The mtDNA mutation spectrum in the PolG mutator mouse reveals germline and somatic selection

Abstract Background Mitochondrial DNA (mtDNA) codes for products necessary for electron transport and mitochondrial gene translation. mtDNA mutations can lead to human disease and influence organismal fitness. The PolG mutator mouse lacks mtDNA proofreading function and rapidly accumulates mtDNA mutations, making it a model for examining the causes and consequences of mitochondrial mutations. Premature aging in PolG mice and their physiology have been examined in depth, but the location, frequency, and diversity of their mtDNA mutations remain understudied. Identifying the locations and spectra of mtDNA mutations in PolG mice can shed light on how selection shapes mtDNA, both within and across organisms. Results Here, we characterized somatic and germline mtDNA mutations in brain and liver tissue of PolG mice to quantify mutation count (number of unique mutations) and frequency (mutation prevalence). Overall, mtDNA mutation count and frequency were the lowest in the D-loop, where an mtDNA origin of replication is located, but otherwise uniform across the mitochondrial genome. Somatic mtDNA mutations have a higher mutation count than germline mutations. However, germline mutations maintain a higher frequency and were also more likely to be silent. Cytosine to thymine mutations characteristic of replication errors were the plurality of basepair changes, and missense C to T mutations primarily resulted in increased protein hydrophobicity. Unlike wild type mice, PolG mice do not appear to show strand asymmetry in mtDNA mutations. Indel mutations had a lower count and frequency than point mutations and tended to be short, frameshift deletions. Conclusions Our results provide strong evidence that purifying selection plays a major role in the mtDNA of PolG mice. Missense mutations were less likely to be passed down in the germline, and they were less likely to spread to high frequencies. The D-loop appears to have resistance to mutations, either through selection or as a by-product of replication processes. Missense mutations that decrease hydrophobicity also tend to be selected against, reflecting the membrane-bound nature of mtDNA-encoded proteins. The abundance of mutations from polymerase errors compared with reactive oxygen species (ROS) damage supports previous studies suggesting ROS plays a minimal role in exacerbating the PolG phenotype, but our findings on strand asymmetry provide discussion for the role of polymerase errors in wild type organisms. Our results provide further insight on how selection shapes mtDNA mutations and on the aging mechanisms in PolG mice.

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The mtDNA Mutation Spectrum in the Polg Mutator Mouse Reveals Germline and Somatic Selection

10.21203/rs.3.rs-659185/v1 ◽

2021 ◽

Author(s):

Kendra D Maclaine ◽

Kevin A Stebbings ◽

Justin C Havird

Keyword(s):

Mitochondrial Gene ◽

Point Mutations ◽

Purifying Selection ◽

Germline Mutations ◽

Premature Aging ◽

Missense Mutations ◽

Mtdna Mutation ◽

Mtdna Mutations ◽

D Loop ◽

Aging Mechanisms

Abstract Background:Mitochondrial DNA (mtDNA) codes for products necessary for electron transport and mitochondrial gene translation. mtDNA mutations can lead to human disease and influence organismal fitness. The PolG mutator mouse lacks mtDNA proofreading function and rapidly accumulates mtDNA mutations, making it a model for examining the causes and consequences of mitochondrial mutations. Premature aging in PolG mice and their physiology have been examined in depth, but the location, frequency, and diversity of their mtDNA mutations remain understudied. Identifying the locations and spectra of mtDNA mutations in PolG mice can shed light on how selection shapes mtDNA, both within and across organisms.Results:Here, we characterized somatic and germline mtDNA mutations in brain and liver tissue of PolG mice to quantify mutation count (number of unique mutations) and frequency (mutation prevalence). Overall, mtDNA mutation count and frequency were the lowest in the D-loop, where an mtDNA origin of replication is located, but otherwise uniform across the mitochondrial genome. Somatic mtDNA mutations have a higher mutation count than germline mutations. However, germline mutations maintain a higher frequency and were also more likely to be silent. Cytosine to thymine mutations characteristic of replication errors were the plurality of basepair changes, and missense C to T mutations primarily resulted in increased protein hydrophobicity. Indel mutations had a lower count and frequency than point mutations and tended to be short, frameshift deletions.Conclusions:Our results provide strong evidence that purifying selection plays a major role in the mtDNA of PolG mice. Missense mutations were less likely to be passed down in the germline, and they were less likely to spread to high frequencies. The D-loop appears to have resistance to mutations, either through selection or as a by-product of replication processes. Missense mutations that decrease hydrophobicity also tend to be selected against, reflecting the membrane-bound nature of mtDNA-encoded proteins. The abundance of mutations from polymerase errors compared with reactive oxygen species (ROS) damage supports previous studies suggesting ROS plays a minimal role in exacerbating the PolG phenotype. Our results provide further insight on how selection shapes mtDNA mutations and on the aging mechanisms in PolG mice.

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Analysis of phage-resistant mechanisms inStaphylococcus aureusSA003 reveals a different binding mechanism for the closely related Twort-like phages ϕSA012 and ϕSA039

10.1101/339549 ◽

2018 ◽

Cited By ~ 3

Author(s):

Aa Haeruman Azam ◽

Fumiya Hoshiga ◽

Ippei Takeuchi ◽

Kazuhiko Miyanaga ◽

Yasunori Tanji

Keyword(s):

Teichoic Acid ◽

Point Mutations ◽

In Silico Analysis ◽

Close Relative ◽

Phenotypic Change ◽

Missense Mutations ◽

Wild Type ◽

Wall Teichoic Acid ◽

Whole Genome Analysis ◽

Specific Phage

ABSTRACTWe have previously generated strains ofStaphylococcus aureusSA003 resistant to its specific phage ϕSA012 through long-term coevolution experiment. However, the DNA mutations responsible for the phenotypic change of phage resistance are unknown. Whole-genome analysis revealed six genes that acquired unique point mutations: five missense mutations and one nonsense mutation. Moreover, one deletion, 1.779-bp, resulted in the deletion of the genes encoding glycosyltransferase, TarS, and iron-sulfure repair protein, ScdA. The deletion occurred from the second round of coculture (SA003R2) and remained through the last round. The ϕSA012 infection toward SA003R2 had decreased to 79.77±7.50% according to plating efficiency. Complementation of the phage-resistant strain by the wild-type allele showed two mutated host genes were linked to the inhibition of post-adsorption, and five genes were linked to phage adsorption of ϕSA012. Unlike ϕSA012, infection by ϕSA039, a close relative of ϕSA012, onto SA003R2 was impaired drastically. Complementation of SA003R2 by wild-typetarSrestores the infectivity of ϕSA039. Thus, we concluded that ϕSA039 requires β-GlcNAc in Wall Teichoic Acid (WTA) for its binding. In silico analysis of the ϕSA039 genome revealed that several proteins in the tail and baseplate region were different from ϕSA012; notably the partial deletion oforf96of ϕSA039, a homolog oforf99of ϕSA012.Orf100of ϕSA039, a homolog ofOrf103of ϕSA012, a previously reported receptor binding protein (RBP), had low similarity (86%) to that of ϕSA012. The difference in tail and baseplate proteins might be the factor for specificity difference between ϕSA012 and ϕSA039.

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Modulation of mtDNA copy number ameliorates the pathological consequences of a heteroplasmic mtDNA mutation in the mouse

Science Advances ◽

10.1126/sciadv.aav9824 ◽

2019 ◽

Vol 5 (4) ◽

pp. eaav9824 ◽

Cited By ~ 15

Author(s):

R. Filograna ◽

C. Koolmeister ◽

M. Upadhyay ◽

A. Pajak ◽

P. Clemente ◽

...

Keyword(s):

Copy Number ◽

Important Determinant ◽

Threshold Level ◽

Wild Type ◽

Mtdna Copy Number ◽

Mtdna Mutation ◽

Mtdna Mutations ◽

Selective Elimination ◽

The Absolute ◽

Or Gene

Heteroplasmic mtDNA mutations typically act in a recessive way and cause mitochondrial disease only if present above a certain threshold level. We have experimentally investigated to what extent the absolute levels of wild-type (WT) mtDNA influence disease manifestations by manipulating TFAM levels in mice with a heteroplasmic mtDNA mutation in the tRNAAla gene. Increase of total mtDNA levels ameliorated pathology in multiple tissues, although the levels of heteroplasmy remained the same. A reduction in mtDNA levels worsened the phenotype in postmitotic tissues, such as heart, whereas there was an unexpected beneficial effect in rapidly proliferating tissues, such as colon, because of enhanced clonal expansion and selective elimination of mutated mtDNA. The absolute levels of WT mtDNA are thus an important determinant of the pathological manifestations, suggesting that pharmacological or gene therapy approaches to selectively increase mtDNA copy number provide a potential treatment strategy for human mtDNA mutation disease.

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Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site

Blood ◽

10.1182/blood-2007-11-122457 ◽

2008 ◽

Vol 111 (12) ◽

pp. 5712-5720 ◽

Cited By ~ 34

Author(s):

Massimiliano Gaetani ◽

Sara Mootien ◽

Sandra Harper ◽

Patrick G. Gallagher ◽

David W. Speicher

Keyword(s):

Clinical Symptoms ◽

Point Mutations ◽

Titration Calorimetry ◽

Missense Mutations ◽

Binding Affinities ◽

Wild Type ◽

Tetramer Binding ◽

Structural And Functional Properties ◽

Recombinant Peptides ◽

Self Association

Abstract The most common hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) mutations are α-spectrin missense mutations in the dimer-tetramer self-association site. In this study, we systematically compared structural and functional properties of the 14 known HE/HPP mutations located in the α-spectrin tetramer binding site. All mutant α-spectrin recombinant peptides were well folded, stable structures, with only the R34W mutant exhibiting a slight structural destabilization. In contrast, binding affinities measured by isothermal titration calorimetry were greatly variable, ranging from no detectable binding observed for I24S, R28C, R28H, R28S, and R45S to approximately wild-type binding for R34W and K48R. Binding affinities for the other 7 mutants were reduced by approximately 10- to 100-fold relative to wild-type binding. Some sites, such as R28, were hot spots that were very sensitive to even relatively conservative substitutions, whereas other sites were only moderately perturbed by nonconservative substitutions. The R34W and K48R mutations were particularly intriguing mutations that apparently either destabilize tetramers through mechanisms not probed by the univalent tetramer binding assay or represent polymorphisms rather than the pathogenic mutations responsible for observed clinical symptoms. All α0 HE/HPP mutations studied here appear to exert their destabilizing effects through molecular recognition rather than structural mechanisms.

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Maladaptive nutrient signalling sustains the m.3243A>G mtDNA mutation

10.1101/2020.06.18.159103 ◽

2020 ◽

Author(s):

Chih-Yao Chung ◽

Kritarth Singh ◽

Vassilios N Kotiadis ◽

Jee Hwan Ahn ◽

Lida Kabir ◽

...

Keyword(s):

Mitochondrial Genome ◽

Lactic Acidosis ◽

Treatment Options ◽

Disease Phenotype ◽

Wild Type ◽

Mtdna Mutation ◽

Potential Therapeutic Target ◽

Mtdna Mutations ◽

Cellular Bioenergetics ◽

Clinical Conditions

ABSTRACTMutations of the mitochondrial genome (mtDNA) cause a range of profoundly debilitating clinical conditions for which treatment options are limited. Most mtDNA diseases show heteroplasmy - tissues express both wild-type and mutant mtDNA. The relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are poorly understood. We have extensively characterised changes in bioenergetic, metabolomic, lipidomic and RNAseq profiles in heteroplasmic patient-derived cells carrying the m.3243A>G mtDNA mutation, the cause of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). These studies reveal that the mutation promotes upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 activated mitophagy, reduced mtDNA mutant load and rescued cellular bioenergetics cell-autonomously. The rescue was prevented by inhibition of mitophagy. These findings suggest that activation of the PI3K-Akt-mTORC1 axis is maladaptive and represents a potential therapeutic target for people suffering from the consequences of the m.3243A>G mutation.

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Do Somatic Mitochondrial DNA Mutations Contribute to Parkinson's Disease?

Parkinson s Disease ◽

10.4061/2011/659694 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Joanne Clark ◽

Ying Dai ◽

David K. Simon

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Mitochondrial Dna ◽

Mitochondrial Dysfunction ◽

Point Mutations ◽

Premature Aging ◽

Mtdna Mutations ◽

Dna Mutations ◽

Mitochondrial Dna Polymerase ◽

Mitochondrial Defect

A great deal of evidence supports a role for mitochondrial dysfunction in the pathogenesis of Parkinson's disease (PD), although the origin of the mitochondrial dysfunction in PD remains unclear. Expression of mitochondrial DNA (mtDNA) from PD patients in “cybrid” cell lines recapitulates the mitochondrial defect, implicating a role for mtDNA mutations, but the specific mutations responsible for the mitochondrial dysfunction in PD have been difficult to identify. Somatic mtDNA point mutations and deletions accumulate with age and reach high levels in substantia nigra (SN) neurons. Mutations in mitochondrial DNA polymeraseγ(POLG) that lead to the accumulation of mtDNA mutations are associated with a premature aging phenotype in “mutator” mice, although overt parkinsonism has not been reported in these mice, and with parkinsonism in humans. Together these data support, but do not yet prove, the hypothesis that the accumulation of somatic mtDNA mutations in SN neurons contribute to the pathogenesis of PD.

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TBIO-12. THE SPECTRUM OF MITOCHONDRIAl DNA (mtDNA) MUTATIONS IN PEDIATRIC CENTRAL NERVOUS SYSTEM (CNS) TUMORS

Neuro-Oncology ◽

10.1093/neuonc/noaa222.839 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii468-iii469

Author(s):

Kristiyana Kaneva ◽

Petr Triska ◽

Daria Merkurjev ◽

Moiz Bootwalla ◽

Jennifer Cotter ◽

...

Keyword(s):

Mitochondrial Dna ◽

Cns Tumors ◽

Whole Genome Sequencing Data ◽

Low Grade ◽

Missense Mutations ◽

Loss Of Function ◽

Sequencing Data ◽

Mtdna Mutation ◽

Tumor Subtypes ◽

Mtdna Mutations

Abstract To explore the role of mitochondrial DNA mutations in pediatric CNS tumors, we analyzed 749 tumor-normal paired whole genome sequencing data sets from the Children’s Brain Tumor Tissue Consortium (CBTTC). We detected 307 somatic mtDNA mutations in 222 CNS tumors (29.6%). Most frequently observed were missense mutations (38.1%). We also detected 34 loss-of-function mutations. Different pediatric CNS tumor subtypes have distinct mtDNA mutation profiles. For categorical comparisons, we analyzed subtypes with at least 15 samples. The highest number of mtDNA mutations per tumor sample was in meningiomas (0.85), while atypical teratoid rhabdoid tumors (ATRTs) had the lowest number per sample (0.18). High-grade gliomas had a higher number of mtDNA mutations per sample than low-grade gliomas (0.56 vs. 0.31) (p = 0.0011), with almost twice as many missense mtDNA mutations per sample (0.22 vs. 0.13) (p < 0.001), and higher average heteroplasmy levels (11% vs. 9%). The average heteroplasmy was 10.1%, ranging from 15.6% in medulloblastoma to 6.36% in schwannoma suggesting that these are clonal alterations and not artifacts. Intriguingly, the two chordoma patients in the CBTTC database had an identical heteroplasmic m.10971G>A MT-ND4 nonsense mutation. Similarly, our patient with recurrent gliofibroma harbored the same somatic MT-ND4 synonymous variant (m.10700A>G) detected at 53% heteroplasmy in the initial tumor, 79% in the first recurrence, and 97% in the second recurrence. Although the functional consequences of these alterations are not yet understood, our findings suggest that sequencing the mtDNA genome may be used to characterize CNS tumors at diagnosis and monitor disease progression.

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Packpred: Predicting the functional effect of missense mutations

10.1101/2020.12.30.424909 ◽

2021 ◽

Author(s):

Kuan Pern Tan ◽

Tejashree Rajaram Kanitkar ◽

Kwoh Chee Keong ◽

M.S. Madhusudhan

Keyword(s):

Amino Acid ◽

Single Point ◽

Point Mutations ◽

Substitution Matrix ◽

Saturation Mutagenesis ◽

Data Sets ◽

Missense Mutations ◽

Wild Type ◽

Data Set ◽

Functional Consequences

1.AbstractPredicting the functional consequences of single point mutations has relevance to protein function annotation and to clinical analysis/diagnosis. We developed and tested Packpred that makes use of a multi-body clique statistical potential in combination with a depth dependent amino acid substitution matrix (FADHM) and positional Shannon Entropy to predict the functional consequences of point mutations in proteins. Parameters were trained over a saturation mutagenesis data set of T4-lysozyme (1966 mutations). The method was tested over another saturation mutagenesis data set (CcdB; 1534 mutations) and the Missense3D data set (4099 mutations). The performance of Packpred was compared against those of six other contemporary methods. With MCC values of 0.42, 0.47 and 0.36 on the training and testing data sets respectively, Packpred outperforms all method in all data sets, with the exception of marginally underperforming to FADHM in the CcdB data set. On analyzing the results, we could build meta servers that chose best performing methods of wild type amino acids and for wild type-mutant amino acid pairs. This lead to an increase of MCC value of 0.40 and 0.51 for the two meta predictors respectively on the Missense3D data set. We conjecture that it is possible to improve accuracy with better meta predictors as among the 7 methods compared, at the least one method or another is able to correctly predict ∼99% of the data.

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5 NEUTRAL SEGREGATION OF DONOR CELL MITOCHONDRIA IN FETAL AND ADULT TISSUES OF SOMATIC CELL CLONES IN CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab5 ◽

2005 ◽

Vol 17 (2) ◽

pp. 153

Author(s):

F. Viramontes ◽

F. Filion ◽

L.C. Smith

Keyword(s):

Blood Cells ◽

Bos Taurus ◽

White Blood Cells ◽

Bos Indicus ◽

Pcr Amplification ◽

Donor Cell ◽

Wild Type ◽

Mtdna Mutation ◽

D Loop ◽

The Mean

Until now, animal cloning has been extremely inefficient: only 1–2% of nuclear transfer (NT) clones survive to birth. Some of these anomalies may be related to an incompatibility between nuclear and mitochondrial genes (Cummins JM 2001 Hum. Reprod. Update 7, 217–228). Controversy exists as to the levels of donor cell mitochondrial DNA (mtDNA) inheritance in somatic clones (heteroplasmy). Whereas some researchers found very low quantities (0.1–4%) (Steinborn R et al. 2000 Nat. Genet. 25, 255–257), others found levels of heteroplasmy ranging from 6 to 40% (Takeda et al. Mol. Reprod. Dev. 64, 429–437). Since it remains unclear whether mtDNA segregation is neutral or selective, the purpose of this study was to analyze the transmission of the mtDNA from donor somatic cells in fetal and adult clones using a particular mtDNA marker (mtDNA Bos taurus with one mutation in the D-loop of 40 base pairs plus than the wild type). Fibroblasts from a fetus of 60 days were used as donor cells. The fetus was produced by artificial insemination of a Holstein (Bos taurus) heifer carrying an mtDNA mutation with semen from a Zebu (Bos indicus) bull. Oocytes derived from slaughterhouse ovaries of Holstein cows carrying wild-type mtDNA were used as recipient cells. The presence of the mutated mtDNA from the donor cell (heteroplasmy) was analyzed in a male cloned fetus of 60 days and in three adult male clones at 18 months of age. Heteroplasmy was detected in 7 tissues in the foetus: muscle, skin, stomach, testicle, thymus, tongue, and umbilical cord. Three tissues were analyzed from the adult clones: semen, skin, and white blood cells. Heteroplasmy was detected in all the tissues by nested PCR amplification of the D-loop and analyzed by ANOVA and Tukey-Kramer multiple comparison test. The mean (%) of the mutated mtDNA of the donor cell in the seven tissues of the60-day-old fetus was 1.14 ± 0.34 (SEM). There was no differences in the means of heteroplasmy (%) between the tissues of the fetus (P > 0.05). The mean level of heteroplasmy in the three adult clones analyzed (clones A, B, and C) was 1.41 ± 0.18 (SEM). Analysis of heteroplasmy between the tissues of each clone showed no differences (P > 0.05) with the exception of clone B, where semen was different (P < 0.05) from white blood cells. There were significant differences (P < 0.05) between some clones (taking together all the results of all tissues of each clone). The heteroplasmy in clone B (%) (2.59 ± 0.18 SEM) was different (P < 0.05) from that of both clone A (1.04 ± 0.18) and clone C (1.46 ± 0.18). There was no difference between the heteroplasmy (%) of clone A and that of clone C (P > 0.05). These results show that the tissues of the fetus and the adult clones were heteroplasmic at similar levels, suggesting neutral segregation of the donor cell mtDNA during development and tissue differentiation.

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Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243 A > G mtDNA mutation

Nature Communications ◽

10.1038/s41467-021-26746-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chih-Yao Chung ◽

Kritarth Singh ◽

Vassilios N. Kotiadis ◽

Gabriel E. Valdebenito ◽

Jee Hwan Ahn ◽

...

Keyword(s):

Treatment Options ◽

Disease Phenotype ◽

Wild Type ◽

Mtdna Mutation ◽

Potential Therapeutic Target ◽

Constitutive Activation ◽

Mtdna Mutations ◽

Prevalent Disease ◽

Mtorc1 Pathway ◽

Clinical Conditions

AbstractMutations of the mitochondrial genome (mtDNA) cause a range of profoundly debilitating clinical conditions for which treatment options are very limited. Most mtDNA diseases show heteroplasmy – tissues express both wild-type and mutant mtDNA. While the level of heteroplasmy broadly correlates with disease severity, the relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are poorly understood. We have carried out extensive bioenergetic, metabolomic and RNAseq studies on heteroplasmic patient-derived cells carrying the most prevalent disease related mtDNA mutation, the m.3243 A > G. These studies reveal that the mutation promotes changes in metabolites which are associated with the upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 reduced mtDNA mutant load and partially rescued cellular bioenergetic function. The PI3K-Akt-mTORC1 axis thus represents a potential therapeutic target that may benefit people suffering from the consequences of the m.3243 A > G mutation.

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