A p.R369G POLG2 mutation associated with adPEO and multiple mtDNA deletions causes decreased affinity between polymerase γ subunits

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.     

Inherited Mendelian defects of nuclear-mitochondrial communication affecting the stability of mitochondrial DNA

The presence of mtDNA abnormalities inherited as Mendelian traits indicates the existence of mutations in nuclear genes affecting the integrity of the mitochondrial genome. Two groups of nucleus-driven abnormalities have been described: qualitative alterations of mtDNA, i.e. multiple large-scale deletions of mtDNA, and quantitative decrease of the mtDNA copy number, i.e. tissue-specific depletion of mtDNA. Autosomal dominant or recessive (adPEO), progressive ophthalmoplegia and autosomal-recessive mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), are three neurodegenerative disorders associated with the coexistence of wild-type mtDNA with several deletion-containing mtDNA species. Heterozygous mutations of the genes encoding the muscle-heart isoform of the adenosine diphosphate/adenosine triphosphate mitochondrial translocator (ANT1), the main subunit of polymerase gamma (POLG1), and of the putative mtDNA helicase (Twinkle) have been found in adPEO families linked to three different loci, on chromosomes 4q34-35, 10q24, and 15q25, respectively. Mutations in the gene encoding thymidine phosphorylase have been identified in several MNGIE patients. Severe, tissue-specific depletion of mtDNA is the molecular hallmark of rapidly progressive hepatopathies or myopathies of infancy and childhood. Two genes, deoxyguanosine kinase and thymidine kinase type 2, both involved in the mitochondrion-specific salvage pathways of deoxynucleotide pools, have been associated with depletion syndromes in selected families.     

Defects in mitochondrial DNA replication and human disease

Mitochondrial DNA (mtDNA) is replicated by the DNA polymerase g in concert with accessory proteins such as the mtDNA helicase, single stranded DNA binding protein, topoisomerase, and initiating factors. Nucleotide precursors for mtDNA replication arise from the mitochondrial salvage pathway originating from transport of nucleosides, or alternatively from cytoplasmic reduction of ribonucleotides. Defects in mtDNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mtDNA deletions, point mutations, or depletion which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mtDNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders, such as progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). This review focuses on our current knowledge of genetic defects of mtDNA replication (POLG, POLG2, C10orf2) and nucleotide metabolism (TYMP, TK2, DGOUK, and RRM2B) that cause instability of mtDNA and mitochondrial disease.     

Mutant POLG2 disrupts DNA polymerase gamma subunits and causes progressive external ophthalmoplegia

DNA polymerase gamma (pol gamma ) is required to maintain the genetic integrity of the 16,569-bp human mitochondrial genome (mtDNA). Mutation of the nuclear gene for the catalytic subunit of pol gamma (POLG) has been linked to a wide range of mitochondrial diseases involving mutation, deletion, and depletion of mtDNA. We describe a heterozygous dominant mutation (c.1352G-->A/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol gamma , that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)-deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype.     

Defects in mitochondrial DNA replication and human disease

Mitochondrial DNA (mtDNA) is replicated by the DNA polymerase g in concert with accessory proteins such as the mtDNA helicase, single stranded DNA binding protein, topoisomerase, and initiating factors. Nucleotide precursors for mtDNA replication arise from the mitochondrial salvage pathway originating from transport of nucleosides, or alternatively from cytoplasmic reduction of ribonucleotides. Defects in mtDNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mtDNA deletions, point mutations, or depletion which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mtDNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders, such as progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). This review focuses on our current knowledge of genetic defects of mtDNA replication (POLG, POLG2, C10orf2) and nucleotide metabolism (TYMP, TK2, DGOUK, and RRM2B) that cause instability of mtDNA and mitochondrial disease.     

Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA

Autosomal dominant and/or recessive progressive external ophthalmoplegia (ad/arPEO) is associated with mtDNA mutagenesis. It can be caused by mutations in three nuclear genes, encoding the adenine nucleotide translocator 1, the mitochondrial helicase Twinkle or DNA polymerase γ (POLG). How mutations in these genes result in progressive accumulation of multiple mtDNA deletions in post- mitotic tissues is still unclear. A recent hypothesis suggested that mtDNA replication infidelity could promote slipped mispairing, thereby stimulating deletion formation. This hypothesis predicts that mtDNA of ad/arPEO patients will contain frequent mutations throughout; in fact, our analysis of muscle from ad/arPEO patients revealed an age-dependent, enhanced accumulation of point mutations in addition to deletions, but specifically in the mtDNA control region. Both deleted and non-deleted mtDNA molecules showed increased point mutation levels, as did mtDNAs of patients with a single mtDNA deletion, suggesting that point mutations do not cause multiple deletions. Deletion breakpoint analysis showed frequent breakpoints around homopolymeric runs, which could be a signature of replication stalling. Therefore, we propose replication stalling as the principal cause of deletion formation.     

Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase,POLG1)

These tables list both published and a number of unpublished mutations in genes associated with early onset defects in mitochondrial DNA (mtDNA) maintenance including C10orf2, SUCLG1, SUCLA2, TYMP, RRM2B, MPV17, DGUOK and TK2. The list should not be taken as evidence that any particular mutation is pathogenic. We have included genes known to cause mtDNA depletion, excluding POLG1, because of the existing database (http://tools.niehs.nih.gov/polg/). We have also excluded mutations in C10orf2 associated with dominant adult onset disorders.     

An autosomal locus predisposing to multiple deletions of mtDNA on chromosome 3p.

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a disorder characterized by ptosis, progressive weakness of the external eye muscles, and general muscle weakness. The patients have multiple deletions of mtDNA on Southern blots or in PCR analysis of muscle DNA and a mild deficiency of one or more respiratory-chain enzymes carrying mtDNA-encoded subunits. The pattern of inheritance indicates a nuclear gene defect predisposing to secondary mtDNA deletions. Recently, in one Finnish family, we assigned an adPEO locus to chromosome 10q 23.3-24.3 but also excluded linkage to this same locus in two Italian adPEO families with a phenotype closely resembling the Finnish one. We applied a random mapping approach to informative non-10q-linked Italian families to assign the second locus for adPEO and found strong evidence for linkage on chromosome 3p 14.1-21.2 in three Italian families, with a maximum two-point lod score of 4.62 at a recombination fraction of .0. However, in three additional families, linkage to the same chromosomal region was clearly absent, indicating further genetic complexity of the adPEO trait.     

Modeling pathogenic mutations of human twinkle in Drosophila suggests an apoptosis role in response to mitochondrial defects

The human gene C10orf2 encodes the mitochondrial replicative DNA helicase Twinkle, mutations of which are responsible for a significant fraction of cases of autosomal dominant progressive external ophthalmoplegia (adPEO), a human mitochondrial disease caused by defects in intergenomic communication. We report the analysis of orthologous mutations in the Drosophila melanogaster mitochondrial DNA (mtDNA) helicase gene, d-mtDNA helicase. Increased expression of wild type d-mtDNA helicase using the UAS-GAL4 system leads to an increase in mtDNA copy number throughout adult life without any noteworthy phenotype, whereas overexpression of d-mtDNA helicase containing the K388A mutation in the helicase active site results in a severe depletion of mtDNA and a lethal phenotype. Overexpression of two d-mtDNA helicase variants equivalent to two human adPEO mutations shows differential effects. The A442P mutation exhibits a dominant negative effect similar to that of the active site mutant. In contrast, overexpression of d-mtDNA helicase containing the W441C mutation results in a slight decrease in mtDNA copy number during the third instar larval stage, and a moderate decrease in life span in the adult population. Overexpression of d-mtDNA helicase containing either the K388A or A442P mutations causes a mitochondrial oxidative phosphorylation (OXPHOS) defect that significantly reduces cell proliferation. The mitochondrial impairment caused by these mutations promotes apoptosis, arguing that mitochondria regulate programmed cell death in Drosophila. Our study of d-mtDNA helicase overexpression provides a tractable Drosophila model for understanding the cellular and molecular effects of human adPEO mutations.     

POLG mutations cause decreased mitochondrial DNA repopulation rates following induced depletion in human fibroblasts

Disorders of mitochondrial DNA (mtDNA) maintenance have emerged as an important cause of human genetic disease, but demonstrating the functional consequences of de novo mutations remains a major challenge. We studied the rate of depletion and repopulation of mtDNA in human fibroblasts exposed to ethidium bromide in patients with heterozygous POLG mutations, POLG2 and TK2 mutations. Ethidium bromide induced mtDNA depletion occurred at the same rate in human fibroblasts from patients and healthy controls. By contrast, the restoration of mtDNA levels was markedly delayed in fibroblasts from patients with compound heterozygous POLG mutations. Specific POLG2 and TK2 mutations did not delay mtDNA repopulation rates. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation within intact cells, and provide a potential method of demonstrating the functional consequences of putative pathogenic alleles causing a defect of mtDNA synthesis.     

POLG mutations cause decreased mitochondrial DNA repopulation rates following induced depletion in human fibroblasts

Disorders of mitochondrial DNA (mtDNA) maintenance have emerged as an important cause of human genetic disease, but demonstrating the functional consequences of de novo mutations remains a major challenge. We studied the rate of depletion and repopulation of mtDNA in human fibroblasts exposed to ethidium bromide in patients with heterozygous POLG mutations, POLG2 and TK2 mutations. Ethidium bromide induced mtDNA depletion occurred at the same rate in human fibroblasts from patients and healthy controls. By contrast, the restoration of mtDNA levels was markedly delayed in fibroblasts from patients with compound heterozygous POLG mutations. Specific POLG2 and TK2 mutations did not delay mtDNA repopulation rates. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation within intact cells, and provide a potential method of demonstrating the functional consequences of putative pathogenic alleles causing a defect of mtDNA synthesis.     

Structure–function defects of the twinkle amino-terminal region in progressive external ophthalmoplegia

TWINKLE is a DNA helicase needed for mitochondrial DNA replication. In lower eukaryotes the protein also harbors a primase activity, which is lost from TWINKLE encoded by mammalian cells. Mutations in TWINKLE underlie autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with multiple deletions in the mtDNA. Four different adPEO-causing mutations (W315L, K319T, R334Q, and P335L) are located in the N-terminal domain of TWINKLE. The mutations cause a dramatic decrease in ATPase activity, which is partially overcome in the presence of single-stranded DNA. The mutated proteins have defects in DNA helicase activity and cannot support normal levels of DNA replication. To explain the phenotypes, we use a molecular model of TWINKLE based on sequence similarities with the phage T7 gene 4 protein. The four adPEO-causing mutations are located in a region required to bind single-stranded DNA. These mutations may therefore impair an essential element of the catalytic cycle in hexameric helicases, i.e. the interplay between single-stranded DNA binding and ATP hydrolysis.     

Mitochondriale Erkrankungen im Erwachsenenalter

The most frequent manifestation of mitochondrial disease in adults is chronic progressive external ophthalmoplegia (CPEO) that can present with variable multisystemic involvement. Molecular genetically single mtDNA deletions are identified in more than half of the patients associated with mainly sporadic CPEO. There are also autosomal dominant and recessive forms of CPEO due to mutations in nuclear genes that are important for mtDNA replication resulting in multiple mtDNA deletions. Other common multisystemic disorders are MELAS syndrome and MERRF syndrome due to maternally inherited mtDNA point mutations. Leber??s hereditary optic neuropathy is a frequent mitochondrial disorder without multisystemic involvement, which is also due to mtDNA point mutations. In addition to classical mitochondrial disorders there are patients with mitochondrial disease showing non-characteristic sometimes monosymptomic phenotypes (e.g. myopathy or epilepsy) that are difficult to recognize.     

Mitochondrial DNA Polymerase POLG1 Disease Mutations and Germline Variants Promote Tumorigenic Properties

Germline mutations in mitochondrial DNA polymerase gamma (POLG1) induce mitochondrial DNA (mtDNA) mutations, depletion, and decrease oxidative phosphorylation. Earlier, we identified somatic mutations in POLG1 and the contribution of these mutations in human cancer. However, a role for germline variations in POLG1 in human cancers is unknown. In this study, we examined a role for disease associated germline variants of POLG1, POLG1 gene expression, copy number variation and regulation in human cancers. We analyzed the mutations, expression and copy number variation in POLG1 in several cancer databases and validated the analyses in primary breast tumors and breast cancer cell lines. We discovered 5-aza-2''-deoxycytidine led epigenetic regulation of POLG1, mtDNA-encoded genes and increased mitochondrial respiration. We conducted comprehensive race based bioinformatics analyses of POLG1 gene in more than 33,000 European-Americans and 5,000 African-Americans. We identified a mitochondrial disease causing missense variation in polymerase domain of POLG1 protein at amino acid 1143 (E1143G) to be 25 times more prevalent in European-Americans (allele frequency 0.03777) when compared to African-American (allele frequency 0.00151) population. We identified T251I and P587L missense variations in exonuclease and linker region of POLG1 also to be more prevalent in European-Americans. Expression of these variants increased glucose consumption, decreased ATP production and increased matrigel invasion. Interestingly, conditional expression of these variants revealed that matrigel invasion properties conferred by these germline variants were reversible suggesting a role of epigenetic regulators. Indeed, we identified a set of miRNA whose expression was reversible after variant expression was turned off. Together, our studies demonstrate altered genetic and epigenetic regulation of POLG1 in human cancers and suggest a role for POLG1 germline variants in promoting tumorigenic properties.     

Mitochondrial medicine

After reviewing the history of mitochondrial diseases, I follow a genetic classification to discuss new developments and old conundrums. In the field of mitochondrial DNA (mtDNA) mutations, I argue that we are not yet scraping the bottom of the barrel because: (i) new mtDNA mutations are still being discovered, especially in protein-coding genes; (ii) the pathogenicity of homoplasmic mutations is being revisited; (iii) some genetic dogmas are chipped but not broken; (iv) mtDNA haplotypes are gaining interest in human pathology; (v) pathogenesis is still largely enigmatic. In the field of nuclear DNA (nDNA) mutations, there has been good progress in our understanding of disorders due to faulty intergenomic communication. Of the genes responsible for multiple deletions and depletion of mtDNA, mutations in POLG have been associated with a great variety of clinical phenotypes in humans and to precocious aging in mice. Novel pathogenetic mechanisms include alterations in the lipid milieu of the inner mitochondrial membrane and mutations in genes controlling mitochondrial motility, fission, and fusion.     

Exome sequencing identifies a novel missense variant in <Emphasis Type="Italic">RRM2B</Emphasis> associated with autosomal recessive progressive external ophthalmoplegia

Background  

Whole-exome sequencing using next-generation technologies has been previously demonstrated to be able to detect rare disease-causing variants. Progressive external ophthalmoplegia (PEO) is an inherited mitochondrial disease that follows either autosomal dominant or recessive forms of inheritance (adPEO or arPEO). AdPEO is a genetically heterogeneous disease and several genes, including POLG1 and C10orf2/Twinkle, have been identified as responsible genes. On the other hand, POLG1 was the only established gene causing arPEO with mitochondrial DNA deletions. We previously reported a case of PEO with unidentified genetic etiology. The patient was born of a first-cousin marriage. Therefore, the recessive form of inheritance was suspected.     

Reconstitution of a minimal mtDNA replisome in vitro

We here reconstitute a minimal mammalian mitochondrial DNA (mtDNA) replisome in vitro. The mtDNA polymerase (POLgamma) cannot use double-stranded DNA (dsDNA) as template for DNA synthesis. Similarly, the TWINKLE DNA helicase is unable to unwind longer stretches of dsDNA. In combination, POLgamma and TWINKLE form a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The addition of the mitochondrial ssDNA-binding protein stimulates the reaction further, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. The observed DNA synthesis rate is 180 base pairs (bp)/min, corresponding closely to the previously calculated value of 270 bp/min for in vivo DNA replication. Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. Furthermore, mutations in TWINKLE and POLgamma cause autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with deletions in mitochondrial DNA. The functional interactions between TWINKLE and POLgamma thus explain why mutations in these two proteins cause an identical syndrome.     

Compound mutations of PEO1 and TYMP in a progressive external ophthalmoplegia patient with incomplete mitochondrial neurogastrointestinal encephalomyopathy phenotype

The Mendelian inherited progressive external ophthalmoplegia (PEO) and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) are genetically heterogeneous mitochondrial diseases caused by nuclear-mitochondrial intergenomic defects. The PEO1 and TYMP nuclear genes are closely related in the machinery of the mitochondrial DNA (mtDNA) replication. Mutations in PEO1 and TYMP genes usually cause autosomal dominant PEO, and autosomal recessive MNGIE. We identified a PEO family of Korean origin with additional phenotype of incomplete MNGIE symptom (Family ID: MT16). The entire mitochondrial genome and all coding exons of PEO1, TYMP, ANT1, POLG1, POLG2, DGUOK, and TK2 nuclear genes were sequenced. Clinical information was obtained through history taking, physical examinations, clinical observations, and electrophysiological investigations. Muscle biopsy of left biceps brachii and shoulder magnetic resonance imaging (MRI) were undertaken. We found two heterozygous mutations, Arg374Gln in PEO1 and Glu106Gln in TYMP from the proband who showed complex phenotypes of a typical PEO and late-onset incomplete MNGIE. The PEO1 Arg374Gln has been reported in several PEO patients, but TYMP Glu106Gln has not been reported. Neither large deletion nor causative point mutations were observed in the mtDNA. We suggest that the heterozygous TYMP mutation might affect complex phenotypes as a secondary genetic cause in the co-presence of PEO1 mutation.     

Defects of intergenomic communication: autosomal disorders that cause multiple deletions and depletion of mitochondrial DNA

Depletion and multiple deletions of mitochondrial DNA (mtDNA) have been associated with a growing number of autosomal diseases that have been classified as defects of intergenomic communication. MNGIE, an autosomal recessive disorder associated with mtDNA alterations is due to mutations in thymidine phosphorylase that may cause imbalance of the mitochondrial nucleotide pool. Subsequently, mutations in the mitochondrial proteins adenine nucleotide translocator 1, Twinkle, and polymerase gamma have been found to cause autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA. Uncovering the molecular bases of intergenomic communication defects will enhance our understanding of the mechanisms responsible for maintaining mtDNA integrity.