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William C Copeland - One of the best experts on this subject based on the ideXlab platform.
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Consequences of compromised mitochondrial genome integrity.
DNA repair, 2020Co-Authors: Margaret A. Gustafson, Eric D. Sullivan, William C CopelandAbstract:Maintenance and replication of the mitochondrial genome (mtDNA) is essential to mitochondrial function and eukaryotic energy production through the electron transport chain. mtDNA is replicated by a core set of proteins: Pol γ, Twinkle, and the single-stranded DNA binding protein. Fewer pathways exist for repair of mtDNA than nuclear DNA, and unrepaired damage to mtDNA may accumulate and lead to dysfunctional mitochondria. The mitochondrial genome is susceptible to damage by both endogenous and exogenous sources. Missense mutations to the nuclear genes encoding the core mtDNA replisome (POLG, POLG2, TWNK, and SSBP1) cause changes to the biochemical functions of their protein products. These protein variants can damage mtDNA and perturb oxidative phosphorylation. Ultimately, these mutations cause a diverse set of diseases that can affect virtually every system in the body. Here, we briefly review the mechanisms of mtDNA damage and the clinical consequences of disease variants of the core mtDNA replisome.
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POLG-related disorders and their neurological manifestations
Nature Reviews Neurology, 2019Co-Authors: Shamima Rahman, William C CopelandAbstract:POLG encodes the catalytic subunit of DNA polymerase γ, the enzyme responsible for replicating the mitochondrial DNA (mtDNA). Mutations in POLG are associated with a clinical continuum of heterogeneous syndromes, ranging from infantile-onset epilepsies and liver failure to late-onset ophthalmoplegia and muscle weakness. POLG mutations are a frequent cause of mitochondrial disease, particularly mitochondrial epilepsy, polyneuropathy, ataxia and progressive external ophthalmoplegia. POLG mutations can lead to depletion of the mtDNA and/or accumulation of multiple mtDNA deletions. To a limited extent, clinical phenotypes correlate with the mtDNA phenotype (depletion or deletions). No effective disease-modifying therapies are currently available for POLG -related disease, and symptomatic therapies are the mainstay of treatment. The POLG gene encodes the mitochondrial DNA polymerase that is responsible for replication of the mitochondrial genome. Mutations in POLG can cause early childhood mitochondrial DNA (mtDNA) depletion syndromes or later-onset syndromes arising from mtDNA deletions. POLG mutations are the most common cause of inherited mitochondrial disorders, with as many as 2% of the population carrying these mutations. POLG -related disorders comprise a continuum of overlapping phenotypes with onset from infancy to late adulthood. The six leading disorders caused by POLG mutations are Alpers–Huttenlocher syndrome, which is one of the most severe phenotypes; childhood myocerebrohepatopathy spectrum, which presents within the first 3 years of life; myoclonic epilepsy myopathy sensory ataxia; ataxia neuropathy spectrum; autosomal recessive progressive external ophthalmoplegia; and autosomal dominant progressive external ophthalmoplegia. This Review describes the clinical features, pathophysiology, natural history and treatment of POLG -related disorders, focusing particularly on the neurological manifestations of these conditions. Pathogenic variants in POLG , which encodes the catalytic subunit of DNA polymerase γ, cause a spectrum of overlapping disease phenotypes. This Review describes the clinical features, pathophysiology, natural history and treatment of POLG -related disorders, focusing particularly on the neurological manifestations.
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characterization of the human homozygous r182w POLG2 mutation in mitochondrial dna depletion syndrome
PLOS ONE, 2018Co-Authors: Kirsten E Hoff, Michio Hirano, Karen L Debalsi, Maria J Sanchezquintero, Matthew J Longley, Ali Naini, William C CopelandAbstract:Mutations in mitochondrial DNA (mtDNA) have been linked to a variety of metabolic, neurological and muscular diseases which can present at any time throughout life. MtDNA is replicated by DNA polymerase gamma (Pol γ), twinkle helicase and mitochondrial single-stranded binding protein (mtSSB). The Pol γ holoenzyme is a heterotrimer consisting of the p140 catalytic subunit and a p55 homodimeric accessory subunit encoded by the nuclear genes POLG and POLG2, respectively. The accessory subunits enhance DNA binding and promote processive DNA synthesis of the holoenzyme. Mutations in either POLG or POLG2 are linked to disease and adversely affect maintenance of the mitochondrial genome, resulting in depletion, deletions and/or point mutations in mtDNA. A homozygous mutation located at Chr17: 62492543G>A in POLG2, resulting in R182W substitution in p55, was previously identified to cause mtDNA depletion and fatal hepatic liver failure. Here we characterize this homozygous R182W p55 mutation using in vivo cultured cell models and in vitro biochemical assessments. Compared to control fibroblasts, homozygous R182W p55 primary dermal fibroblasts exhibit a two-fold slower doubling time, reduced mtDNA copy number and reduced levels of POLG and POLG2 transcripts correlating with the reported disease state. Expression of R182W p55 in HEK293 cells impairs oxidative-phosphorylation. Biochemically, R182W p55 displays DNA binding and association with p140 similar to WT p55. R182W p55 mimics the ability of WT p55 to stimulate primer extension, support steady-state nucleotide incorporation, and suppress the exonuclease function of Pol γ in vitro. However, R182W p55 has severe defects in protein stability as determined by differential scanning fluorimetry and in stimulating function as determined by thermal inactivation. These data demonstrate that the Chr17: 62492543G>A mutation in POLG2, R182W p55, severely impairs stability of the accessory subunit and is the likely cause of the disease phenotype.
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Synergistic Effects of the in cis T251I and P587L Mitochondrial DNA Polymerase γ Disease Mutations.
The Journal of biological chemistry, 2017Co-Authors: Karen L Debalsi, Kirsten E Hoff, Matthew J Longley, William C CopelandAbstract:Human mitochondrial DNA (mtDNA) polymerase γ (Pol γ) is the only polymerase known to replicate the mitochondrial genome. The Pol γ holoenzyme consists of the p140 catalytic subunit (POLG) and the p55 homodimeric accessory subunit (POLG2), which enhances binding of Pol γ to DNA and promotes processivity of the holoenzyme. Mutations within POLG impede maintenance of mtDNA and cause mitochondrial diseases. Two common POLG mutations usually found in cis in patients primarily with progressive external ophthalmoplegia generate T251I and P587L amino acid substitutions. To determine whether T251I or P587L is the primary pathogenic allele or whether both substitutions are required to cause disease, we overproduced and purified WT, T251I, P587L, and T251I + P587L double variant forms of recombinant Pol γ. Biochemical characterization of these variants revealed impaired DNA binding affinity, reduced thermostability, diminished exonuclease activity, defective catalytic activity, and compromised DNA processivity, even in the presence of the p55 accessory subunit. However, physical association with p55 was unperturbed, suggesting intersubunit affinities similar to WT. Notably, although the single mutants were similarly impaired, a dramatic synergistic effect was found for the double mutant across all parameters. In conclusion, our analyses suggest that individually both T251I and P587L substitutions functionally impair Pol γ, with greater pathogenicity predicted for the single P587L variant. Combining T251I and P587L induces extreme thermal lability and leads to synergistic nucleotide and DNA binding defects, which severely impair catalytic activity and correlate with presentation of disease in patients.
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Human mitochondrial DNA replication machinery and disease.
Current opinion in genetics & development, 2016Co-Authors: Matthew J. Young, William C CopelandAbstract:The human mitochondrial genome is replicated by DNA polymerase γ in concert with key components of the mitochondrial DNA (mtDNA) replication machinery. Defects in mtDNA replication or nucleotide metabolism cause deletions, point mutations, or depletion of mtDNA. The resulting loss of cellular respiration ultimately induces mitochondrial genetic diseases, including mtDNA depletion syndromes (MDS) such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. Here we review the current literature regarding human mtDNA replication and heritable disorders caused by genetic changes of the POLG, POLG2, Twinkle, RNASEH1, DNA2, and MGME1 genes.
Andreina Bordoni - One of the best experts on this subject based on the ideXlab platform.
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mutations in dna2 link progressive myopathy to mitochondrial dna instability
American Journal of Human Genetics, 2013Co-Authors: Dario Ronchi, Andreina Bordoni, Alessio Di Fonzo, Weiqiang Lin, Changwei Liu, E Fassone, Serena Pagliarani, Mafalda Rizzuti, Li Zheng, Massimiliano FilostoAbstract:Syndromes associated with multiple mtDNA deletions are due to different molecular defects that can result in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia (PEO) to multisystemic disorders of variable severity. The autosomal-dominant form of PEO is genetically heterogeneous. Recently, causative mutations have been reported in several nuclear genes that encode proteins of the mtDNA replisome machinery (POLG, POLG2, and C10orf2) or that are involved in pathways for the synthesis of deoxyribonuclotides (ANT1 and RRM2B). Despite these findings, putative mutations remain unknown in half of the subjects with PEO. We report the identification, by exome sequencing, of mutations in DNA2 in adult-onset individuals with a form of mitochondrial myopathy featuring instability of muscle mtDNA. DNA2 encodes a helicase/nuclease family member that is most likely involved in mtDNA replication, as well as in the long-patch base-excision repair (LP-BER) pathway. In vitro biochemical analysis of purified mutant proteins revealed a severe impairment of nuclease, helicase, and ATPase activities. These results implicate human DNA2 and the LP-BER pathway in the pathogenesis of adult-onset disorders of mtDNA maintenance.
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POLG1 mutations and stroke like episodes: a distinct clinical entity rather than an atypical MELAS syndrome
BMC Neurology, 2013Co-Authors: Antonella Cheldi, Dario Ronchi, Andreina Bordoni, Bianca Bordo, Silvia Lanfranconi, Maria Grazia Bellotti, Stefania Corti, Valeria Lucchini, Monica Sciacco, M. MoggioAbstract:Background POLG1 mutations have been associated with MELAS-like phenotypes. However given several clinical differences it is unknown whether POLG1 mutations are possible causes of MELAS or give raise to a distinct clinical and genetic entity, named POLG1 -associated encephalopathy. Case presentation We describe a 74 years old man carrying POLG1 mutations presenting with strokes, myopathy and ragged red fibers with some atypical aspects for MELAS such as late onset, lack of cerebral calcification and presence of frontal and occipital MRI lesions better consistent with the POLG associated-encephalopathy spectrum. Conclusion The lack of available data hampers a definite diagnosis in our patient as well as makes it difficult to compare MELAS, which is a clearly defined clinical syndrome, with POLG1 -associated encephalopathy, which is so far a purely molecularly defined syndrome with a quite heterogeneous clinical picture. However, the present report contributes to expand the phenotypic spectrum of POLG1 mutations underlining the importance of searching POLG1 mutations in patients with mitochondrial signs and MELAS like phenotypes but negative for common mtDNA mutations.
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polg1 mutations and stroke like episodes a distinct clinical entity rather than an atypical melas syndrome
BMC Neurology, 2013Co-Authors: Antonella Cheldi, Dario Ronchi, Andreina Bordoni, Bianca Bordo, Silvia Lanfranconi, Maria Grazia Bellotti, Stefania Corti, Valeria Lucchini, Monica Sciacco, M. MoggioAbstract:Background POLG1 mutations have been associated with MELAS-like phenotypes. However given several clinical differences it is unknown whether POLG1 mutations are possible causes of MELAS or give raise to a distinct clinical and genetic entity, named POLG1-associated encephalopathy.
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Novel Twinkle (PEO1) gene mutations in mendelian progressive external ophthalmoplegia
Journal of Neurology, 2008Co-Authors: R. Virgilio, Dario Ronchi, Andreina Bordoni, G. M. Hadjigeorgiou, F. Saladino, M. Moggio, L. Adobbati, D. Kafetsouli, E. Tsironi, S. PrevitaliAbstract:Multiple deletions of mitochondrial DNA (mtDNA) are associated with different mitochondrial disorders inherited as autosomal dominant and recessive traits. Causative mutations have been found in five genes, mainly involved in mtDNA replication and stability. They include POLG1 , the gene encoding the catalytic subunit of mtDNA polymerase (polγ), POLG2 encoding its accessory subunit, ANT1 coding the adenine nucleotide translocator and PEO1 which codes for Twinkle, the mitochondrial helicase. Finally OPA1 missense mutations are involved in phenotypes presenting optic atrophy as a major feature. To define the relative contribution of POLG1, POLG2, ANT1 and PEO1 genes to the mtDNA multiple deletion syndromes, we analysed them in a cohort of 67 probands showing accumulation of multiple mtDNA deletions in muscle. The patients were predominantly affected with a mitochondrial myopathy with or without progressive external ophthalmoplegia (PEO). Genetic analysis revealed that 1) PEO1 has a major role in determining familial PEO, since it accounts for 26.8 % of familial cases, followed by ANT1 (14.6 %) and POLG1 (9.8 %); 2) no mutations in any of the known genes were found in 53.7 % of probands of this series. Six novel missense mutations contributing to the mutational load of PEO1 gene (p.R334P, p.W315S, p. S426N, p.W474S, p.F478I, p.E479K) were associated with an adult onset PEO phenotype.
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Novel Twinkle (PEO1) gene mutations in mendelian progressive external ophthalmoplegia.
Journal of neurology, 2008Co-Authors: R. Virgilio, Dario Ronchi, Andreina Bordoni, G. M. Hadjigeorgiou, F. Saladino, M. Moggio, L. Adobbati, D. Kafetsouli, E. Tsironi, Stefano C. PrevitaliAbstract:Multiple deletions of mitochondrial DNA (mtDNA) are associated with different mitochondrial disorders inherited as autosomal dominant and recessive traits. Causative mutations have been found in five genes, mainly involved in mtDNA replication and stability. They include POLG1, the gene encoding the catalytic subunit of mtDNA polymerase (polγ), POLG2 encoding its accessory subunit, ANT1 coding the adenine nucleotide translocator and PEO1 which codes for Twinkle, the mitochondrial helicase. Finally OPA1 missense mutations are involved in phenotypes presenting optic atrophy as a major feature.
Anu Suomalainen - One of the best experts on this subject based on the ideXlab platform.
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Diseases of DNA Polymerase Gamma
Diagnosis and Management of Mitochondrial Disorders, 2019Co-Authors: Omar Hikmat, Pirjo Isohanni, Anu Suomalainen, Laurence A. BindoffAbstract:Polymerase gamma (Polγ) is the DNA-dependent DNA polymerase responsible for replicating mitochondrial DNA. The enzyme is a trimer and comprises one catalytic subunit (POLG), which contains the polymerase activity together with proofreading exonuclease activity, and two accessory subunits (POLG2) that promote DNA binding and processivity. Replication of mtDNA requires several additional proteins: in vitro studies have shown that the minimal replication machinery consists of the helicase Twinkle, mitochondrial RNA polymerase and single-stranded binding protein [1], but others may be required in vivo [2]. Mutations in POLG are one of the most common causes of mitochondrial disease and responsible for a wide range of phenotypes. Mutations in POLG2 are rare. Disease caused by mutations in Twinkle gives a similar spectrum of disease to those caused by POLG and we will discuss them together where this is appropriate.
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A complex genomic locus drives mtDNA replicase POLG expression to its disease‐related nervous system regions
Wiley, 2018Co-Authors: Joni Nikkanen, Pirjo Isohanni, Juan Cruz Landoni, Diego Balboa, Maarja Haugas, Juha Partanen, Anders Paetau, Virginia Brilhante, Anu SuomalainenAbstract:Abstract DNA polymerase gamma (POLG), the mtDNA replicase, is a common cause of mitochondrial neurodegeneration. Why POLG defects especially cause central nervous system (CNS) diseases is unknown. We discovered a complex genomic regulatory locus for POLG, containing three functional CNS‐specific enhancers that drive expression specifically in oculomotor complex and sensory interneurons of the spinal cord, completely overlapping with the regions showing neuronal death in POLG patients. The regulatory locus also expresses two functional RNAs, LINC00925‐RNA and MIR9‐3, which are coexpressed with POLG. The MIR9‐3 targets include NR2E1, a transcription factor maintaining neural stem cells in undifferentiated state, and MTHFD2, the regulatory enzyme of mitochondrial folate cycle, linking POLG expression to stem cell differentiation and folate metabolism. Our evidence suggests that distant genomic non‐coding regions contribute to regulation of genes encoding mitochondrial proteins. Such genomic arrangement of POLG locus, driving expression to CNS regions affected in POLG patients, presents a potential mechanism for CNS‐specific manifestations in POLG disease
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a complex genomic locus drives mtdna replicase polg expression to its disease related nervous system regions
Embo Molecular Medicine, 2018Co-Authors: Joni Nikkanen, Pirjo Isohanni, Juan Cruz Landoni, Diego Balboa, Maarja Haugas, Juha Partanen, Anders Paetau, Virginia Brilhante, Anu SuomalainenAbstract:Abstract DNA polymerase gamma (POLG), the mtDNA replicase, is a common cause of mitochondrial neurodegeneration. Why POLG defects especially cause central nervous system (CNS) diseases is unknown. We discovered a complex genomic regulatory locus for POLG , containing three functional CNS‐specific enhancers that drive expression specifically in oculomotor complex and sensory interneurons of the spinal cord, completely overlapping with the regions showing neuronal death in POLG patients. The regulatory locus also expresses two functional RNAs, LINC00925‐ RNA and MIR9‐3, which are coexpressed with POLG . The MIR9‐3 targets include NR2E1, a transcription factor maintaining neural stem cells in undifferentiated state, and MTHFD2, the regulatory enzyme of mitochondrial folate cycle, linking POLG expression to stem cell differentiation and folate metabolism. Our evidence suggests that distant genomic non‐coding regions contribute to regulation of genes encoding mitochondrial proteins. Such genomic arrangement of POLG locus, driving expression to CNS regions affected in POLG patients, presents a potential mechanism for CNS‐specific manifestations in POLG disease.
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polg1 polyglutamine tract variants associated with parkinson s disease
Neuroscience Letters, 2010Co-Authors: J Eerola, Anu Suomalainen, Petri Luoma, Terhi Peuralinna, Sonja W Scholz, Coro Paisanruiz, Andrew B Singleton, Pentti J TienariAbstract:A possible role of allelic variation of the mitochondrial DNA polymerase gamma (POLG I) gene in Parkinson's disease (PD) has been suggested. First, POLG I missense mutations have been found in patients with familial parkinsonism and mitochondrial myopathy. Second, increased frequency of rare alleles of the POLG1 CAG-repeat (poly-Q) has been found in Finnish idiopathic apparently sporadic PD patients, but conflicting reports exist. The POLG1 poly-Q exhibits one major allele with 10 repeats (10Q, frequency >= 80%) and several less common alleles such as 11Q (frequency 6-9%), 6Q-9Q and 12Q-14Q (frequencies <4%). It is not known, whether the poly-Q variation modulates POLG1 function. Here we sequenced the poly-Q in 641 North American Caucasian PD patients and 292 controls. Caucasian literature controls were also used. Normal allele was defined either as 10/11Q or as 10Q according to the previous literature. The frequency of the non-10/11Q alleles in cases was not significantly different from the controls. Variant alleles defined as non-10Q were significantly increased in the PD patients compared to the North American controls (17.6% vs. 12.3%, p=0.004) as well as compared to the larger set of 897 controls (17.6% vs. 13.2%, p=0.0007). These results suggest that POLG1 poly-Q alleles other than the conserved 10Q allele may increase susceptibility to PD. This finding may be attributable to a beneficial function of the 10Q repeat protein or linkage disequilibrium between the 10Q allele and another variation within or close to POLG1. Other large case-control studies and analyses on functional differences of POLG1 poly-Q variants are warranted. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
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a heterozygous truncating mutation in rrm2b causes autosomal dominant progressive external ophthalmoplegia with multiple mtdna deletions
American Journal of Human Genetics, 2009Co-Authors: Henna Tyynismaa, Anu Suomalainen, Emil Ylikallio, Mehul Patel, Maria Judit Molnar, Ronald G HallerAbstract:Autosomal-dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disorder that is characterized by accumulation of multiple mitochondrial DNA (mtDNA) deletions in postmitotic tissues. The disorder is heterogeneous, with five known nuclear disease genes that encode the proteins ANT1, Twinkle, POLG, POLG2, and OPA1. Defects in these proteins affect mtDNA maintenance, probably leading to stalled replication forks, consequent mtDNA deletion formation, and progressive respiratory chain deficiency. Here we present a large adPEO family with multiple mtDNA deletions, whose disease was not explained by mutations in any of the known adPEO loci. We mapped the disease locus in this family to chromosome 8q22.1-q23.3. The critical linkage region contained the RRM2B gene, which encodes the small subunit of the ribonucleotide reductase p53R2, which has previously been shown to be essential for the maintenance of mtDNA copy number. Mutation screening of RRM2B revealed a heterozygous nonsense mutation in exon 9 (c.979C→T [p.R327X]) in all affected individuals that was absent in 380 control chromosomes. The same mutation was found to segregate in another adPEO family. The mutant mRNA escaped nonsense-mediated decay and resulted in a protein with truncation of 25 highly conserved C-terminal amino acids essential for the interaction with the ribonucleotide reductase subunit R1. We conclude that dominant-negative or gain-of-function mutations in RRM2B are a cause of multiple mtDNA deletions and adPEO.
Matthew J Longley - One of the best experts on this subject based on the ideXlab platform.
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characterization of the human homozygous r182w POLG2 mutation in mitochondrial dna depletion syndrome
PLOS ONE, 2018Co-Authors: Kirsten E Hoff, Michio Hirano, Karen L Debalsi, Maria J Sanchezquintero, Matthew J Longley, Ali Naini, William C CopelandAbstract:Mutations in mitochondrial DNA (mtDNA) have been linked to a variety of metabolic, neurological and muscular diseases which can present at any time throughout life. MtDNA is replicated by DNA polymerase gamma (Pol γ), twinkle helicase and mitochondrial single-stranded binding protein (mtSSB). The Pol γ holoenzyme is a heterotrimer consisting of the p140 catalytic subunit and a p55 homodimeric accessory subunit encoded by the nuclear genes POLG and POLG2, respectively. The accessory subunits enhance DNA binding and promote processive DNA synthesis of the holoenzyme. Mutations in either POLG or POLG2 are linked to disease and adversely affect maintenance of the mitochondrial genome, resulting in depletion, deletions and/or point mutations in mtDNA. A homozygous mutation located at Chr17: 62492543G>A in POLG2, resulting in R182W substitution in p55, was previously identified to cause mtDNA depletion and fatal hepatic liver failure. Here we characterize this homozygous R182W p55 mutation using in vivo cultured cell models and in vitro biochemical assessments. Compared to control fibroblasts, homozygous R182W p55 primary dermal fibroblasts exhibit a two-fold slower doubling time, reduced mtDNA copy number and reduced levels of POLG and POLG2 transcripts correlating with the reported disease state. Expression of R182W p55 in HEK293 cells impairs oxidative-phosphorylation. Biochemically, R182W p55 displays DNA binding and association with p140 similar to WT p55. R182W p55 mimics the ability of WT p55 to stimulate primer extension, support steady-state nucleotide incorporation, and suppress the exonuclease function of Pol γ in vitro. However, R182W p55 has severe defects in protein stability as determined by differential scanning fluorimetry and in stimulating function as determined by thermal inactivation. These data demonstrate that the Chr17: 62492543G>A mutation in POLG2, R182W p55, severely impairs stability of the accessory subunit and is the likely cause of the disease phenotype.
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Synergistic Effects of the in cis T251I and P587L Mitochondrial DNA Polymerase γ Disease Mutations.
The Journal of biological chemistry, 2017Co-Authors: Karen L Debalsi, Kirsten E Hoff, Matthew J Longley, William C CopelandAbstract:Human mitochondrial DNA (mtDNA) polymerase γ (Pol γ) is the only polymerase known to replicate the mitochondrial genome. The Pol γ holoenzyme consists of the p140 catalytic subunit (POLG) and the p55 homodimeric accessory subunit (POLG2), which enhances binding of Pol γ to DNA and promotes processivity of the holoenzyme. Mutations within POLG impede maintenance of mtDNA and cause mitochondrial diseases. Two common POLG mutations usually found in cis in patients primarily with progressive external ophthalmoplegia generate T251I and P587L amino acid substitutions. To determine whether T251I or P587L is the primary pathogenic allele or whether both substitutions are required to cause disease, we overproduced and purified WT, T251I, P587L, and T251I + P587L double variant forms of recombinant Pol γ. Biochemical characterization of these variants revealed impaired DNA binding affinity, reduced thermostability, diminished exonuclease activity, defective catalytic activity, and compromised DNA processivity, even in the presence of the p55 accessory subunit. However, physical association with p55 was unperturbed, suggesting intersubunit affinities similar to WT. Notably, although the single mutants were similarly impaired, a dramatic synergistic effect was found for the double mutant across all parameters. In conclusion, our analyses suggest that individually both T251I and P587L substitutions functionally impair Pol γ, with greater pathogenicity predicted for the single P587L variant. Combining T251I and P587L induces extreme thermal lability and leads to synergistic nucleotide and DNA binding defects, which severely impair catalytic activity and correlate with presentation of disease in patients.
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A p.R369G POLG2 mutation associated with adPEO and multiple mtDNA deletions causes decreased affinity between polymerase γ subunits
Mitochondrion, 2011Co-Authors: Kate Craig, William C Copeland, Matthew J. Young, Matthew J Longley, D M Turnbull, Emma L. Blakely, Robert W. TaylorAbstract:Human mitochondrial DNA (mtDNA) polymerase γ (pol γ) is the sole enzyme required to replicate and maintain the integrity of the mitochondrial genome. It comprises two subunits, a catalytic p140 subunit and a smaller p55 accessory subunit encoded by the POLG2 gene. We describe the molecular characterization of a potential dominant POLG2 mutation (p.R369G) in a patient with adPEO and multiple mtDNA deletions. Biochemical studies of the recombinant mutant p55 protein showed a reduced affinity to the pol γ p140 subunit, leading to impaired processivity of the holoenzyme complex but did not show sensitivity to N-ethylmalaimide (NEM) inhibition, inferring a novel disease mechanism.
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Biochemical analysis of human POLG2 variants associated with mitochondrial disease
Human molecular genetics, 2011Co-Authors: Matthew J. Young, Leejun C Wong, Matthew J Longley, Rajesh Kasiviswanathan, William C CopelandAbstract:Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, eight heterozygous mutations (six novel) in POLG2 were identified in one control and eight patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations, we biochemically characterized seven variants [c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)] that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic.
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Progressive External Ophthalmoplegia and Vision and Hearing Loss in a Patient With Mutations in POLG2 and OPA1
Archives of neurology, 2008Co-Authors: S. Ferraris, Matthew J Longley, Susanna Clark, Emanuela Garelli, Guido Davidzon, Steven A. Moore, Randy H. Kardon, Rachelle J. Bienstock, Michelangelo Mancuso, Purificacion Gutierrez RiosAbstract:Progressive external ophthalmoplegia (PEO), with ptosis and weakness of extraocular muscles, is a common manifestation of mitochondrial diseases and is often associated with multisystem involvement.1 Progressive external ophthalmoplegia is best classified based on genetic transmission into sporadic, maternally inherited, or mendelian traits. Sporadic cases include Kearns-Sayre syndrome and adult-onset PEO with myopathy. In Kearns-Sayre syndrome, PEO is the clinical hallmark, together with pigmentary retinopathy and onset before the age of 20 years.2 Additional symptoms include ataxia and defects of cardiac conduction. Adult-onset PEO is characterized by the exclusive or prevalent involvement of skeletal muscle, although other clinical features may include deafness and cataracts. These patients harbor single heteroplasmic deletions of mitochondrial DNA (mtDNA). Patients with maternal inheritance have point mutations in mtDNA, most commonly the A3243G transition, typically seen in mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes.1 The mendelian forms of PEO can be autosomal dominant or recessive, occur in about 15% of all cases, and are associated with multiple mtDNA deletions that are due to nuclear gene mutations.3,4 Autosomal dominant PEO has been associated with mutations in genes encoding adenine nucleotide translocator 1 (ANT1)5 and Twinkle, a putative mtDNA helicase (PEO1).6 Both protein products are involved in mtDNA maintenance. Mutations in the polymerase γ (polγ) gene (POLG), encoding the α subunit of mtDNA polγ, were identified in patients with either autosomal dominant PEO or autosomal recessive PEO,7,8 but approximately one-fourth of patients with PEO and multiple deletions do not have a positive family history of PEO. Of these sporadic cases, only 36% carry mutations in 1 of the 3 genes associated with familial autosomal recessive PEO or autosomal dominant PEO, and the remaining cases have no recognized molecular defect.9 Herein, we describe a sporadic adult patient with an unusual clinical history characterized by PEO, hearing loss, macrocytic anemia, hypogonadism, central vision loss, and mild ataxia. Skeletal muscle histochemistry revealed variation in fiber size because of the presence of atrophic fibers. There were no ragged-red fibers, but several fibers had no cytochrome-c oxidase activity. Because we detected multiple mtDNA deletions in muscle, we looked for a mutation in ANT1, PEO1, and POLG1 (the gene encoding the catalytic subunit of polγ) but found none. Genetic testing for Leber hereditary optic neuropathy excluded the typical mtDNA mutations. The recent report10 of a similar patient harboring a single mutation in the gene encoding the accessory subunit (p55) of polγ (POLG2) prompted us to sequence this gene: we identified a different heterozygous mutation, which, however, did not significantly impair enzyme function. We, therefore, sequenced OPA1, which encodes a dynamin-related GTPase, and identified a novel heterozygous missense mutation that better explains this patient’s features.
Dario Ronchi - One of the best experts on this subject based on the ideXlab platform.
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mutations in dna2 link progressive myopathy to mitochondrial dna instability
American Journal of Human Genetics, 2013Co-Authors: Dario Ronchi, Andreina Bordoni, Alessio Di Fonzo, Weiqiang Lin, Changwei Liu, E Fassone, Serena Pagliarani, Mafalda Rizzuti, Li Zheng, Massimiliano FilostoAbstract:Syndromes associated with multiple mtDNA deletions are due to different molecular defects that can result in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia (PEO) to multisystemic disorders of variable severity. The autosomal-dominant form of PEO is genetically heterogeneous. Recently, causative mutations have been reported in several nuclear genes that encode proteins of the mtDNA replisome machinery (POLG, POLG2, and C10orf2) or that are involved in pathways for the synthesis of deoxyribonuclotides (ANT1 and RRM2B). Despite these findings, putative mutations remain unknown in half of the subjects with PEO. We report the identification, by exome sequencing, of mutations in DNA2 in adult-onset individuals with a form of mitochondrial myopathy featuring instability of muscle mtDNA. DNA2 encodes a helicase/nuclease family member that is most likely involved in mtDNA replication, as well as in the long-patch base-excision repair (LP-BER) pathway. In vitro biochemical analysis of purified mutant proteins revealed a severe impairment of nuclease, helicase, and ATPase activities. These results implicate human DNA2 and the LP-BER pathway in the pathogenesis of adult-onset disorders of mtDNA maintenance.
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POLG1 mutations and stroke like episodes: a distinct clinical entity rather than an atypical MELAS syndrome
BMC Neurology, 2013Co-Authors: Antonella Cheldi, Dario Ronchi, Andreina Bordoni, Bianca Bordo, Silvia Lanfranconi, Maria Grazia Bellotti, Stefania Corti, Valeria Lucchini, Monica Sciacco, M. MoggioAbstract:Background POLG1 mutations have been associated with MELAS-like phenotypes. However given several clinical differences it is unknown whether POLG1 mutations are possible causes of MELAS or give raise to a distinct clinical and genetic entity, named POLG1 -associated encephalopathy. Case presentation We describe a 74 years old man carrying POLG1 mutations presenting with strokes, myopathy and ragged red fibers with some atypical aspects for MELAS such as late onset, lack of cerebral calcification and presence of frontal and occipital MRI lesions better consistent with the POLG associated-encephalopathy spectrum. Conclusion The lack of available data hampers a definite diagnosis in our patient as well as makes it difficult to compare MELAS, which is a clearly defined clinical syndrome, with POLG1 -associated encephalopathy, which is so far a purely molecularly defined syndrome with a quite heterogeneous clinical picture. However, the present report contributes to expand the phenotypic spectrum of POLG1 mutations underlining the importance of searching POLG1 mutations in patients with mitochondrial signs and MELAS like phenotypes but negative for common mtDNA mutations.
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polg1 mutations and stroke like episodes a distinct clinical entity rather than an atypical melas syndrome
BMC Neurology, 2013Co-Authors: Antonella Cheldi, Dario Ronchi, Andreina Bordoni, Bianca Bordo, Silvia Lanfranconi, Maria Grazia Bellotti, Stefania Corti, Valeria Lucchini, Monica Sciacco, M. MoggioAbstract:Background POLG1 mutations have been associated with MELAS-like phenotypes. However given several clinical differences it is unknown whether POLG1 mutations are possible causes of MELAS or give raise to a distinct clinical and genetic entity, named POLG1-associated encephalopathy.
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Novel Twinkle (PEO1) gene mutations in mendelian progressive external ophthalmoplegia
Journal of Neurology, 2008Co-Authors: R. Virgilio, Dario Ronchi, Andreina Bordoni, G. M. Hadjigeorgiou, F. Saladino, M. Moggio, L. Adobbati, D. Kafetsouli, E. Tsironi, S. PrevitaliAbstract:Multiple deletions of mitochondrial DNA (mtDNA) are associated with different mitochondrial disorders inherited as autosomal dominant and recessive traits. Causative mutations have been found in five genes, mainly involved in mtDNA replication and stability. They include POLG1 , the gene encoding the catalytic subunit of mtDNA polymerase (polγ), POLG2 encoding its accessory subunit, ANT1 coding the adenine nucleotide translocator and PEO1 which codes for Twinkle, the mitochondrial helicase. Finally OPA1 missense mutations are involved in phenotypes presenting optic atrophy as a major feature. To define the relative contribution of POLG1, POLG2, ANT1 and PEO1 genes to the mtDNA multiple deletion syndromes, we analysed them in a cohort of 67 probands showing accumulation of multiple mtDNA deletions in muscle. The patients were predominantly affected with a mitochondrial myopathy with or without progressive external ophthalmoplegia (PEO). Genetic analysis revealed that 1) PEO1 has a major role in determining familial PEO, since it accounts for 26.8 % of familial cases, followed by ANT1 (14.6 %) and POLG1 (9.8 %); 2) no mutations in any of the known genes were found in 53.7 % of probands of this series. Six novel missense mutations contributing to the mutational load of PEO1 gene (p.R334P, p.W315S, p. S426N, p.W474S, p.F478I, p.E479K) were associated with an adult onset PEO phenotype.
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Novel Twinkle (PEO1) gene mutations in mendelian progressive external ophthalmoplegia.
Journal of neurology, 2008Co-Authors: R. Virgilio, Dario Ronchi, Andreina Bordoni, G. M. Hadjigeorgiou, F. Saladino, M. Moggio, L. Adobbati, D. Kafetsouli, E. Tsironi, Stefano C. PrevitaliAbstract:Multiple deletions of mitochondrial DNA (mtDNA) are associated with different mitochondrial disorders inherited as autosomal dominant and recessive traits. Causative mutations have been found in five genes, mainly involved in mtDNA replication and stability. They include POLG1, the gene encoding the catalytic subunit of mtDNA polymerase (polγ), POLG2 encoding its accessory subunit, ANT1 coding the adenine nucleotide translocator and PEO1 which codes for Twinkle, the mitochondrial helicase. Finally OPA1 missense mutations are involved in phenotypes presenting optic atrophy as a major feature.