Application of Genome Sequencing from Blood to Diagnose Mitochondrial Diseases

Mitochondrial diseases can be caused by pathogenic variants in nuclear or mitochondrial DNA-encoded genes that often lead to multisystemic symptoms and can have any mode of inheritance. Using a single test, Genome Sequencing (GS) can effectively identify variants in both genomes, but it has not yet been universally used as a first-line approach to diagnosing mitochondrial diseases due to related costs and challenges in data analysis. In this article, we report three patients with mitochondrial disease molecularly diagnosed through GS performed on DNA extracted from blood to demonstrate different diagnostic advantages of this technology, including the detection of a low-level heteroplasmic pathogenic variant, an intragenic nuclear DNA deletion, and a large mtDNA deletion. Current technical improvements and cost reductions are likely to lead to an expanded routine diagnostic usage of GS and of the complementary “Omic” technologies in mitochondrial diseases.

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Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes

Biochemistry and Cell Biology ◽

10.1139/o02-135 ◽

2002 ◽

Vol 80 (5) ◽

pp. 645-654 ◽

Cited By ~ 57

Author(s):

William Y Tsang ◽

Bernard D Lemire

Keyword(s):

Mitochondrial Dna ◽

Caenorhabditis Elegans ◽

Copy Number ◽

Mitochondrial Diseases ◽

Wild Type ◽

Mtdna Copy Number ◽

Mitochondrial Dna Deletion ◽

Dna Deletion ◽

Average Proportion ◽

Mtdna Deletion

Many human mitochondrial diseases are associated with defects in the mitochondrial DNA (mtDNA). Mutated and wild-type forms of mtDNA often coexist in the same cell in a state called heteroplasmy. Here, we report the isolation of a Caenorhabditis elegans strain bearing the 3.1-kb uaDf5 deletion that removes 11 genes from the mtDNA. The uaDf5 deletion is maternally transmitted and has been maintained for at least 100 generations in a stable heteroplasmic state in which it accounts for ~60% of the mtDNA content of each developmental stage. Heteroplasmy levels vary between individual animals (from ~20 to 80%), but no observable phenotype is detected. The total mtDNA copy number in the uaDf5 mutant is approximately twice that of the wild type. The maternal transmission of the uaDf5 mtDNA is controlled by at least two competing processes: one process promotes the increase in the average proportion of uaDf5 mtDNA in the offspring, while the second promotes a decrease. These two forces prevent the segregation of the mtDNAs to homoplasmy.Key words: mtDNA deletion, Caenorhabditis elegans, heteroplasmy, inheritance, mtDNA copy number.

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Treatment for mitochondrial diseases

Reviews in the Neurosciences ◽

10.1515/revneuro-2020-0034 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Tongling Liufu ◽

Zhaoxia Wang

Keyword(s):

Clinical Trials ◽

Nuclear Dna ◽

Mitochondrial Diseases ◽

Cochrane Collaboration ◽

Potential Bias ◽

Leber Hereditary Optic Neuropathy ◽

Pharmacological Agents ◽

Unpublished Studies ◽

New Treatment ◽

Allotopic Expression

AbstractMitochondrial diseases are predominantly caused by mutations of mitochondrial or nuclear DNA, resulting in multisystem defects. Current treatments are largely supportive, and the disorders progress relentlessly. Nutritional supplements, pharmacological agents and physical therapies have been used in different clinical trials, but the efficacy of these interventions need to be further evaluated. Several recent reviews discussed some of the interventions but ignored bias in those trials. This review was conducted to discover new studies and grade the original studies for potential bias with revised Cochrane Collaboration guidelines. We focused on seven published studies and three unpublished studies; eight of these studies showed improvement in outcome measurements. In particular, two of the interventions have been tested in studies with strict design, which we believe deserve further clinical trials with a large sample. Additionally, allotopic expression of the ND4 subunit seemed to be an effective new treatment for patients with Leber hereditary optic neuropathy.

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Mitochondrial DNA Replacement Techniques to Prevent Human Mitochondrial Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms22020551 ◽

2021 ◽

Vol 22 (2) ◽

pp. 551

Author(s):

Luis Sendra ◽

Alfredo García-Mares ◽

María José Herrero ◽

Salvador F. Aliño

Keyword(s):

Mitochondrial Dna ◽

Nuclear Dna ◽

Genetic Disorders ◽

Mitochondrial Diseases ◽

Legal Regulation ◽

Donor Oocyte ◽

Legal Position ◽

Ethical And Legal Issues ◽

Mitochondrial Replacement Techniques ◽

Do So

Background: Mitochondrial DNA (mtDNA) diseases are a group of maternally inherited genetic disorders caused by a lack of energy production. Currently, mtDNA diseases have a poor prognosis and no known cure. The chance to have unaffected offspring with a genetic link is important for the affected families, and mitochondrial replacement techniques (MRTs) allow them to do so. MRTs consist of transferring the nuclear DNA from an oocyte with pathogenic mtDNA to an enucleated donor oocyte without pathogenic mtDNA. This paper aims to determine the efficacy, associated risks, and main ethical and legal issues related to MRTs. Methods: A bibliographic review was performed on the MEDLINE and Web of Science databases, along with searches for related clinical trials and news. Results: A total of 48 publications were included for review. Five MRT procedures were identified and their efficacy was compared. Three main risks associated with MRTs were discussed, and the ethical views and legal position of MRTs were reviewed. Conclusions: MRTs are an effective approach to minimizing the risk of transmitting mtDNA diseases, but they do not remove it entirely. Global legal regulation of MRTs is required.

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Understanding mitochondrial myopathies: a review

PeerJ ◽

10.7717/peerj.4790 ◽

2018 ◽

Vol 6 ◽

pp. e4790 ◽

Cited By ~ 6

Author(s):

Abhimanyu S. Ahuja

Keyword(s):

Speech Therapy ◽

Nuclear Dna ◽

Severe Disease ◽

Mitochondrial Diseases ◽

The Body ◽

Genetic Mutations ◽

Mitochondrial Myopathies ◽

Positive Side ◽

Organ Systems ◽

New Research

Mitochondria are small, energy-producing structures vital to the energy needs of the body. Genetic mutations cause mitochondria to fail to produce the energy needed by cells and organs which can cause severe disease and death. These genetic mutations are likely to be in the mitochondrial DNA (mtDNA), or possibly in the nuclear DNA (nDNA). The goal of this review is to assess the current understanding of mitochondrial diseases. This review focuses on the pathology, causes, risk factors, symptoms, prevalence data, symptomatic treatments, and new research aimed at possible preventions and/or treatments of mitochondrial diseases. Mitochondrial myopathies are mitochondrial diseases that cause prominent muscular symptoms such as muscle weakness and usually present with a multitude of symptoms and can affect virtually all organ systems. There is no cure for these diseases as of today. Treatment is generally supportive and emphasizes symptom management. Mitochondrial diseases occur infrequently and hence research funding levels tend to be low in comparison with more common diseases. On the positive side, quite a few genetic defects responsible for mitochondrial diseases have been identified, which are in turn being used to investigate potential treatments. Speech therapy, physical therapy, and respiratory therapy have been used in mitochondrial diseases with variable results. These therapies are not curative and at best help with maintaining a patient’s current abilities to move and function.

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The heart in neuromuscular disease: primary mitochondrial diseases

ESC CardioMed ◽

10.1093/med/9780198784906.003.0369 ◽

2018 ◽

pp. 1528-1530

Author(s):

Denis Duboc

Keyword(s):

Neuromuscular Disease ◽

Nuclear Dna ◽

Single Cells ◽

Mitochondrial Diseases ◽

Respiratory Chain Complexes ◽

Base Pairs ◽

Daughter Cells ◽

Dna Molecule ◽

Chain Complexes ◽

Genes Encoding

Mitochondria are responsible for energy production in most eukaryotic cells. Each cell contains at least one mitochondrion and every mitochondrion contains two to ten copies of a circular DNA molecule (mitochondrial DNA or mtDNA). Cardiomyocytes contain approximately 10,000 mtDNA copies. MtDNA is composed of around 16,500 base pairs and 37 genes encoding 13 subunits of the respiratory chain complexes I, III, IV, and V, 22 mitochondrial tRNAs and 2 rRNAs. With each cell division, mitochondria and mtDNA are randomly distributed to daughter cells. In humans, mitochondria are inherited exclusively from the mother. In healthy people mtDNA copies are usually identical at birth (homoplasmy) but with ageing, mtDNA is particularly prone to somatic mutation because, unlike nuclear DNA, it is continuously replicated, even in non-dividing tissues such as myocardium. This can lead to the propagation of somatic mutations within single cells by a process called clonal expansion. In addition, mtDNA lacks an extensive DNA repair mechanism.

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4478 Not just GLUT1: genome sequencing reveals genetic heterogeneity in Doose syndrome

Journal of Clinical and Translational Science ◽

10.1017/cts.2020.84 ◽

2020 ◽

Vol 4 (s1) ◽

pp. 13-13

Author(s):

Jeffrey Dennis Calhoun ◽

Jonathan Gunti ◽

Katie Angione ◽

Elizabeth Geiger ◽

Krista Eschbach ◽

...

Keyword(s):

Genome Sequencing ◽

Genetic Heterogeneity ◽

Subject Matter ◽

De Novo ◽

Structural Abnormality ◽

Short Read ◽

Coding Regions ◽

Initial Analysis ◽

Pathogenic Variants ◽

Unrelated Subject

OBJECTIVES/GOALS: Epilepsy with myoclonic-atonic seizures (EMAS) is a childhood onset epilepsy disorder characterized by seizures with sudden loss of posture, or drop seizures. Our objective was to use short-read genome sequencing in 40 EMAS trios to better understand variants contributing to the development of EMAS. METHODS/STUDY POPULATION: Eligibility for the cohort included a potential diagnosis of EMAS by child neurology faculty at Children’s Hospital Colorado. Exclusion criteria included lack of drop seizures upon chart review or structural abnormality on MRI. Some individuals had prior genetic testing and priority for genome sequencing was given to individuals without clear genetic diagnosis based on previous testing. We analyzed single nucleotide variants (SNVs), small insertions and deletions (INDELs), and larger structural variants (SVs) from trio genomes and determined those that were likely contributory based on standardized American College of Medical Genetics (ACMG) criteria. RESULTS/ANTICIPATED RESULTS: Our initial analysis focused on variants in coding regions of known epilepsy-associated genes. We identified pathogenic or likely pathogenic variants in 6 different individuals involving 6 unique genes. Of these, 5 are de novo SNVs or INDELs and 1 is a de novo SV. One of these involve a de novo heterozygous variant in an X-linked gene (ARHGEF9) in a female individual. We hypothesize the skewed X-inactivation may result in primarily expression of the pathogenic variant. We anticipate identifying additional candidate variants in coding regions of genes previously not associated with EMAS or pediatric epilepsies as well as in noncoding regions of the genome. DISCUSSION/SIGNIFICANCE OF IMPACT: Despite the genetic heterogeneity of EMAS, our initial analysis identified de novo pathogenic or likely pathogenic variants in 15% (6/40) of our cohort. As the cost continues to decline, short read genome sequencing represents a promising diagnostic tool for EMAS and other pediatric onset epilepsy syndromes. CONFLICT OF INTEREST DESCRIPTION: The authors have no conflicts of interest to disclose. SD has consulted for Upsher-Smith, Biomarin and Neurogene on an unrelated subject matter. GLC holds a research collaborative grant with Stoke therapeutics on unrelated subject matter.

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A combined RNA-seq and whole genome sequencing approach for identification of non-coding pathogenic variants in single families

Human Molecular Genetics ◽

10.1093/hmg/ddaa016 ◽

2020 ◽

Vol 29 (6) ◽

pp. 967-979 ◽

Cited By ~ 6

Author(s):

Revital Bronstein ◽

Elizabeth E Capowski ◽

Sudeep Mehrotra ◽

Alex D Jansen ◽

Daniel Navarro-Gomez ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genetic Diagnosis ◽

Genetic Diagnostics ◽

Whole Genome ◽

Unique Challenge ◽

Coding Regions ◽

Pathogenic Variants ◽

Number Of Patients ◽

Coding Variants

Abstract Inherited retinal degenerations (IRDs) are at the focus of current genetic therapeutic advancements. For a genetic treatment such as gene therapy to be successful, an accurate genetic diagnostic is required. Genetic diagnostics relies on the assessment of the probability that a given DNA variant is pathogenic. Non-coding variants present a unique challenge for such assessments as compared to coding variants. For one, non-coding variants are present at much higher number in the genome than coding variants. In addition, our understanding of the rules that govern the non-coding regions of the genome is less complete than our understanding of the coding regions. Methods that allow for both the identification of candidate non-coding pathogenic variants and their functional validation may help overcome these caveats allowing for a greater number of patients to benefit from advancements in genetic therapeutics. We present here an unbiased approach combining whole genome sequencing (WGS) with patient-induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) transcriptome analysis. With this approach, we identified and functionally validated a novel pathogenic non-coding variant in a small family with a previously unresolved genetic diagnosis.

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Mechanisms of replication and repair in mitochondrial DNA deletion formation

Nucleic Acids Research ◽

10.1093/nar/gkaa804 ◽

2020 ◽

Vol 48 (20) ◽

pp. 11244-11258

Author(s):

Gabriele A Fontana ◽

Hailey L Gahlon

Keyword(s):

Dna Damage ◽

Mitochondrial Dna ◽

Large Scale ◽

Molecular Mechanisms ◽

Mitochondrial Dna Deletion ◽

Deletion Formation ◽

Dna Deletion ◽

Mtdna Deletions ◽

Mtdna Deletion

Abstract Deletions in mitochondrial DNA (mtDNA) are associated with diverse human pathologies including cancer, aging and mitochondrial disorders. Large-scale deletions span kilobases in length and the loss of these associated genes contributes to crippled oxidative phosphorylation and overall decline in mitochondrial fitness. There is not a united view for how mtDNA deletions are generated and the molecular mechanisms underlying this process are poorly understood. This review discusses the role of replication and repair in mtDNA deletion formation as well as nucleic acid motifs such as repeats, secondary structures, and DNA damage associated with deletion formation in the mitochondrial genome. We propose that while erroneous replication and repair can separately contribute to deletion formation, crosstalk between these pathways is also involved in generating deletions.

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