scholarly journals Multiplex analysis of mitochondrial DNA pathogenic and polymorphic sequence variants

2010 ◽  
Vol 391 (10) ◽  
Author(s):  
Jason C. Poole ◽  
Vincent Procaccio ◽  
Martin C. Brandon ◽  
Greg Merrick ◽  
Douglas C. Wallace
Keyword(s):  
Mitochondrial Dna ◽  
Pcr Amplification ◽  
Integrated System ◽  
Sequence Variants ◽  
Coding Region ◽  
Single Nucleotide Variants ◽  
Standard Curve ◽  
Mtdna Mutations ◽  
Pathogenic Mutations ◽  
Optimal Extension

Abstract The mitochondrial DNA (mtDNA) encompasses two classes of functionally important sequence variants: recent pathogenic mutations and ancient adaptive polymorphisms. To rapidly and cheaply evaluate both classes of single nucleotide variants (SNVs), we have developed an integrated system in which mtDNA SNVs are analyzed by multiplex primer extension using the SNaPshot system. A multiplex PCR amplification strategy was used to amplify the entire mtDNA, a computer program identifies optimal extension primers, and a complete global haplotyping system is also proposed. This system genotypes SNVs on multiplexed mtDNA PCR products or directly from enriched mtDNA samples and can quantify heteroplasmic variants down to 0.8% using a standard curve. With this system, we have developed assays for testing the common pathogenic mutations in four multiplex panels: two genotype the 13 most common pathogenic mtDNA mutations and two genotype the 10 most common Leber Hereditary Optic Neuropathy mutations along with haplogroups J and T. We use a hierarchal system of 140 SNVs to delineate the major global mtDNA haplogroups based on a global phylogenetic tree of coding region polymorphisms. This system should permit rapid and inexpensive genotyping of pathogenic and lineage-specific mtDNA SNVs by clinical and research laboratories.

scholarly journals Unbiased PCR-free spatio-temporal mapping of the mtDNA mutation spectrum reveals brain region-specific responses to replication instability

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Emilie Kristine Bagge ◽  
Noriko Fujimori-Tonou ◽  
Mie Kubota-Sakashita ◽  
Takaoki Kasahara ◽  
Tadafumi Kato
Keyword(s):  
Pcr Amplification ◽  
Brain Regions ◽  
Mutation Spectrum ◽  
Mtdna Mutation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Mtdna Mutations ◽  
Proof Reading ◽  
Spatio Temporal ◽  
Mitochondrial Dna Polymerase

Abstract Background The accumulation of mtDNA mutations in different tissues from various mouse models has been widely studied especially in the context of mtDNA mutation-driven ageing but has been confounded by the inherent limitations of the most widely used approaches. By implementing a method to sequence mtDNA without PCR amplification prior to library preparation, we map the full unbiased mtDNA mutation spectrum across six distinct brain regions from mice. Results We demonstrate that ageing-induced levels of mtDNA mutations (single nucleotide variants and deletions) reach stable levels at 50 weeks of age but can be further elevated specifically in the cortex, nucleus accumbens (NAc), and paraventricular thalamic nucleus (PVT) by expression of a proof-reading-deficient mitochondrial DNA polymerase, PolgD181A. The increase in single nucleotide variants increases the fraction of shared SNVs as well as their frequency, while characteristics of deletions remain largely unaffected. In addition, PolgD181A also induces an ageing-dependent accumulation of non-coding control-region multimers in NAc and PVT, a feature that appears almost non-existent in wild-type mice. Conclusions Our data provide a novel view of the spatio-temporal accumulation of mtDNA mutations using very limited tissue input. The differential response of brain regions to a state of replication instability provides insight into a possible heterogenic mitochondrial landscape across the brain that may be involved in the ageing phenotype and mitochondria-associated disorders.

scholarly journals Mitochondrial DNA variation of Leber’s Hereditary Optic Neuropathy (LHON) in Western Siberia

2019 ◽  
Author(s):  
E.B. Starikovskaya ◽  
S.A. Shalaurova ◽  
S.V. Dryomov ◽  
A.M. Nazhmidenova ◽  
N.V. Volodko ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Optic Neuropathy ◽  
Western Siberia ◽  
Vision Loss ◽  
Leber’S Hereditary Optic Neuropathy ◽  
Inherited Disease ◽  
Leber's Hereditary Optic Neuropathy ◽  
Mtdna Mutations ◽  
Pathogenic Mutations ◽  
Hereditary Optic Neuropathy

AbstractLeber’s hereditary optic neuropathy (LHON) is a form of disorder caused by pathogenic mutations in a mitochondrial DNA. LHON is maternally inherited disease, which manifests mainly in young adults, affecting predominantly males. Clinically LHON has a manifestation as painless central vision loss, resulting in early onset of disability. Epidemiology of LHON has not been fully investigated yet. In this study, we report 44 genetically unrelated families with LHON manifestation. We performed whole mtDNA genome sequencing and provided genealogical and molecular genetic data on mutations and haplogroup background of LHON patients in the Western Siberia population. Known “primary” pathogenic mtDNA mutations (MITOMAP) were found in 32 families: m.11778G>A represents 53,10% (17/32), m.3460G>A – 21,90% (7/32), m.14484T>C – 18,75% (6/32), and rare m.10663T>C and m.3635G>A represent 6,25% (2/32). We describe potentially pathogenic m.4659G>A in one subject without known pathogenic mutations, and potentially pathogenic m.9444C>T, m.6261G>A, m.9921G>A, m.8551T>C, m.8412T>C, m.15077G>A in families with known pathogenic mutations confirmed. We suppose these mutations could contribute to the pathogenesis of optic neuropathy development. Our results indicate that haplogroup affiliation and mutational spectrum of the Western Siberian LHON cohort substantially deviate from those of European populations.

scholarly journals Deep sequencing shows that accumulation of potentially pathogenic mtDNA mutations rather than mtDNA copy numbers may be associated with early embryonic loss

2020 ◽  
Vol 37 (9) ◽  
pp. 2181-2188
Author(s):  
Licheng Ji ◽  
Tingting Liao ◽  
Juan Yang ◽  
Houming Su ◽  
Jianyuan Song ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Spontaneous Abortion ◽  
Missense Mutations ◽  
Mtdna Copy Number ◽  
Mtdna Mutations ◽  
Pathogenic Mutations ◽  
Copy Numbers ◽  
Significant Difference ◽  
Embryonic Loss ◽  
Dna Copy Numbers

Abstract Purpose To explore the relationship between mitochondrial DNA quantity and heteroplasmy and early embryonic loss. Methods A total of 150 villous samples from patients with spontaneous abortion (SA, n = 75) or induced abortion (IA, n = 75) were collected. qPCR and next-generation sequencing (NGS) were used to test mitochondrial DNA quantity and heteroplasmy. Missense mutations with a CADD score > 15 and heteroplasmy ≥ 70% were defined as potentially pathogenic mutations. Results With respect to mitochondrial DNA copy numbers, there was no significant difference between the SA and IA groups (median (IQR), 566 (397–791) vs. 614 (457–739); P = 0.768) or between the euploid and aneuploid groups (median (IQR), 516 (345–730) vs. 599 (423–839); P = 0.107). mtDNA copy numbers were not associated with spontaneous abortion using logistic regression analysis (P = 0.196, 95% CI 1.000–1.001). In addition, more patients harbored possibly pathogenic mtDNA mutations in their chorionic villi in the SA group (70.7%, 53/75) compared with the IA group (54.7%, 41/75; P < 0.05). However, there was no statistical difference between the euploid (80%, 24/30) and aneuploid groups (64.4%, 29/45; p = 0.147). Conclusion Early embryonic loss and the formation of aneuploidy were not related to mtDNA copy number. Patients with spontaneous abortion were more likely to have possibly pathogenic mutations in their mtDNA, and this may assist in purifying pathogenic mtDNA. However, whether the accumulation of these potentially morbific mtDNA mutations caused early embryonic loss requires further investigation.

scholarly journals Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours

Oncogene ◽  
2000 ◽  
Vol 19 (16) ◽  
pp. 2060-2066 ◽  
Author(s):  
Jen Jen Yeh ◽  
Kathryn L Lunetta ◽  
Nathalie J van Orsouw ◽  
Francis D Moore ◽  
George L Mutter ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Sequence Variants ◽  
Papillary Thyroid ◽  
Thyroid Carcinomas ◽  
Mtdna Mutations ◽  
Mtdna Sequence

scholarly journals Design and Use of a Peptide Nucleic Acid for Detection of the Heteroplasmic Low-Frequency Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes (MELAS) Mutation in Human Mitochondrial DNA

2002 ◽  
Vol 48 (12) ◽  
pp. 2155-2163 ◽  
Author(s):  
Diane K Hancock ◽  
Frederick P Schwarz ◽  
Fenhong Song ◽  
Lee-Jun C Wong ◽  
Barbara C Levin
Keyword(s):  
Mitochondrial Dna ◽  
Lactic Acidosis ◽  
Low Frequency ◽  
Pcr Amplification ◽  
Mitochondrial Encephalomyopathy ◽  
Nucleotide Position ◽  
Wild Type ◽  
Human Mitochondrial Dna ◽  
Mtdna Mutations ◽  
A3243g Mutation

Abstract Background: Most pathogenic human mitochondrial DNA (mtDNA) mutations are heteroplasmic (i.e., mutant and wild-type mtDNA coexist in the same individual) and are difficult to detect when their concentration is a small proportion of that of wild-type mtDNA molecules. We describe a simple methodology to detect low proportions of the single base pair heteroplasmic mutation, A3243G, that has been associated with the disease mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) in total DNA extracted from blood. Methods: Three peptide nucleic acids (PNAs) were designed to bind to the wild-type mtDNA in the region of nucleotide position 3243, thus blocking PCR amplification of the wild-type mtDNA while permitting the mutant DNA to become the dominant product and readily discernable. DNA was obtained from both apparently healthy and MELAS individuals. Optimum PCR temperatures were based on the measured ultraviolet thermal stability of the DNA/PNA duplexes. The presence or absence of the mutation was determined by sequencing. Results: In the absence of PNAs, the heteroplasmic mutation was either difficult to detect or undetectable by PCR and sequencing. Only PNA 3 successfully inhibited amplification of the wild-type mtDNA while allowing the mutant mtDNA to amplify. In the presence of PNA 3, we were able to detect the heteroplasmic mutation when its concentration was as low as 0.1% of the concentration of the wild-type sequence. Conclusion: This methodology permits easy detection of low concentrations of the MELAS A3243G mutation in blood by standard PCR and sequencing methods.

scholarly journals Mitochondrial DNA Variation of Leber’s Hereditary Optic Neuropathy in Western Siberia

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1574 ◽  
Author(s):  
Elena Starikovskaya ◽  
Sofia Shalaurova ◽  
Stanislav Dryomov ◽  
Azhar Nazhmidenova ◽  
Natalia Volodko ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Optic Neuropathy ◽  
Western Siberia ◽  
Vision Loss ◽  
Molecular Genetic ◽  
Leber’S Hereditary Optic Neuropathy ◽  
Leber's Hereditary Optic Neuropathy ◽  
Mtdna Mutations ◽  
Pathogenic Mutations ◽  
Hereditary Optic Neuropathy

Our data first represent the variety of Leber’s hereditary optic neuropathy (LHON) mutations in Western Siberia. LHON is a disorder caused by pathogenic mutations in the mitochondrial DNA (mtDNA), inherited maternally and presents mainly in young adults, predominantly males. Clinically, LHON manifests itself as painless central vision loss, resulting in early onset of disability. The epidemiology of LHON has not been fully investigated yet. In this study, we report 44 genetically unrelated families with LHON manifestation. We performed whole mtDNA genome sequencing and provided genealogical and molecular genetic data on mutations and haplogroup background of LHON patients. Known “primary” pathogenic mtDNA mutations (MITOMAP) were found in 32 families: m.11778G>A represents 53.10% (17/32), m.3460G>A—21.90% (7/32), m.14484T>C–18.75% (6/32), and rare m.10663T>C and m.3635G>A represent 6.25% (2/32). We describe potentially pathogenic m.4659G>A in one subject without known pathogenic mutations, and potentially pathogenic m.6261G>A, m.8412T>C, m.8551T>C, m.9444C>T, m.9921G>A, and m.15077G>A in families with known pathogenic mutations confirmed. We suppose these mutations could contribute to the pathogenesis of optic neuropathy development. Our results indicate that haplogroup affiliation and mutational spectrum of the Western Siberian LHON cohort substantially deviate from those of European populations.

Do mitochondrial DNA mutations play the key role in chronification of sterile inflammation? Special focus on atherosclerosis

2020 ◽  
Vol 26 ◽  
Author(s):  
Alexander N. Orekhov ◽  
Elena V. Gerasimova ◽  
Vasily N. Sukhorukov ◽  
Anastasia V. Poznyak ◽  
Nikita G. Nikiforov
Keyword(s):  
Innate Immunity ◽  
Mitochondrial Dna ◽  
Low Density Lipoprotein ◽  
Atherosclerotic Lesion ◽  
Density Lipoprotein ◽  
Sterile Inflammation ◽  
Special Focus ◽  
Mitochondrial Dysfunctions ◽  
Mtdna Mutations ◽  
Dna Mutations

Background: The elucidation of mechanisms implicated in the chronification of inflammation is able to shed the light on the pathogenesis of disorders that are responsible for the majority of the incidence of disease and deaths, and also causes of ageing. Atherosclerosis is an example of the most significant inflammatory pathology. The inflammatory response of innate immunity is implicated in the development of atherosclerosis arising locally or focally. Modified low-density lipoprotein (LDL) was regarded as the trigger for this response. No atherosclerotic changes in the arterial wall occur due to the quick decrease in inflammation rate. Nonetheless, the atherosclerotic lesion formation can be a result of the chronification of local inflammation, which, in turn, is caused by alteration of the response of innate immunity. Objective: In this review, we discussed potential mechanisms of the altered response of the immunity in atherosclerosis with a particular emphasis on mitochondrial dysfunctions. Conclusion: A few mitochondrial dysfunctions can be caused by the mitochondrial DNA (mtDNA) mutations. Moreover, mtDNA mutations were found to affect the development of defective mitophagy. Modern investigations have demonstrated the controlling mitophagy function in the response of the immune system. Therefore, we hypothesized that impaired mitophagy, as a consequence of mutations in mtDNA, can raise a disturbed innate immunity response resulting in the chronification of inflammation in atherosclerosis.

Sequence variants in the FcεRI alpha chain gene

2002 ◽  
Vol 93 (1) ◽  
pp. 37-41 ◽  
Author(s):  
Toshiki Shikanai ◽  
Eric S. Silverman ◽  
Brian W. Morse ◽  
Craig M. Lilly ◽  
Hiroshi Inoue ◽  
...  
Keyword(s):  
Promoter Region ◽  
Population Substructure ◽  
Chain Gene ◽  
Polymorphism Analysis ◽  
Sequence Variants ◽  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Alpha Chain ◽  
Asthmatic Patients ◽  
Single Stranded Conformational Polymorphism

There is a relationship between IgE levels and expression of high-affinity IgE receptors (FcεRI). Because the alpha chain is the only portion of the receptor that binds directly to IgE, we reasoned that sequence variants in the FcεRI alpha gene may exist that alter these binding events. We screened all of the exons and the promoter region of the FcεRI alpha chain gene with genomic DNA from 389 asthmatic and 341 normal control subjects for mutations by using single-stranded conformational polymorphism analysis. No nonsynonomous single nucleotide polymorphisms (SNPs) were identified in the coding region. Three SNPs were found in the promoter region: an A/C transversion at −770 from the translation start site; a G/A transition at −664; and a T/C transition at −335. No differences in allele frequencies were detected between asthmatic subjects and controls. Homozygosity for the C variant at locus −335 was more common in Caucasian asthmatic patients with IgE levels in the lower quartile than in the upper quartile ( P = 0.032). An analysis of highly polymorphic SNPs indicated that this association is unlikely to be due to population substructure. We conclude that homozygosity for the C allele of FcεRI alpha chain variant is associated with lower IgE levels.

Abstract 17097: Non-Synonymous Mitochondrial DNA Variants Are Common in Myocardial Tissue of Heart Failure Patients

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Pappu Ananya ◽  
Michael Binder ◽  
Yang Wanjun ◽  
Rebecca McClellan ◽  
Brittney Murray ◽  
...  
Keyword(s):  
Heart Failure ◽  
Mitochondrial Dna ◽  
Nuclear Dna ◽  
Left Ventricular ◽  
Individual Risk ◽  
Myocardial Tissue ◽  
Mtdna Mutation ◽  
Mtdna Mutations ◽  
Ventricular Tissue ◽  
Mtdna Variants

Introduction: Mitochondrial heart disease due to pathogenic mitochondrial DNA (mtDNA) mutations can present as hypertrophic or dilated cardiomyopathy, ventricular arrhythmias and conduction disease. It is estimated that the mutation rate of mtDNA is 10 to 20-fold higher than that of nuclear DNA genes due to damage from reactive oxygen species released as byproducts during oxidative phosphorylation. When a new mtDNA mutation arises, it creates an intracellular heteroplasmic mixture of mutant and normal mtDNAs, called heteroplasmy. Heteroplasmy levels can vary in various tissues and examining mtDNA variants in blood may not be representative for the heart. The frequency of pathogenic mtDNA variants in myocardial tissues in unknown. Hypothesis: Human ventricular tissue may contain mtDNA mutations which can lead to alterations in mitochondrial function and increase individual risk for heart failure. Methods: Mitochondrial DNA was isolated from 61 left ventricular myocardial samples obtained from failing human hearts at the time of transplantation. mtDNA was sequenced with 23 primer pairs. In silico prediction of non-conservative missense variants was performed via PolyPhen-2. Heteroplasmy levels of variants predicted to be pathogenic were quantified using allele-specific ARMS-PCR. Results: We identified 21 mtDNA non-synonymous variants predicted to be pathogenic in 17 hearts. Notably, one heart contained four pathogenic mtDNA variants (ATP6: p.M104; ND5: p.P265S; ND4: p.N390S and p.L445F). Heteroplasmy levels exceeded 90% for all four variants in myocardial tissue and were significantly lower in blood. No pathogenic mtDNA variants were identified in 44 hearts. Hearts with mtDNA mutations had higher levels of myocardial GDF-15 (growth differentiation factor-15; 6.2±2.3 vs. 1.3±0.18, p=0.045), an established serum biomarker in various mitochondrial diseases. Conclusions: Non-synonymous mtDNA variants predicted to be pathogenic are common in human left ventricular tissue and may be an important modifier of the heart failure phenotype. Future studies are necessary to correlate myocardial mtDNA mutations with cardiovascular outcomes and to assess whether serum GDF-15 allows identifying patients with myocardial mtDNA mutations.

Abstract 206: Accumulation of Mitochondrial DNA Mutations Impairs Survival, Proliferation, and Differentiation of Cardiac Progenitor Cells

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Amabel M Orogo ◽  
Dieter A Kubli ◽  
Anne N Murphy ◽  
Åsa B Gustafsson
Keyword(s):  
Mitochondrial Dna ◽  
Progenitor Cells ◽  
Mitochondrial Biogenesis ◽  
Nuclear Dna ◽  
Critical Role ◽  
Chemotherapeutic Agents ◽  
Cardiac Progenitor Cells ◽  
Mtdna Mutations ◽  
Differentiation Medium ◽  
Proliferation And Differentiation

Activation and participation of cardiac progenitor cells (CPCs) in regeneration are critical for effective repair in the wake of pathologic injury. Stem cell activation and commitment involve increased energy demand and mitochondrial biogenesis. To date, little attention has been paid to the importance of mitochondria in CPC survival, proliferation and differentiation. CPC function is reduced with age but the underlying mechanism is still unclear. Mitochondrial DNA (mtDNA) is more susceptible to oxidative attacks than nuclear DNA due to its proximity to the mitochondrial respiratory chain and lack of protective histone-like proteins. With age, mtDNA accumulates mutations that can impair mitochondrial respiration and increase ROS production. In this study, we examined the effects of accumulating mtDNA mutations on CPC proliferation and survival. We have found that incubation of uncommitted c-kit+ CPCs in differentiation medium increased mitochondrial mass and expansion of the mitochondrial network, which correlated with increased cell size and expression of cardiac lineage commitment markers. Differentiation activated mitochondrial biogenesis, increased mtDNA copy number, and enhanced oxidative capacity and cellular ATP levels in CPCs. To investigate the effect of mtDNA mutations and aging on CPC survival and function, we utilized a mouse model in which a mutation in the mtDNA polymerase γ (POLG m/m ) leads to accumulation of mtDNA mutations, mitochondrial dysfunction, and accelerated aging. Isolated CPCs from hearts of 2-month old POLG m/m mice had reduced proliferation and were more susceptible to oxidative stress and chemotherapeutic agents compared to WT CPCs. The majority of POLG m/m CPCs contained fragmented mitochondria as shown by immunostaining. Incubation in differentiation medium resulted in fewer GATA-4 positive POLG m/m CPCs compared to WT CPCs. The reduced differentiation in these POLG m/m CPCs correlated with reduced PGC-1α expression and OXPHOS protein levels, suggesting that mitochondrial biogenesis is impaired. These data demonstrate that mitochondria play a critical role in CPC function, and accumulation of mtDNA mutations impairs CPC function and reduces their repair potential.

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