Yeast model analysis of novel polymerase gamma variants found in patients with autosomal recessive mitochondrial disease

Kaliszewska, Magdalena; Kruszewski, Jakub; Kierdaszuk, Biruta; Kostera‐Pruszczyk, Anna; Nojszewska, Monika; Łusakowska, Anna; Vizueta, Joel; Sabat, D.; Lutyk, Dorota; Lower, Michal; Piekutowska‐Abramczuk, Dorota; Kaniak-Golik, Aneta; Pronicka, Ewa; Kamińska, Anna; Bartnik, Ewa; Golik, Paweł; Tońska, Katarzyna

doi:10.1007/s00439-015-1578-x

Cited by 17 publications

(12 citation statements)

References 67 publications

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“…Because mutations in Polg resulting in deficient polymerase activity can result in mtDNA depletion ( 39 ), we hypothesized that qPCR analysis of mtDNA content from polg −/− zebrafish would reveal a gradual decrease in mtDNA until their death at 3 wpf. Equal amounts of purified DNA were used in order to make comparisons about mtDNA levels between animals and time points, and the difference in Cts for mtDNA and nuclear DNA were calculated to give the dCt.…”

Section: Resultsmentioning

confidence: 99%

Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage, despite rapid and sustained mitochondrial DNA depletion, altered energetics and growth

Rahn¹,

Bestman²,

Stackley³

et al. 2015

Nucleic Acids Res

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DNA polymerase gamma (POLG) is essential for replication and repair of mitochondrial DNA (mtDNA). Mutations in POLG cause mtDNA instability and a diverse range of poorly understood human diseases. Here, we created a unique Polg animal model, by modifying polg within the critical and highly conserved polymerase domain in zebrafish. polg+/− offspring were indistinguishable from WT siblings in multiple phenotypic and biochemical measures. However, polg−/− mutants developed severe mtDNA depletion by one week post-fertilization (wpf), developed slowly and had regenerative defects, yet surprisingly survived up to 4 wpf. An in vivo mtDNA polymerase activity assay utilizing ethidium bromide (EtBr) to deplete mtDNA, showed that polg+/− and WT zebrafish fully recover mtDNA content two weeks post-EtBr removal. EtBr further reduced already low levels of mtDNA in polg−/− animals, but mtDNA content did not recover following release from EtBr. Despite significantly decreased respiration that corresponded with tissue-specific levels of mtDNA, polg−/− animals had WT levels of ATP and no increase in lactate. This zebrafish model of mitochondrial disease now provides unique opportunities for studying mtDNA instability from multiple angles, as polg−/− mutants can survive to juvenile stage, rather than lose viability in embryogenesis as seen in Polg mutant mice.

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Section: Resultsmentioning

confidence: 99%

Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage, despite rapid and sustained mitochondrial DNA depletion, altered energetics and growth

Rahn¹,

Bestman²,

Stackley³

et al. 2015

Nucleic Acids Res

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show abstract

“…Comparable phenotypes (the harmful effects observed in yeast reproduce the severity of the phenotypes in humans), the possibility to study variants in heteroallelic states, and easy and fast analysis make budding yeast an excellent model for testing mutations in POLG. In POLG-associated diseases, yeast has enabled researchers to distinguish pathogenic mutations from other single-nucleotide polymorphisms; to show that some polymorphisms may act as phenotypic modifiers; and has demonstrated that certain mutations are not the only cause of a pathology, highlighting the need for further genetic analysis [75][76][77][78][79][80][81].…”

Section: Tnnt2mentioning

confidence: 99%

A Yeast-Based Model for Hereditary Motor and Sensory Neuropathies: A Simple System for Complex, Heterogeneous Diseases

Rzepnikowska

Kamińska

Kabzińska

et al. 2020

IJMS

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Charcot–Marie–Tooth (CMT) disease encompasses a group of rare disorders that are characterized by similar clinical manifestations and a high genetic heterogeneity. Such excessive diversity presents many problems. Firstly, it makes a proper genetic diagnosis much more difficult and, even when using the most advanced tools, does not guarantee that the cause of the disease will be revealed. Secondly, the molecular mechanisms underlying the observed symptoms are extremely diverse and are probably different for most of the disease subtypes. Finally, there is no possibility of finding one efficient cure for all, or even the majority of CMT diseases. Every subtype of CMT needs an individual approach backed up by its own research field. Thus, it is little surprise that our knowledge of CMT disease as a whole is selective and therapeutic approaches are limited. There is an urgent need to develop new CMT models to fill the gaps. In this review, we discuss the advantages and disadvantages of yeast as a model system in which to study CMT diseases. We show how this single-cell organism may be used to discriminate between pathogenic variants, to uncover the mechanism of pathogenesis, and to discover new therapies for CMT disease.

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“…We have grouped together on the server mutations that would result in a putatively non-functional (PNF) POLG. Several of them have been characterized biochemically, corroborating such a non-functional status, though some may retain limited DNA binding capability or other partial functionality [17], [34]. Patients with compound heterozygous PNF mutations that carry the three most commonly reported POLG mutations typically manifest with very severe, infantile onset conditions.…”

Section: Resultsmentioning

confidence: 99%

Pathogenicity in POLG syndromes: DNA polymerase gamma pathogenicity prediction server and database

2017

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DNA polymerase gamma (POLG) is the replicative polymerase responsible for maintaining mitochondrial DNA (mtDNA). Disorders related to its functionality are a major cause of mitochondrial disease. The clinical spectrum of POLG syndromes includes Alpers-Huttenlocher syndrome (AHS), childhood myocerebrohepatopathy spectrum (MCHS), myoclonic epilepsy myopathy sensory ataxia (MEMSA), the ataxia neuropathy spectrum (ANS) and progressive external ophthalmoplegia (PEO). We have collected all publicly available POLG-related patient data and analyzed it using our pathogenic clustering model to provide a new research and clinical tool in the form of an online server. The server evaluates the pathogenicity of both previously reported and novel mutations. There are currently 176 unique point mutations reported and found in mitochondrial patients in the gene encoding the catalytic subunit of POLG, POLG. The mutations are distributed nearly uniformly along the length of the primary amino acid sequence of the gene. Our analysis shows that most of the mutations are recessive, and that the reported dominant mutations cluster within the polymerase active site in the tertiary structure of the POLG enzyme. The POLG Pathogenicity Prediction Server (http://polg.bmb.msu.edu) is targeted at clinicians and scientists studying POLG disorders, and aims to provide the most current available information regarding the pathogenicity of POLG mutations.

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Yeast model analysis of novel polymerase gamma variants found in patients with autosomal recessive mitochondrial disease

Cited by 17 publications

References 67 publications

Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage, despite rapid and sustained mitochondrial DNA depletion, altered energetics and growth

Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage, despite rapid and sustained mitochondrial DNA depletion, altered energetics and growth

A Yeast-Based Model for Hereditary Motor and Sensory Neuropathies: A Simple System for Complex, Heterogeneous Diseases

Pathogenicity in POLG syndromes: DNA polymerase gamma pathogenicity prediction server and database

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