Multiple Mitochondrial DNA Research Articles

Multiple Maternal Lineages of <i>Strauchbufo raddei</i> (Strauch, 1876) in Mongolia Revealed by Mitochondrial DNA

The Mongolian toad Strauchbufo raddei (Strauch, 1876) is one of widely distributed and dominated amphibian species in eastern Asian, including Mongolia. In this study, we amplified and sequenced a total of 50 samples of S. raddei, from three main regions in its Mongolian distribution range namely, Northern, Eastern, and Gobi regions. Our phylogenetic trees (Bayesian inference; BI and time-calibrated) and network analysis clearly support presence of multiple maternal mitochondrial DNA lineages for S. raddei in Mongolia. As expected, samples from these three regions were clustered separately in BI tree (Bayesian posterior probability (BPP 0.52). In addition, the population specific haplotypes, estimated genetic distances (e.g. uncorrelated p distance; ranged from 1% to 25%) and hierarchical AMOVA suggested relatively strong population genetic structure of S. raddei across Mongolia. Time-calibrated tree and ancestral range analysis showed that the first colonization of S. raddei may have occurred in Northern part of Mongolia approximately 9.63 mya, then dispersed over other parts of the country. Estimated approximate time for crossing event of S. raddei over dry steppe and semi-desert regions in Mongolia was 5.02 – 2.50 mya. Finally, high level of genetic diversity (overall; haplotype diversity, Hd = 0.93; nucleotide diversity, π = 0.011) of S. raddei were observed in Mongolia. Importantly, through this study, we contributed to closing sampling gaps in distribution range of S. raddei in Eastern Asia, which is key to better understanding species history, as well as describing cryptic lineages.

Multiple distinct evolutionary mechanisms govern the dynamics of selfish mitochondrial genomes in Caenorhabditis elegans

Cells possess multiple mitochondrial DNA (mtDNA) copies, which undergo semi-autonomous replication and stochastic inheritance. This enables mutant mtDNA variants to arise and selfishly compete with cooperative (wildtype) mtDNA. Selfish mitochondrial genomes are subject to selection at different levels: they compete against wildtype mtDNA directly within hosts and indirectly through organism-level selection. However, determining the relative contributions of selection at different levels has proven challenging. We overcome this challenge by combining mathematical modeling with experiments designed to isolate the levels of selection. Applying this approach to many selfish mitochondrial genotypes in Caenorhabditis elegans reveals an unexpected diversity of evolutionary mechanisms. Some mutant genomes persist at high frequency for many generations, despite a host fitness cost, by aggressively outcompeting cooperative genomes within hosts. Conversely, some mutant genomes persist by evading inter-organismal selection. Strikingly, the mutant genomes vary dramatically in their susceptibility to genetic drift. Although different mechanisms can cause high frequency of selfish mtDNA, we show how they give rise to characteristically different distributions of mutant frequency among individuals. Given that heteroplasmic frequency represents a key determinant of phenotypic severity, this work outlines an evolutionary theoretic framework for predicting the distribution of phenotypic consequences among individuals carrying a selfish mitochondrial genome.

Lysosomal dysfunction and overload of nucleosides in thymidine phosphorylase deficiency of MNGIE

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.Graphical

Evidence for hybridization-driven heteroplasmy maintained across generations in a ricefish endemic to a Wallacean ancient lake.

Heteroplasmy, the presence of multiple mitochondrial DNA (mtDNA) haplotypes within cells of an individual, is caused by mutation or paternal leakage. However, heteroplasmy is usually resolved to homoplasmy within a few generations because of germ-line bottlenecks; therefore, instances of heteroplasmy are limited in nature. Here, we report heteroplasmy in the ricefish species Oryzias matanensis, endemic to Lake Matano, an ancient lake in Sulawesi Island, in which one individual was known to have many heterozygous sites in the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene. In this study, we cloned the ND2 gene for some additional individuals with heterozygous sites and demonstrated that they are truly heteroplasmic. Phylogenetic analysis revealed that the extra haplotype within the heteroplasmic O. matanensis individuals clustered with haplotypes of O. marmoratus, a congeneric species inhabiting adjacent lakes. This indicated that the heteroplasmy originated from paternal leakage due to interspecific hybridization. The extra haplotype was unique and contained two non-synonymous substitutions. These findings demonstrate that this hybridization-driven heteroplasmy was maintained across generations for a long time to the extent that the extra mitochondria evolved within the new host.

Mitochondrial myopathies diagnosed in adulthood: clinico-genetic spectrum and long-term outcomes.

Mitochondrial myopathies are frequently recognized in childhood as part of a broader multisystem disorder and often overlooked in adulthood. Herein, we describe the phenotypic and genotypic spectrum and long-term outcomes of mitochondrial myopathies diagnosed in adulthood, focusing on neuromuscular features, electrodiagnostic and myopathological findings and survival. We performed a retrospective chart review of adult patients diagnosed with mitochondrial myopathy at Mayo Clinic (2005-21). We identified 94 patients. Median time from symptom onset to diagnosis was 11 years (interquartile range 4-21 years). Median age at diagnosis was 48 years (32-63 years). Primary genetic defects were identified in mitochondrial DNA in 48 patients (10 with single large deletion, 38 with point mutations) and nuclear DNA in 29. Five patients had multiple mitochondrial DNA deletions or depletion without nuclear DNA variants. Twelve patients had histopathological features of mitochondrial myopathy without molecular diagnosis. The most common phenotypes included multisystem disorder (n = 30); mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (14); limb myopathy (13); chronic progressive external ophthalmoplegia (12); and chronic progressive external ophthalmoplegia-plus (12). Isolated skeletal muscle manifestations occurred in 27%. Sixty-nine per cent had CNS and 21% had cardiac involvement. Mutations most frequently involved MT-TL1 (27) and POLG (17); however, a wide spectrum of established and novel molecular defects, with overlapping phenotypes, was identified. Electrodiagnostic studies identified myopathy (77%), fibrillation potentials (27%) and axonal peripheral neuropathy (42%, most common with nuclear DNA variants). Among 42 muscle biopsies available, median percentage counts were highest for cytochrome C oxidase negative fibres (5.1%) then ragged blue (1.4%) and ragged red fibres (0.5%). Skeletal muscle weakness was mild and slowly progressive (decline in strength summated score of 0.01/year). Median time to gait assistance was 5.5 years from diagnosis and 17 years from symptom onset. Thirty patients died, with median survival of 33.4 years from symptom onset and 10.9 years from diagnosis. Median age at death was 55 years. Cardiac involvement was associated with increased mortality [hazard ratio 2.36 (1.05, 5.29)]. There was no difference in survival based on genotype or phenotype. Despite the wide phenotypic and genotypic spectrum, mitochondrial myopathies in adults share similar features with slowly progressive limb weakness, contrasting with common multiorgan involvement and high mortality.

Myopathy and Ophthalmologic Abnormalities in Association With Multiple Skeletal Muscle Mitochondrial DNA Deletions.

Establishing a molecular diagnosis of mitochondrial diseases due to pathogenic mitochondrial DNA (mtDNA) variants can be difficult because of varying levels of tissue heteroplasmy, and identifying these variants is important for clinical management. Here, we present clinical and molecular findings in 8 adult patients with classical features of mitochondrial ophthalmologic and/or muscle disease and multiple mtDNA deletions isolated to muscle. The patients were identified via a retrospective review of patients seen in both a tertiary ophthalmology center and a genetics clinic with a clinical diagnosis of chronic progressive external ophthalmoplegia, optic nerve abnormalities, and/or mitochondrial myopathy. Age at onset of symptoms ranged from 18 to 61 years. Ocular manifestations included bilateral optic neuropathy in one patient, bilateral optic disc cupping without optic neuropathy in 2 patients, ptosis in 4 patients, and ocular motility deficits in 2 patients. Five patients had generalized weakness. Pathogenic variants in mtDNA were not found in the blood or buccal sample from any patient, but 7 of 8 patients had multiple mtDNA deletions identified in muscle tissue. One patient had a single mtDNA deletion identified in the muscle. Heteroplasmy was less than 15% for all of the identified deletions, with the exception of one deletion that had a heteroplasmy of 50%-60%. None of the patients were found to have a nuclear gene variant known to be associated with mitochondrial DNA maintenance. mtDNA deletions were identified in adult patients with ophthalmologic and/or musle abnormalities and may underlie their clinical presentations.

Data-driven subgroups of newly diagnosed type 2 diabetes and the relationship with cardiovascular diseases at genetic and clinical levels in Chinese adults

BackgroundTo subgroup Chinese patients with newly diagnosed type 2 diabetes (T2D) by K-means cluster analysis on clinical indicators, and to explore whether these subgroups represent different genetic features and calculated cardiovascular risks. MethodsThe K-means clustering analysis was performed on two cohorts (n = 590 and 392), both consisting of Chinese participants with newly diagnosed T2D. To assess genetic risks, multiple polygenic risk scores (PRSs) and mitochondrial DNA copy numbers (mtDNA-CN) were calculated for all participants. Furthermore, Framingham risk scores (FRS) of cardiovascular diseases in two cohorts were also calculated to verify the genetic risks. ResultsFour clusters were identified including the mild age-related diabetes (MARD)(35.08%), mild obesity-related diabetes (MOD) (34.41%), severe autoimmune diabetes (SAID) 19.15%, and severe insulin-resistant diabetes (SIRD) 11.36% subgroups in the MARCH (metformin, and acarbose in Chinese patients as the initial hypoglycemic treatment) cohort. There was a significant difference in PRS for cardiovascular diseases (CVD) across four subgroups in the MARCH cohort (p < 0.05). Compared with the SIDD and SIRD subgroups, patients in the MOD subgroup had a relatively lower PRS for CVD (p < 0.05) in the MARCH cohort. Females had a higher PRS compared to males, with no significant difference in FRS across the four clusters. The MOD subgroup had a significantly lower FRS which was consistent with the results of PRS. Similar results of PRS and FRS were also replicated in the CONFIDENCE (comparison of glycemic control and b-cell function among newly diagnosed patients with type 2 diabetes treated with exenatide, insulin or pioglitazone) cohort. ConclusionThere are different CVD risks in diabetic subgroups based on clinical and genetic evidence which may promote precision medicine.

Nuclear DNA segments homologous to mitochondrial DNA are obstacles for detecting heteroplasmy in sugar beet (Beta vulgaris L.).

Heteroplasmy, the coexistence of multiple mitochondrial DNA (mtDNA) sequences in a cell, is well documented in plants. Next-generation sequencing technology (NGS) has made it feasible to sequence entire genomes. Thus, NGS has the potential to detect heteroplasmy; however, the methods and pitfalls in heteroplasmy detection have not been fully investigated and identified. One obstacle for heteroplasmy detection is the sequence homology between mitochondrial-, plastid-, and nuclear DNA, of which the influence of nuclear DNA segments homologous to mtDNA (numt) need to be minimized. To detect heteroplasmy, we first excluded nuclear DNA sequences of sugar beet (Beta vulgaris) line EL10 from the sugar beet mtDNA sequence. NGS reads were obtained from single plants of sugar beet lines NK-195BRmm-O and NK-291BRmm-O and mapped to the unexcluded mtDNA regions. More than 1000 sites exhibited intra-individual polymorphism as detected by genome browsing analysis. We focused on a 309-bp region where 12 intra-individual polymorphic sites were closely linked to each other. Although the existence of DNA molecules having variant alleles at the 12 sites was confirmed by PCR amplification from NK-195BRmm-O and NK-291BRmm-O, these variants were not always called by six variant-calling programs, suggesting that these programs are inappropriate for intra-individual polymorphism detection. When we changed the nuclear DNA reference, a numt absent from EL10 was found to include the 309-bp region. Genetic segregation of an F2 population from NK-195BRmm-O x NK-291BRmm-O supported the numt origin of the variant alleles. Using four references, we found that numt detection exhibited reference dependency, and extreme polymorphism of numts exists among sugar beet lines. One of the identified numts absent from EL10 is also associated with another intra-individual polymorphic site in NK-195mm-O. Our data suggest that polymorphism among numts is unexpectedly high within sugar beets, leading to confusion about the true degree of heteroplasmy.

Short-term exposure to multiple metals mixture and mitochondrial DNA copy number among children: A panel study

BackgroundLittle is known regarding the individual and overall associations of short-term co-exposure to metals mixture with mitochondrial DNA copy number (mtDNAcn) among healthy children. MethodsWe conducted a panel study across three seasons among 144 children aged 4 to 12 years in Guangzhou. For each season, we collected the first-morning urine for four consecutive days and fasting blood on the 4th day to detect 23 urinary metals and blood leukocyte mtDNAcn, respectively. Linear mixed-effect (LME) models and multiple informant models were used to examine the relations of individual metals with mtDNAcn over different lag days, and the least absolute shrinkage and selection operator (LASSO) regression was applied to determine the most important metal. We further employed weighted quantile sum (WQS) regression to investigate the overall association of metals mixture with mtDNAcn. ResultsNickel (Ni), manganese (Mn) and antimony (Sb) were independently associated with mtDNAcn in a linear dose-response manner. Each 1-fold increase in Ni at lag 0 day, Mn and Sb at lag 2 day was associated with respective decrements of 8.74 %, 6.93 % and 3.98 % in mtDNAcn in multi-metal LME models. LASSO regression also selected Ni, Mn and Sb as the most significant metals at the corresponding lag day. WQS regression showed overall inverse associations between metals mixture and mtDNAcn both at lag 0 and lag 2 day, with mtDNAcn decreased by 2.75 % and 3.14 % in response to a quartile increase in the WQS index. Additionally, the associations of Ni and Mn with decreased mtDNAcn were stronger among children younger than 7 years, girls and those having less vegetables and fruit intake. ConclusionWe found an overall association between metals mixture and decreased mtDNAcn among healthy children, in which Ni, Mn and Sb were the major contributors. Younger children, girls and those with less vegetables and fruit intake were more susceptible.

Possible frequent multiple mitochondrial DNA copies in a single nucleoid in HeLa cells

Previously, a number of ~ 1.4 of mitochondrial DNA (mtDNA) molecules in a single nucleoid was reported, which would reflect a minimum nucleoid division. We applied 3D-double-color direct stochastic optical reconstruction microscopy (dSTORM), i.e. nanoscopy with ~ 25–40 nm x,y-resolution, together with our novel method of Delaunay segmentation of 3D data to identify unbiased 3D-overlaps. Noncoding D-loops were recognized in HeLa cells by mtDNA fluorescence in situ hybridization (mtFISH) 7S-DNA 250-bp probe, containing biotin, visualized by anti-biotin/Cy3B-conjugated antibodies. Other mtFISH probes with biotin or Alexa Fluor 647 (A647) against ATP6-COX3 gene overlaps (1,100 bp) were also used. Nucleoids were imaged by anti-DNA/(A647-)-Cy3B-conjugated antibodies. Resulting histograms counting mtFISH-loci/nucleoid overlaps demonstrated that 45% to 70% of visualized nucleoids contained two or more D-loops or ATP6-COX3-loci, indicating two or more mtDNA molecules per nucleoid. With increasing number of mtDNA per nucleoid, diameters were larger and their distribution histograms peaked at ~ 300 nm. A wide nucleoid diameter distribution was obtained also using 2D-STED for their imaging by anti-DNA/A647. At unchanged mtDNA copy number in osteosarcoma 143B cells, TFAM expression increased nucleoid spatial density 1.67-fold, indicating expansion of existing mtDNA and its redistribution into more nucleoids upon the higher TFAM/mtDNA stoichiometry. Validation of nucleoid imaging was also done with two TFAM mutants unable to bend or dimerize, respectively, which reduced both copy number and nucleoid spatial density by 80%. We conclude that frequently more than one mtDNA molecule exists within a single nucleoid in HeLa cells and that mitochondrial nucleoids do exist in a non-uniform size range.

DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing

Expanding mitochondrial base editing tools with broad sequence compatibility is of high need for both research and therapeutic purposes. In this study, we identify a DddA homolog from Simiaoa sunii (Ddd_Ss) which can efficiently deaminate cytosine in DC context in double-stranded DNA (dsDNA). We successfully develop Ddd_Ss-derived cytosine base editors (DdCBE_Ss) and introduce mutations at multiple mitochondrial DNA (mtDNA) loci including disease-associated mtDNA mutations in previously inaccessible GC context. Finally, by introducing a single amino acid substitution from Ddd_Ss, we successfully improve the activity and sequence compatibility of DdCBE derived from DddA of Burkholderia cenocepacia (DdCBE_Bc). Our study expands mtDNA editing tool boxes and provides resources for further screening and engineering dsDNA base editors for biological and therapeutic applications.

P581: Multiple mitochondrial DNA deletions in patients with myopathy

P581: Multiple mitochondrial DNA deletions in patients with myopathy

The “southeastern Europe” lineage of the brown dog tick Rhipicephalus sanguineus (sensu lato) identified as Rhipicephalus rutilus Koch, 1844: Comparison with holotype and generation of mitogenome reference from Israel

The brown dog tick Rhipicephalus sanguineus (sensu lato) in the southeastern Mediterranean region and the Middle East is difficult to identify due to the presence of multiple mitochondrial DNA haplogroup lineages. The purpose of this study was to clarify the identity of the “southeastern Europe” lineage of this tick species complex. Our research shows that female ticks of the “southeastern Europe” lineage correspond to the morphology of R. rutilus Koch, 1844 as found in type-material at the Museum für Naturkunde Berlin in Germany. We characterised the complete mitogenomes of R. rutilus, R. turanicus Pomerantsev, 1940 and Rhipicephalus sanguineus (Latreille, 1806) in order to improve our understanding of the phylogenetic relationships among species within the R. sanguineus (sensu lato) complex. The material associated with the morphology of R. rutilus was previously labelled as the “southeastern Europe” lineage and found in Israel and Egypt, including Lower Egypt and the Nile Delta, where the original type-material was collected. Based on the morphology, genetic identity, and geographical distribution of the species, we conclude that the name R. rutilus is correctly linked to the “southeastern Europe” lineage of R. sanguineus (sensu lato).

Multiple metals exposure and blood mitochondrial DNA copy number: A cross-sectional study from the Dongfeng-Tongji cohort

ObjectiveMitochondria are essential organelles that execute fundamental biological processes, while mitochondrial DNA is vulnerable to environmental insults. The aim of this study was to investigate the individual and mixture effect of plasma metals on blood mitochondria DNA copy number (mtDNAcn). MethodsThis study involved 1399 randomly selected subcohort participants from the Dongfeng-Tongji cohort. The blood mtDNAcn and plasma levels of 23 metals were determined by using quantitative real-time polymerase chain reaction (qPCR) and inductively coupled plasma mass spectrometer (ICP-MS), respectively. The multiple linear regression was used to explore the association between each metal and mtDNAcn, and the LASSO penalized regression was performed to select the most significant metals. We also used the quantile g-computation analysis to assess the mixture effect of multiple metals. ResultsBased on multiple linear regression models, each 1% increase in plasma concentration of copper (Cu), rubidium (Rb), and titanium (Ti) was associated with a separate 0.16% [β(95% CI) = 0.158 (0.066, 0.249), P = 0.001], 0.20% [β(95% CI) = 0.196 (0.073, 0.318), P = 0.002], and 0.25% [β(95% CI) = 0.245 (0.081, 0.409), P = 0.003] increase in blood mtDNAcn. The LASSO regression also confirmed Cu, Rb, and Ti as significant predictors for mtDNAcn. There was a significant mixture effect of multiple metals on increasing mtDNAcn among the elder participants (aged ≥65), with an approximately 11% increase in mtDNAcn for each quartile increase in all metal concentrations [β(95% CI) = 0.146 (0.048, 0.243), P = 0.004]. ConclusionsOur results show that plasma Cu, Rb and Ti were associated with increased blood mtDNA, and we further revealed a significant mixture effect of all metals on mtDNAcn among elder population. These findings may provide a novel perspective on the effect of metals on mitochondrial dysfunction.

Multi-mtDNA Variants May Be a Factor Contributing to Mitochondrial Function Variety in the Skin-Derived Fibroblasts of Leber's Hereditary Optic Neuropathy Patients.

Heterogeneity is a major feature of Leber's hereditary optic neuropathy (LHON) and has a significant impact on the manifestation and diagnosis of the disease. This study explored whether multiple variations in mitochondrial genes were associated with the heterogeneity, mainly phenotypic heterogeneity. Ophthalmic examinations were conducted in two probands with LHON with G11778A and multiple mitochondrial DNA gene (mtDNA) variants. Skin fibroblast cell lines were generated from patients and age- and sex-matched controls. ROS levels, mitochondrial membrane potential, cell energy respiration, and metabolic functions were measured. Flow cytometry and cell viability tests were performed to evaluate the cell apoptosis levels and fate. We found that cells with more mtDNA variants had higher ROS levels, lower mitochondrial membrane potential, and weaker respiratory function. Flow cytometry and cell viability testing showed that multiple mtDNA variants are associated with different levels of cell viability and apoptosis. In conclusion, we found that skin-derived fibroblast cells from G11778A LHON patients could be used as models for LHON research. Multi-mtDNA variants contribute to mitochondrial function variety, which may be associated with heterogeneity in patients with LHON.

Complete Sequence of a 641-kb Insertion of Mitochondrial DNA in the Arabidopsis thaliana Nuclear Genome.

Intracellular transfers of mitochondrial DNA continue to shape nuclear genomes. Chromosome 2 of the model plant Arabidopsis thaliana contains one of the largest known nuclear insertions of mitochondrial DNA (numts). Estimated at over 600 kb in size, this numt is larger than the entire Arabidopsis mitochondrial genome. The primary Arabidopsis nuclear reference genome contains less than half of the numt because of its structural complexity and repetitiveness. Recent data sets generated with improved long-read sequencing technologies (PacBio HiFi) provide an opportunity to finally determine the accurate sequence and structure of this numt. We performed a de novo assembly using sequencing data from recent initiatives to span the Arabidopsis centromeres, producing a gap-free sequence of the Chromosome 2 numt, which is 641 kb in length and has 99.933% nucleotide sequence identity with the actual mitochondrial genome. The numt assembly is consistent with the repetitive structure previously predicted from fiber-based fluorescent in situ hybridization. Nanopore sequencing data indicate that the numt has high levels of cytosine methylation, helping to explain its biased spectrum of nucleotide sequence divergence and supporting previous inferences that it is transcriptionally inactive. The original numt insertion appears to have involved multiple mitochondrial DNA copies with alternative structures that subsequently underwent an additional duplication event within the nuclear genome. This work provides insights into numt evolution, addresses one of the last unresolved regions of the Arabidopsis reference genome, and represents a resource for distinguishing between highly similar numt and mitochondrial sequences in studies of transcription, epigenetic modifications, and de novo mutations.

Mitochondrial Haplogroup Classification of Ancient DNA Samples Using Haplotracker.

Mitochondrial DNA haplogroup classification is used to study maternal lineage of ancient human populations. The haplogrouping of ancient DNA is not easy because the DNA is usually found in small pieces in limited quantities. We have developed Haplotracker, a straightforward and efficient high-resolution haplogroup classification tool optimized specifically for ancient DNA samples. Haplotracker offers a user-friendly input interface for multiple mitochondrial DNA sequence fragments in a sample. It provides accurate haplogroup classification with full-length mitochondrial genome sequences and provides high-resolution haplogroup predictions for some fragmented control region sequences using a novel algorithm built on Phylotree mtDNA Build 17 (Phylotree) and our haplotype database (n = 118,869). Its performance for accuracy was demonstrated to be high through haplogroup classification using 8,216 Phylotree full-length and control region mitochondrial DNA sequences compared with HaploGrep 2, one of the most accurate current haplogroup classifiers. Haplotracker provides a novel haplogroup tracking solution for fragmented sequences to track subhaplogroups or verify the haplogroups efficiently. Using Haplotracker, we classified mitochondrial haplogroups to the final subhaplogroup level in nine ancient DNA samples extracted from human skeletal remains found in 2,000-year-old elite Xiongnu cemetery in Northeast Mongolia. Haplotracker can be freely accessed at https://haplotracker.cau.ac.kr.

Benchmarking the Effectiveness and Accuracy of Multiple Mitochondrial DNA Variant Callers: Practical Implications for Clinical Application.

Mitochondrial DNA (mtDNA) mutations contribute to human disease across a range of severity, from rare, highly penetrant mutations causal for monogenic disorders to mutations with milder contributions to phenotypes. mtDNA variation can exist in all copies of mtDNA or in a percentage of mtDNA copies and can be detected with levels as low as 1%. The large number of copies of mtDNA and the possibility of multiple alternative alleles at the same DNA nucleotide position make the task of identifying allelic variation in mtDNA very challenging. In recent years, specialized variant calling algorithms have been developed that are tailored to identify mtDNA variation from whole-genome sequencing (WGS) data. However, very few studies have systematically evaluated and compared these methods for the detection of both homoplasmy and heteroplasmy. A publicly available synthetic gold standard dataset was used to assess four mtDNA variant callers (Mutserve, mitoCaller, MitoSeek, and MToolBox), and the commonly used Genome Analysis Toolkit “best practices” pipeline, which is included in most current WGS pipelines. We also used WGS data from 126 trios and calculated the percentage of maternally inherited variants as a metric of calling accuracy, especially for homoplasmic variants. We additionally compared multiple pathogenicity prediction resources for mtDNA variants. Although the accuracy of homoplasmic variant detection was high for the majority of the callers with high concordance across callers, we found a very low concordance rate between mtDNA variant callers for heteroplasmic variants ranging from 2.8% to 3.6%, for heteroplasmy thresholds of 5% and 1%. Overall, Mutserve showed the best performance using the synthetic benchmark dataset. The analysis of mtDNA pathogenicity resources also showed low concordance in prediction results. We have shown that while homoplasmic variant calling is consistent between callers, there remains a significant discrepancy in heteroplasmic variant calling. We found that resources like population frequency databases and pathogenicity predictors are now available for variant annotation but still need refinement and improvement. With its peculiarities, the mitochondria require special considerations, and we advocate that caution needs to be taken when analyzing mtDNA data from WGS data.

Neuropathic Pain as Main Manifestation of POLG-Related Disease: A Case Report

Mutations in nuclear-encoded genes that are involved in mitochondrial DNA replication and maintenance (e.g., POLG) have been associated with chronic progressive external ophthalmoplegia (CPEO) phenotype. These nuclear genome mutations may lead to multiple mitochondrial DNA deletions or mitochondrial DNA depletion. On the other hand, primary genetic defects of mitochondrial DNA (such as single large-scale deletion or point mutations) have also been associated with the CPEO phenotype. Chronic progressive external ophthalmoplegia (CPEO) may be a manifestation of specific syndromes that, when clinically recognized, prompt clinicians to investigate specific genetic defects. Thus, CPEO, as part of Kearns Sayre syndrome, suggests the presence of a large-scale deletion of mitochondrial DNA. However, in pure CPEO or CPEO plus phenotypes, it is more difficult to know whether causative genetic defects affect the nuclear or mitochondrial DNA. Here, we present a patient with a long-standing history of CPEO plus phenotype, in whom the sequencing of mitochondrial DNA from skeletal muscle was normal, and no other genetic defect was suspected at first. At the time of our evaluation, the presence of polyneuropathy and neuropathic pain prompted us to investigate nuclear genetic defects and, specifically, mutations in the POLG gene. Thus, the sequencing of the POLG gene revealed p.Thr251Ile and p.Pro587Leu mutations in one allele, and p.Ala467Thr mutation in another allele. Although one would expect that mutations in POLG lead to multiple mitochondrial DNA deletions or depletion (loss of copies), the absence of mitochondrial DNA abnormalities in tissue may be explained by heteroplasmy, a lack or no significant involvement of biopsied tissue, or a sampling bias. So, the absence of secondary mitochondrial DNA alterations should not discourage clinicians from further investigating mutations in nuclear-encoded genes. Lastly, mitochondrial point mutations and single mitochondrial DNA deletions very rarely cause CPEO associated with polyneuropathy and neuropathic pain, and POLG-related disease should be considered in this scenario, instead.

Abstract 17081: End Stage Mitochondrial Cardiomyopathy And Heart Transplantation Due To Pathogenic Biallelic C1QBP Variants

Case Presentation: A 29-year-old male with LVH diagnosed in childhood was admitted with acute HF. TTE showed LVEF 5-10% and LV thrombi for which he was anticoagulated. He received inappropriate ICD shocks due to T wave oversensing, leading to cardiogenic shock requiring VA-ECMO support. Serum lactate peaked at 17 mmol/L due to cardiac and metabolic decompensation. He underwent heart transplantation (HT) on hospital day (HD) 8 and tolerated standard immunosuppression. First endomyocardial biopsy showed acute cellular rejection requiring pulse steroids. He was discharged on HD 33. Trio whole exome and mitochondrial genome sequencing revealed biallelic variants in complement component 1Q subcomponent-binding protein ( C1QBP ), due to a maternally inherited likely pathogenic variant c.612C&gt;G (p.F204L in exon 5) and an apparently de novo deletion of 17p13.2, spanning exons 4-6 of C1QBP and exon 6 of the RPAIN gene. Mitochondrial genome sequencing of the explanted heart revealed multiple large-scale mitochondrial DNA deletions at 33% heteroplasmy. Discussion: C1QBP variants are associated with mitochondrial and multi-organ dysfunction. Only 12 patients exhibiting biallelic C1QBP variants are reported. Four died in the peripartum period due to fetal hydrops or HF; 5 exhibited early-onset cardiomyopathy (CM); 3 others had late-onset ophthalmoplegia without CM. The p.F204L variant has been reported in 1 patient with compound C1QBP p.F204L/p.C186S heterozygosity who died from hydrops fetalis and a second with p.F204L homozygosity with late-onset ophthalmoplegia and skeletal myopathy without CM. Differences in the size, heteroplasmy, and tissue distribution of mitochondrial genome secondary deletions may explain variability in disease onset and progression. We present the first patient with biallelic pathogenic C1QBP gene variants with mitochondrial CM to undergo HT and highlight the diagnosis and management of an exceptionally uncommon genetic disorder.