was not cES. cES was performed in a proband-only (60/67) or trio (7/67) strategy depending on available samples and was analysed with an in-house pipeline including software for CNV and mitochondrial-DNA variant detection.ResultsIn 29/67 individuals, cES identified clearly pathogenic variants leading to a 43% positive yield. When performed as a first-tier test, cES identified pathogenic variants for 53% of individuals (10/19). Difficult cases were solved including double diagnoses within a kindred or identification of a neurodegeneration with brain iron accumulation in a patient with encephalopathy of suspected mitochondrial origin.ConclusionThis study shows that cES is a powerful tool for the daily practice of neurogenetics offering an efficient (43%) and appropriate approach for clinically and genetically complex and heterogeneous disorders.

A Method for Authentication of Meat by PCR Amplification of Species-specific Markers of Mitochondrial Origin

Background: Adulteration of meat with their cheaper or inferior counterparts has become a common practice in the meat industry which threatens the feelings as well as the health of the people. Meat adulteration has issues relating to social, religious, economic, and public health. Therefore, it is important to develop simple and reliable techniques for the authentication of species of meat. Mitochondrial markers have been widely used in species identification and authentication as PCR of species-specific markers of mitochondrial origin is relatively rapid, accurate, sensitive and cost-effective as compared to other PCR based assays. The present study was carried out for authentication of raw and cooked meat from different species using PCR amplification of species-specific Cytb and D-loop markers of mitochondrial origin.Methods: In this study, detection of different raw meat viz. beef, carabeef, mutton, chevon, chicken, duck meat and dog meat as well as meat samples subjected to different processing temperatures was done using PCR of species-specific mitochondrial Cytb and D-loop markers. Samples of beef, carabeef, mutton, chevon, chicken, duck meat and dog meat were collected randomly from different locations of the North-Eastern region of India. The meat samples were subjected to heat treatment in hot water (80oC) to have 75oC core temperature. They were also cooked in steam to have the core temperature of 95oC. The samples were also subjected to autoclaving at a temperature of 121oC and 15 lb pressure. Result: The markers used in this study successfully amplified unique fragments for beef, carabeef, mutton, chevon, chicken, duck meat and dog meat. The sizes of the amplified products were 126 bp for beef, 226 bp for carabeef, 254 bp for mutton, 453 bp for chevon, 256 bp for chicken, 292 bp for duck meat and 100 bp for dog meat. The results were consistent in the meat samples which were subjected to different cooking temperatures ranging from 75-121oC. Consequently, these markers were validated for cross-amplification by checking them with other meat samples and no amplifications were observed in non-target species. The results suggested that all the markers were highly specific for the target species. The simplicity, sensitivity and stability of the assay indicated that this method could be very useful for meat authentication and thereby to detect adulteration.

Mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in Down syndrome

Mitochondrial dysfunction is an established hallmark of aging and neurodegenerative disorders such as Down syndrome (DS) and Alzheimer’s disease (AD). Using a high-resolution density gradient separation of extracellular vesicles (EVs) isolated from murine and human DS and diploid control brains, we identify and characterize a previously unknown population of double-membraned EVs containing multiple mitochondrial proteins distinct from previously described EV subtypes, including microvesicles and exosomes. We term these newly identified mitochondria-derived EVs “mitovesicles.” We demonstrate that brain-derived mitovesicles contain a specific subset of mitochondrial constituents and that their levels and cargo are altered during pathophysiological processes where mitochondrial dysfunction occurs, including in DS. The development of a method for the selective isolation of mitovesicles paves the way for the characterization in vivo of biological processes connecting EV biology and mitochondria dynamics and for innovative therapeutic and diagnostic strategies.