Mitochondrial Genetic Research Articles

DdCBE mediates efficient and inheritable modifications in mouse mitochondrial genome

Critical mutations of mitochondrial DNA (mtDNA) generally lead to maternally inheritable diseases that affect multiple organs and systems; however, it was difficult to alter mtDNA in mammalian cells to intervene in or cure mitochondrial disorders. Recently, the discovery of DddA-derived cytosine base editor (DdCBE) enabled the precise manipulation of mtDNA. To test its feasibility for in vivo use, we selected several sites in mouse mtDNA as DdCBE targets to resemble the human pathogenic mtDNA G-to-A mutations. The efficiency of DdCBE-mediated mtDNA editing was first screened in mouse Neuro-2A cells and DdCBE pairs with the best performance were chosen for in vivo targeting. Microinjection of the mRNAs of DdCBE halves in the mouse zygotes or 2-cell embryo successfully generated edited founder mice with a base conversion rate ranging from 2.48% to 28.51%. When backcrossed with wild-type male mice, female founders were able to transmit the mutations to their offspring with different mutation loads. Off-target analyses demonstrated a high fidelity for DdCBE-mediated base editing in mouse mtDNA both in vitro and in vivo. Our study demonstrated that the DdCBE is feasible for generation of mtDNA mutation models to facilitate disease study and for potential treatment of mitochondrial disorders.

Prenatal particulate matter exposure and mitochondrial mutational load at the maternal-fetal interface: Effect modification by genetic ancestry

Prenatal ambient particulate matter (PM2.5) exposure impacts infant development and alters placental mitochondrial DNA abundance. We investigated whether the timing of PM2.5 exposure predicts placental mitochondrial mutational load using NextGen sequencing in 283 multi-ethnic mother-infant dyads. We observed increased PM2.5exposure, particularly during mid- to late-pregnancy and among genes coding for NADH dehydrogenase and subunits of ATP synthase, was associated with a greater amount of nonsynonymous mutations. The strongest associations were observed for participants of African ancestry. Further work is needed to tease out the role of mitochondrial genetics and its impact on offspring development and emerging disease disparities.

MitoScape: A big-data, machine-learning platform for obtaining mitochondrial DNA from next-generation sequencing data.

The growing number of next-generation sequencing (NGS) data presents a unique opportunity to study the combined impact of mitochondrial and nuclear-encoded genetic variation in complex disease. Mitochondrial DNA variants and in particular, heteroplasmic variants, are critical for determining human disease severity. While there are approaches for obtaining mitochondrial DNA variants from NGS data, these software do not account for the unique characteristics of mitochondrial genetics and can be inaccurate even for homoplasmic variants. We introduce MitoScape, a novel, big-data, software for extracting mitochondrial DNA sequences from NGS. MitoScape adopts a novel departure from other algorithms by using machine learning to model the unique characteristics of mitochondrial genetics. We also employ a novel approach of using rho-zero (mitochondrial DNA-depleted) data to model nuclear-encoded mitochondrial sequences. We showed that MitoScape produces accurate heteroplasmy estimates using gold-standard mitochondrial DNA data. We provide a comprehensive comparison of the most common tools for obtaining mtDNA variants from NGS and showed that MitoScape had superior performance to compared tools in every statistically category we compared, including false positives and false negatives. By applying MitoScape to common disease examples, we illustrate how MitoScape facilitates important heteroplasmy-disease association discoveries by expanding upon a reported association between hypertrophic cardiomyopathy and mitochondrial haplogroup T in men (adjusted p-value = 0.003). The improved accuracy of mitochondrial DNA variants produced by MitoScape will be instrumental in diagnosing disease in the context of personalized medicine and clinical diagnostics.

Extensive analysis of mitochondrial DNA quantity and sequence variation in human cumulus cells and assisted reproduction outcomes.

Are relative mitochondrial DNA (mtDNA) content and mitochondrial genome (mtGenome) variants in human cumulus cells (CCs) associated with oocyte reproductive potential and assisted reproductive technology (ART) outcomes? Neither the CC mtDNA quantity nor the presence of specific mtDNA genetic variants was associated with ART outcomes, although associations with patient body mass index (BMI) were detected, and the total number of oocytes retrieved differed between major mitochondrial haplogroups. CCs fulfil a vital role in the support of oocyte developmental competence. As with other cell types, appropriate cellular function is likely to rely upon adequate energy production, which in turn depends on the quantity and genetic competence of the mitochondria. mtDNA mutations can be inherited or they can accumulate in somatic cells over time, potentially contributing to aging. Such mutations may be homoplasmic (affecting all mtDNA in a cell) or they may display varying levels of heteroplasmy (affecting a proportion of the mtDNA). Currently, little is known concerning variation in CC mitochondrial genetics and how this might influence the reproductive potential of the associated oocyte. This was a prospective observational study involving human CCs collected with 541 oocytes from 177 IVF patients. mtDNA quantity was measured in all the samples with a validated quantitative PCR method and the entire mtGenome was sequenced in a subset of 138 samples using a high-depth massively parallel sequencing approach. Associations between relative mtDNA quantity and mtGenome variants in CCs and patient age, BMI (kg/m2), infertility diagnosis and ART outcomes were investigated. Massively parallel sequencing permitted not only the accurate detection of mutations but also the precise quantification of levels of mutations in cases of heteroplasmy. Sequence variants in the mtDNA were evaluated using Mitomaster and HmtVar to predict their potential impact. The relative mtDNA CC content was significantly associated with BMI. No significant associations were observed between CC mtDNA quantity and patient age, female infertility diagnosis or any ART outcome variable. mtGenome sequencing revealed 4181 genetic variants with respect to a reference genome. The COXI locus contained the least number of coding sequence variants, whereas ATPase8 had the most. The number of variants predicted to affect the ATP production differed significantly between mitochondrial macrohaplogroups. The total number of retrieved oocytes was different between the H-V and J-T as well as the U-K and J-T macrohaplogroups. There was a non-significant increase in mtDNA levels in CCs with heteroplasmic mitochondrial mutations. N/A. Although a large number of samples were analysed in this study, it was not possible to analyse all the CCs from every patient. Also, the results obtained with respect to specific clinical outcomes and macrohaplogroups should be interpreted with caution due to the smaller sample sizes when subdividing the dataset. These findings suggest that the analysis of mtDNA in CCs is unlikely to provide an advantage in terms of improved embryo selection during assisted reproduction cycles. Nonetheless, our data raise interesting biological questions, particularly regarding the interplay of metabolism and BMI and the association of mtDNA haplogroup with oocyte yield in ovarian stimulation cycles. This study was funded by National Institutes of Health grant 5R01HD092550-02. D.J.N. and C.R. co-hold patent US20150346100A1 and D.J.N. holds US20170039415A1, both for metabolic imaging methods. D.W. receives support from the NIHR Oxford Biomedical Research Centre. The remaining authors have no conflicts of interest to declare.

The FASTK family proteins fine-tune mitochondrial RNA processing.

Transcription of the human mitochondrial genome and correct processing of the two long polycistronic transcripts are crucial for oxidative phosphorylation. According to the tRNA punctuation model, nucleolytic processing of these large precursor transcripts occurs mainly through the excision of the tRNAs that flank most rRNAs and mRNAs. However, some mRNAs are not punctuated by tRNAs, and it remains largely unknown how these non-canonical junctions are resolved. The FASTK family proteins are emerging as key players in non-canonical RNA processing. Here, we have generated human cell lines carrying single or combined knockouts of several FASTK family members to investigate their roles in non-canonical RNA processing. The most striking phenotypes were obtained with loss of FASTKD4 and FASTKD5 and with their combined double knockout. Comprehensive mitochondrial transcriptome analyses of these cell lines revealed a defect in processing at several canonical and non-canonical RNA junctions, accompanied by an increase in specific antisense transcripts. Loss of FASTKD5 led to the most severe phenotype with marked defects in mitochondrial translation of key components of the electron transport chain complexes and in oxidative phosphorylation. We reveal that the FASTK protein family members are crucial regulators of non-canonical junction and non-coding mitochondrial RNA processing.

Multiple Myeloma Derived Mitochondrial Damps Induce Inflammation in the Bone Marrow Adipose Tissue Which Promotes Tumour Development

Multiple myeloma (MM) is a malignancy of the antibody secreting plasma cells, characterized by the localisation and accumulation of tumour cells in the bone marrow (BM) microenvironment. The mitochondrial DNA (mtDNA) belongs to a group of mitochondrial damage associated molecular patterns (mtDAMPs) and contains islands of unmethylated CpG nucleotide motifs, which have been shown to activate and promote memory B cell antibody secretion and proliferation. Studies have indicated that mitochondrial DNA (mtDNA) is elevated in the circulation of trauma and cancer patients, highlighting a need to explore the functional purpose of mtDNA in the BM microenvironment. Moreover, myeloma relies on the BM microenvironment for survival, proliferation and drug resistance. BM adipocytes are highly secretory cells that support myeloma cells through the release of various factors. It has been shown that myeloma cells induce a shift from osteoblast progenitors towards adipogenesis. Here, we hypothesise that MM cells release mtDAMPs that leads to the promotion of a state of chronic inflammation that supports multiple myeloma disease progression and expansion by inducing adipocyte inflammation.First, we engrafted immunocompromised NSG mice with human myeloma cell line MM1S, blood serum samples were taken and analysed by RT-qPCR to detect the presence of human mtDNA. We found elevated levels of human mtDNA in the serum of engrafted mice correlating to disease progression. Next, we showed that mtDNA was higher in serum patients with MM compared to healthy controls, moreover, we discovered that BM serum from MM patients had significantly higher levels of mtDNA than blood serum. Furthermore, this observation was recapitulated in myeloma engrafted NSG mice. Next, we examined the effect of mtDNA on various cell populations of the BM. We found that in vitro both BM derived macrophages and BM derived adipocytes had increased inflammatory signatures, including upregulation of IL-6, when treated with mtDNA from myeloma cells. MtDNA induced IL-6 expression was attenuated upon blocking of the TLR9 receptor with antagonist ODN 2088. To understand the role of BM adipocyte induced inflammation in MM, we treated adipocytes with mtDNA before culturing them with MM. We found that MM cells had enhanced proliferation on mtDNA activated adipocytes. We further show that MM cells cultured with mtDNA activated adipocytes have increased uptake of free fatty acids and seahorse analysis confirmed an increase in b-oxidation derived metabolism.Here we establish that MM releases mtDNA into the microenvironment promoting the disease progression via a pro-inflammatory BM. We also show that both BM macrophages and BM adipocytes play a supportive role in multiple myeloma expansion and contribute to the pro-inflammatory bone marrow microenvironment. DisclosuresNo relevant conflicts of interest to declare.

TFB2M and POLRMT are essential for mammalian mitochondrial DNA replication

Two classes of replication intermediates have been observed from mitochondrial DNA (mtDNA) in many mammalian tissue and cells with two-dimensional agarose gel electrophoresis. One is assigned to leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long noncoding RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Unexpectedly, removal of either protein resulted in complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for the maintenance of human mtDNA. Moreover, a lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of mtDNA replication initiation.

Gaining Insight into Mitochondrial Genetic Variation and Downstream Pathophysiology: What Can i(PSCs) Do?

Mitochondria are specialized organelles involved in energy production that have retained their own genome throughout evolutionary history. The mitochondrial genome (mtDNA) is maternally inherited and requires coordinated regulation with nuclear genes to produce functional enzyme complexes that drive energy production. Each mitochondrion contains 5–10 copies of mtDNA and consequently, each cell has several hundreds to thousands of mtDNAs. Due to the presence of multiple copies of mtDNA in a mitochondrion, mtDNAs with different variants may co-exist, a condition called heteroplasmy. Heteroplasmic variants can be clonally expanded, even in post-mitotic cells, as replication of mtDNA is not tied to the cell-division cycle. Heteroplasmic variants can also segregate during germ cell formation, underlying the inheritance of some mitochondrial mutations. Moreover, the uneven segregation of heteroplasmic variants is thought to underlie the heterogeneity of mitochondrial variation across adult tissues and resultant differences in the clinical presentation of mitochondrial disease. Until recently, however, the mechanisms mediating the relation between mitochondrial genetic variation and disease remained a mystery, largely due to difficulties in modeling human mitochondrial genetic variation and diseases. The advent of induced pluripotent stem cells (iPSCs) and targeted gene editing of the nuclear, and more recently mitochondrial, genomes now provides the ability to dissect how genetic variation in mitochondrial genes alter cellular function across a variety of human tissue types. This review will examine the origins of mitochondrial heteroplasmic variation and propagation, and the tools used to model mitochondrial genetic diseases. Additionally, we discuss how iPSC technologies represent an opportunity to advance our understanding of human mitochondrial genetics in disease.

An in vitro system to silence mitochondrial gene expression

The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an invitro system to silence translation in purified mitochondria. Invitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.

The population frequency of human mitochondrial DNA variants is highly dependent upon mutational bias.

ABSTRACTNext-generation sequencing can quickly reveal genetic variation potentially linked to heritable disease. As databases encompassing human variation continue to expand, rare variants have been of high interest, since the frequency of a variant is expected to be low if the genetic change leads to a loss of fitness or fecundity. However, the use of variant frequency when seeking genomic changes linked to disease remains very challenging. Here, I explored the role of selection in controlling human variant frequency using the HelixMT database, which encompasses hundreds of thousands of mitochondrial DNA (mtDNA) samples. I found that a substantial number of synonymous substitutions, which have no effect on protein sequence, were never encountered in this large study, while many other synonymous changes are found at very low frequencies. Further analyses of human and mammalian mtDNA datasets indicate that the population frequency of synonymous variants is predominantly determined by mutational biases rather than by strong selection acting upon nucleotide choice. My work has important implications that extend to the interpretation of variant frequency for non-synonymous substitutions.

A benchmarking of human mitochondrial DNA haplogroup classifiers from whole-genome and whole-exome sequence data

The mitochondrial genome (mtDNA) is of interest for a range of fields including evolutionary, forensic, and medical genetics. Human mitogenomes can be classified into evolutionary related haplogroups that provide ancestral information and pedigree relationships. Because of this and the advent of high-throughput sequencing (HTS) technology, there is a diversity of bioinformatic tools for haplogroup classification. We present a benchmarking of the 11 most salient tools for human mtDNA classification using empirical whole-genome (WGS) and whole-exome (WES) short-read sequencing data from 36 unrelated donors. We also assessed the best performing tool in third-generation long noisy read WGS data obtained with nanopore technology for a subset of the donors. We found that, for short-read WGS, most of the tools exhibit high accuracy for haplogroup classification irrespective of the input file used for the analysis. However, for short-read WES, Haplocheck and MixEmt were the most accurate tools. Based on the performance shown for WGS and WES, and the accompanying qualitative assessment, Haplocheck stands out as the most complete tool. For third-generation HTS data, we also showed that Haplocheck was able to accurately retrieve mtDNA haplogroups for all samples assessed, although only after following assembly-based approaches (either based on a referenced-based assembly or a hybrid de novo assembly). Taken together, our results provide guidance for researchers to select the most suitable tool to conduct the mtDNA analyses from HTS data.

Identification of a Nuclear Mitochondrial-Related Multi-Genes Signature to Predict the Prognosis of Bladder Cancer.

IntroductionBladder cancer (BC) is one of the most prevalent urinary cancers, and its management is still a problem causing recurrence and progression, elevating mortality.Materials and MethodsWe aimed at the nuclear mitochondria-related genes (MTRGs), collected from the MITOMAP: A Human Mitochondrial Genome Database. Meanwhile, the expression profiles and clinical information of BC were downloaded from the Cancer Genome Atlas (TCGA) as a training group. The univariate, multivariate, and the least absolute shrinkage and selection operator (LASSO) Cox regression analyses were used to construct a nuclear mitochondrial-related multi-genes signature and the prognostic nomogram.ResultsA total of 17 nuclear MTRGs were identified to be correlated with the overall survival (OS) of BC patients, and a nuclear MTRGs signature based on 16 genes expression was further determined by the LASSO Cox regression analysis. Based on a nuclear MTRGs scoring system, BC patients from the TCGA cohort were divided into high- and low- nuclear MTRGs score groups. Patients with a high nuclear MTRGs score exhibited a significantly poorer outcome (median OS: 92.90 vs 20.20 months, p<0.0001). The nuclear MTRGs signature was further verified in three independent datasets, namely, GSE13507, GSE31684, and GSE32548, from the Gene Expression Omnibus (GEO). The BC patients with a high nuclear MTRGs score had significantly worse survival (median OS in GSE13507: 31.52 vs 98.00 months, p<0.05; GSE31684: 32.85 months vs unreached, p<0.05; GSE32548: unreached vs unreached, p<0.05). Furthermore, muscle-invasive bladder cancer (MIBC) patients had a significantly higher nuclear MTRGs score (p<0.05) than non-muscle-invasive bladder cancer (NMIBC) patients. The integrated signature outperformed each involved MTRG. In addition, a nuclear MTRGs-based nomogram was constructed as a novel prediction prognosis model, whose AUC values for OS at 1, 3, 5 years were 0.76, 0.75, and 0.75, respectively, showing the prognostic nomogram had good and stable predicting ability. Enrichment analyses of the hallmark gene set and KEGG pathway revealed that the E2F targets, G2M checkpoint pathways, and cell cycle had influences on the survival of BC patients. Furthermore, the analysis of tumor microenvironment indicated more CD8+ T cells and higher immune score in patients with high nuclear MTRGs score, which might confer sensitivity to immune checkpoint inhibitors.ConclusionsNot only could the signature and prognostic nomogram predict the prognosis of BC, but it also had potential therapeutic guidance.

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational ‘hot-spots’ or ‘cold-spots’. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.

Nuclear and Mitochondrial Genome, Epigenome and Gut Microbiome: Emerging Molecular Biomarkers for Parkinson's Disease.

Background: Parkinson’s disease (PD) is currently the second most common neurodegenerative disorder, burdening about 10 million elderly individuals worldwide. The multifactorial nature of PD poses a difficult obstacle for understanding the mechanisms involved in its onset and progression. Currently, diagnosis depends on the appearance of clinical signs, some of which are shared among various neurologic disorders, hindering early diagnosis. There are no effective tools to prevent PD onset, detect the disease in early stages or accurately report the risk of disease progression. Hence, there is an increasing demand for biomarkers that may identify disease onset and progression, as treatment-based medicine may not be the best approach for PD. Over the last few decades, the search for molecular markers to predict susceptibility, aid in accurate diagnosis and evaluate the progress of PD have intensified, but strategies aimed to improve individualized patient care have not yet been established. Conclusions: Genomic variation, regulation by epigenomic mechanisms, as well as the influence of the host gut microbiome seem to have a crucial role in the onset and progress of PD, thus are considered potential biomarkers. As such, the human nuclear and mitochondrial genome, epigenome, and the host gut microbiome might be the key elements to the rise of personalized medicine for PD patients.

A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy.

We report a rapid experimental procedure based on high-density in vivo psoralen inter-strand DNA cross-linking coupled to spreading of naked purified DNA, positive staining, low-angle rotary shadowing, and transmission electron microscopy (TEM) that allows quick visualization of the dynamic of heavy strand (HS) and light strand (LS) human mitochondrial DNA replication. Replication maps built on linearized mitochondrial genomes and optimized rotary shadowing conditions enable clear visualization of the progression of the mitochondrial DNA synthesis and visualization of replication intermediates carrying long single-strand DNA stretches. One variant of this technique, called denaturing spreading, allowed the inspection of the fine chromatin structure of the mitochondrial genome and was applied to visualize the in vivo three-strand DNA structure of the human mitochondrial D-loop intermediate with unprecedented clarity.

Library-independent source tracking of fecal contamination in selected stations and tributaries of Laguna Lake, Philippines.

Laguna Lake is the largest inland freshwater body in the Philippines. Although it is classified to be usable for agricultural and recreational purposes by the country's Department of Environment and Natural Resources (DENR), studies looking at lake ecology revealed severe fecal contamination which contributes to the deterioration of water quality. Determining the sources of fecal contamination is necessary for lake protection and management. This study utilized a library-independent method of microbial source tracking (LIM-MST) to identify sources of fecal contamination in selected Laguna Lake stations and tributaries. Genetic markers of the host-associated Escherichia coli, heat-labile toxin (LTIIA) and heat-stable II (STII), were used to identify cattle and swine fecal contaminations, respectively. Meanwhile, human mitochondrial DNA (mtDNA) was used to identify human fecal contamination. Results identified the presence of agricultural and human fecal contamination in Laguna Lake Stations 1 and 5, Mangangate River, and Alabang River. The selected sites are known to be surrounded by residential and industrial complexes, and most of their discharges find their way into the lake. The identification of the specific sources of fecal contamination will guide management practices that aim to regulate the discharges in order to improve the water quality of Laguna Lake.

RNA Granules in the Mitochondria and Their Organization under Mitochondrial Stresses.

The human mitochondrial genome (mtDNA) regulates its transcription products in specialised and distinct ways as compared to nuclear transcription. Thanks to its mtDNA mitochondria possess their own set of tRNAs, rRNAs and mRNAs that encode a subset of the protein subunits of the electron transport chain complexes. The RNA regulation within mitochondria is organised within specialised, membraneless, compartments of RNA-protein complexes, called the Mitochondrial RNA Granules (MRGs). MRGs were first identified to contain nascent mRNA, complexed with many proteins involved in RNA processing and maturation and ribosome assembly. Most recently, double-stranded RNA (dsRNA) species, a hybrid of the two complementary mRNA strands, were found to form granules in the matrix of mitochondria. These RNA granules are therefore components of the mitochondrial post-transcriptional pathway and as such play an essential role in mitochondrial gene expression. Mitochondrial dysfunctions in the form of, for example, RNA processing or RNA quality control defects, or inhibition of mitochondrial fission, can cause the loss or the aberrant accumulation of these RNA granules. These findings underline the important link between mitochondrial maintenance and the efficient expression of its genome.

A globally diverse reference alignment and panel for imputation of mitochondrial DNA variants

BackgroundVariation in mitochondrial DNA (mtDNA) identified by genotyping microarrays or by sequencing only the hypervariable regions of the genome may be insufficient to reliably assign mitochondrial genomes to phylogenetic lineages or haplogroups. This lack of resolution can limit functional and clinical interpretation of a substantial body of existing mtDNA data. To address this limitation, we developed and evaluated a large, curated reference alignment of complete mtDNA sequences as part of a pipeline for imputing missing mtDNA single nucleotide variants (mtSNVs). We call our reference alignment and pipeline MitoImpute.ResultsWe aligned the sequences of 36,960 complete human mitochondrial genomes downloaded from GenBank, filtered and controlled for quality. These sequences were reformatted for use in imputation software, IMPUTE2. We assessed the imputation accuracy of MitoImpute by measuring haplogroup and genotype concordance in data from the 1000 Genomes Project and the Alzheimer’s Disease Neuroimaging Initiative (ADNI). The mean improvement of haplogroup assignment in the 1000 Genomes samples was 42.7% (Matthew’s correlation coefficient = 0.64). In the ADNI cohort, we imputed missing single nucleotide variants.ConclusionThese results show that our reference alignment and panel can be used to impute missing mtSNVs in existing data obtained from using microarrays, thereby broadening the scope of functional and clinical investigation of mtDNA. This improvement may be particularly useful in studies where participants have been recruited over time and mtDNA data obtained using different methods, enabling better integration of early data collected using less accurate methods with more recent sequence data.

Mechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL

The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ∼25nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre-binds to OriL, downstream of the replication origin thus allowing for the primer synthesis. Our data suggest the primosome assembly at OriL-a complex of mtRNAP and PolG-can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases.

Leigh Syndrome: A Tale of Two Genomes.

Leigh syndrome is a rare, complex, and incurable early onset (typically infant or early childhood) mitochondrial disorder with both phenotypic and genetic heterogeneity. The heterogeneous nature of this disorder, based in part on the complexity of mitochondrial genetics, and the significant interactions between the nuclear and mitochondrial genomes has made it particularly challenging to research and develop therapies. This review article discusses some of the advances that have been made in the field to date. While the prognosis is poor with no current substantial treatment options, multiple studies are underway to understand the etiology, pathogenesis, and pathophysiology of Leigh syndrome. With advances in available research tools leading to a better understanding of the mitochondria in health and disease, there is hope for novel treatment options in the future.