Depletion of mitochondrial DNA in fibroblast cultures from patients with POLG1 mutations is a consequence of catalytic mutations

We investigated clinical and cellular phenotypes of 24 children with mutations in the catalytic (alpha) subunit of the mitochondrial DNA (mtDNA) gamma polymerase (POLG1). Twenty-one had Alpers syndrome, the commonest severe POLG1 autosomal recessive phenotype, comprising hepatoencephalopathy and often mtDNA depletion. The cellular mtDNA content reflected the genotype more closely than did clinical features. Patients with tissue depletion of mtDNA all had at least one allele with either a missense mutation in a catalytic domain or a nonsense mutation. Four out of 12 patients exhibited a progressive, mosaic pattern of mtDNA depletion in cultured fibroblasts. All these patients had mutations in a catalytic domain in both POLG1 alleles, in either the polymerase or exonuclease domain or both. The tissue mtDNA content of patients who had two linker mutations was normal, and their phenotypes the mildest. Epilepsy and/or movement disorder were major features in all 21. Previous studies have implicated replication stalling as a mechanism for mtDNA depletion. The mosaic cellular depletion that we have demonstrated in cell cultures may be a manifestation of severe replication stalling. One patient with a severe cellular and clinical phenotype was a compound heterozygote with POLG1 mutations in the polymerase and exonuclease domain intrans. This suggests that POLG1 requires both polymerase and 3'-5' exonuclease activity in the same molecule. This is consistent with current functional models for eukaryotic DNA polymerases, which alternate between polymerizing and editing modes, as determined by competition between these two active sites for the 3' end of the DNA.     

Thymidine kinase 2 mutations in autosomal recessive progressive external ophthalmoplegia with multiple mitochondrial DNA deletions

Autosomal-inherited progressive external ophthalmoplegia (PEO) is an adult-onset disease characterized by the accumulation of multiple mitochondrial DNA (mtDNA) deletions in post-mitotic tissues. Mutations in six different genes have been described to cause the autosomal dominant form of the disease, but only mutations in the DNA polymerase gamma gene are known to cause autosomal recessive PEO (arPEO), leaving the genetic background of arPEO mostly unknown. Here we used whole-exome sequencing and identified compound heterozygous mutations, leading to two amino acid alterations R225W and a novel T230A in thymidine kinase 2 (TK2) in arPEO patients. TK2 is an enzyme of the mitochondrial nucleotide salvage pathway and its loss-of-function mutations have previously been shown to underlie the early-infantile myopathic form of mtDNA depletion syndrome (MDS). Our TK2 activity measurements of patient fibroblasts and mutant recombinant proteins show that the combination of the identified arPEO variants, R225W and T230A, leads to a significant reduction in TK2 activity, consistent with the late-onset phenotype, whereas homozygosity for R225W, previously associated with MDS, leads to near-total loss of activity. Our finding identifies a new genetic cause of arPEO with multiple mtDNA deletions. Furthermore, MDS and multiple mtDNA deletion disorders are manifestations of the same pathogenic pathways affecting mtDNA replication and repair, indicating that MDS-associated genes should be studied when searching for genetic background of PEO disorders.     

Genetic and chemical rescue of the Saccharomyces cerevisiae phenotype induced by mitochondrial DNA polymerase mutations associated with progressive external ophthalmoplegia in humans

The human POLG gene encodes the catalytic subunit of mitochondrial DNA polymerase gamma (pol gamma). Mutations in pol gamma are associated with a spectrum of disease phenotypes including autosomal dominant and recessive forms of progressive external ophthalmoplegia, spino-cerebellar ataxia and epilepsy, and Alpers-Huttenlocher hepatocerebral poliodystrophy. Multiple deletions, or depletion of mtDNA in affected tissues, are the molecular hallmarks of pol gamma mutations. To shed light on the pathogenic mechanisms leading to these phenotypes, we have introduced in MIP1, the yeast homologue of POLG, two mutations equivalent to the human Y955C and G268A mutations, which are associated with dominant and recessive PEO, respectively. Both mutations induced the generation of petite colonies, carrying either rearranged (rho-) or no (rho0) mtDNA. Mutations in genes that control the mitochondrial supply of deoxynucleotides (dNTP) affect the mtDNA integrity in both humans and yeast. To test whether the manipulation of the dNTP pool can modify the effects of pol gamma mutations in yeast, we have overexpressed a dNTP checkpoint enzyme, ribonucleotide reductase, RNR1, or deleted its inhibitor, SML1. In both mutant strains, the petite mutability was dramatically reduced. The same result was obtained by exposing the mutant strains to dihydrolipoic acid, an anti-oxidant agent. Therefore, an increase of the mitochondrial dNTP pool and/or a decrease of reactive oxygen species can prevent the mtDNA damage induced by pol gamma mutations in yeast and, possibly, in humans.     

Infantile-onset spinocerebellar ataxia and mitochondrial recessive ataxia syndrome are associated with neuronal complex I defect and mtDNA depletion

Infantile-onset spinocerebellar ataxia (IOSCA) is a severe neurodegenerative disorder caused by the recessive mutation in PEO1, leading to an Y508C change in the mitochondrial helicase Twinkle, in its helicase domain. However, no mitochondrial dysfunction has been found in this disease. We studied here the consequences of IOSCA for the central nervous system, as well as the in vitro performance of the IOSCA mutant protein. The results of the mtDNA analyses were compared to findings in a similar juvenile or adult-onset ataxia syndrome, mitochondrial recessive ataxia syndrome (MIRAS), caused by the W748S mutation in the mitochondrial DNA polymerase (POLG). We show here that IOSCA brain does not harbor mtDNA deletions or increased amount of mtDNA point mutations, whereas MIRAS brain shows multiple deletions of mtDNA. However, IOSCA, and to a lesser extent also MIRAS, show mtDNA depletion in the brain and the liver. In both diseases, especially large neurons show respiratory chain complex I (CI) deficiency, but also CIV is decreased in IOSCA. Helicase activity, hexamerization and nucleoid structure of the IOSCA mutant were, however, unaffected. The lack of in vitro helicase defect or cell culture phenotype suggest that Twinkle-Y508C dysfunction affects mtDNA maintenance in a highly context and cell-type specific manner. Our results indicate that IOSCA is a new member of the mitochondrial DNA depletion syndromes.     

Defects in maintenance of mitochondrial DNA are associated with intramitochondrial nucleotide imbalances

Defects in mtDNA maintenance range from fatal multisystem childhood diseases, such as Alpers syndrome, to milder diseases in adults, including mtDNA depletion syndromes (MDS) and familial progressive external ophthalmoplegia (AdPEO). Most are associated with defects in genes involved in mitochondrial deoxynucleotide metabolism or utilization, such as mutations in thymidine kinase 2 (TK2) as well as the mtDNA replicative helicase, Twinkle and gamma polymerase (POLG). We have developed an in vitro system to measure incorporation of radiolabelled dNTPs into mitochondria of saponin permeabilized cells. We used this to compare the rates of mtDNA synthesis in cells from 12 patients with diseases of mtDNA maintenance. We observed reduced incorporation of exogenous alpha (32)P-dTTP in fibroblasts from a patient with Alpers syndrome associated with the A467T substitution in POLG, a patient with dGK mutations, and a patient with mtDNA depletion of unknown origin compared to controls. However, incorporation of alpha (32)P-dTTP relative to either cell doubling time or alpha (32)P-dCTP incorporation was increased in patients with thymidine kinase deficiency or PEO as the result of TWINKLE mutations compared with controls. The specific activity of newly synthesized mtDNA depends on the size of the endogenous pool diluting the exogenous labelled nucleotide. Our result is consistent with a deficiency in the intramitochondrial pool of dTTP relative to dCTP in cells from patients with TK2 deficiency and TWINKLE mutations. Such DNA precursor asymmetry could cause pausing of the replication complex and hence exacerbate the propensity for age-related mtDNA mutations. Because deviations from the normal concentrations of dNTPs are known to be mutagenic, we suggest that intramitochondrial nucleotide imbalance could underlie the multiple mtDNA mutations observed in these patients.     

Molecular analysis of ANT1, TWINKLE and POLG in patients with multiple deletions or depletion of mitochondrial DNA by a dHPLC-based assay

ANT1, TWINKLE and POLG genes affect mtDNA stability and are involved in autosomal dominant PEO, while mutations in POLG are responsible for numerous clinical presentations, including autosomal recessive PEO, sensory ataxic neuropathy, dysarthria and ophthalmoparesis (SANDO), spino-cerebellar ataxia and epilepsy (SCAE) or Alpers syndrome. In this study, we report on the mutational analysis of ANT1, TWINKLE and POLG genes in 15 unrelated patients, using a dHPLC-based protocol. This series of patients illustrates the large array of clinical presentations associated with mtDNA stability defects, ranging from isolated benign PEO to fatal Alpers syndrome. A total of seven different mutations were identified in six of 15 patients (40%). Six different recessive mutations were found in POLG, one in TWINKLE while no mutation was identified in ANT1. Among the POLG mutations, three are novel and include two missense and one frameshift changes. Seventeen neutral changes and polymorphisms were also identified, including four novel neutral polymorphisms. Overall, this study illustrates the variability of phenotypes associated with mtDNA stability defects, increases the mutational spectrum of POLG variants and provides an efficient and reliable detection protocol for ANT1, TWINKLE and POLG mutational screening.     

Leber's hereditary optic neuropathy with 3460 mitochondrial DNA mutation

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease causing acute or subacute, bilateral optic atrophy mainly in young men. It is found to be a mitochondrial disorder with the primary mitochondrial DNA (mtDNA) mutations at 11,778, 3460, and 14,484. The incidence of each mutation is reported to be race-dependent. Point mutations at mtDNA nucleotide position 11,778 and 14,484 have been reported in Korean patients with LHON, however there has been no report of mtDNA mutation at nucleotide position 3460. Molecular genetic analyses at four primary sites (11,778, 14,484, 15,257, and 3460) of mitochondrial DNA using the polymerase chain reaction, restriction enzyme digestion, and direct sequencing were performed in a 35-yr-old man with severe visual loss. A point mutation in the mtDNA at nucleotide position 3460 was identified and a conversion of a single alanine to a threonine was confirmed. To our knowledge, this is the first report confirming mtDNA mutation at nucleotide position 3460 in Korean patients with LHON. Detailed molecular analyses would be very helpful for the correct diagnosis of optic neuropathy of unknown etiology and for genetic counseling.     

Low frequency of mtDNA point mutations in patients with PEO associated with POLG1 mutations

Mitochondrial myopathy in progressive external ophthalmoplegia (PEO) has been associated with POLG1 mutations. POLG1 encodes the catalytic alpha subunit of polymerase gamma and is the only polymerase known to be involved in mtDNA replication. It has two functionally different domains, one polymerase domain and one exonuclease domain with proofreading activity. In this study we have investigated whether mtDNA point mutations are involved, directly or indirectly, in the pathogenesis of PEO. Muscle biopsy specimens from patients with POLG1 mutations, affecting either the exonuclease or the polymerase domain, were investigated. Single cytochrome c oxidase (COX)-deficient muscle fibers were dissected and screened for clonally expanded mtDNA point mutations using a sensitive denaturing gradient gel electrophoresis analysis, in which three different regions of mtDNA, including five different tRNA genes, were investigated. To screen for randomly distributed mtDNA point mutations in muscle, two regions of mtDNA including deletion breakpoints were investigated by high-fidelity PCR, followed by cloning and sequencing. Long-range PCR revealed multiple mtDNA deletions in all the patients but not the controls. No point mutations were identified in single COX-deficient muscle fibers. Cloning and sequencing of muscle homogenate identified randomly distributed point mutations at very low frequency in patients and controls (<1:50 000). We conclude that mtDNA point mutations do not appear to be directly or indirectly involved in the pathogenesis of mitochondrial disease in patients with different POLG1 mutations.     

A patient with two mitochondrial DNA mutations causing PEO and LHON

We report a 22-year-old man with PEO and optic atrophy. PEO developed before the onset of optic atrophy. The patient showed mitochondrial myopathy with cytochrome c oxidase deficient fibers.In skeletal muscle the patient was homoplasmic for the mtDNA G11778A Leber hereditary optic neuropathy (LHON) mutation and heteroplasmic for the mtDNA 5 kb “common” deletion mutation. In blood only the homoplasmic LHON mutation was identified.The occurrence of two pathogenic mtDNA mutations is exceedingly rare. The clinical findings in this patient indicate that the combination of the two mtDNA mutations resulted in the expected combined phenotype since the mtDNA deletion mutation accounted for the PEO and the mtDNA G11778A point mutation for the optic atrophy.     

Method for in situ investigation of mitochondrial DNA deletions

A number of mitochondrial DNA (mtDNA) deletions have been recently identified in the tissues of patients with mitochondrial diseases and in elderly individuals. To investigate the distribution of mutant mitochondrial genomes within any particular tissue, we have developed a sensitive method based on indirect in situ PCR. Our experiments have shown that the new method had the advantage of selectively amplifying only mtDNA bearing the 4,977 bp deletion. We show that this method is more sensitive than in situ hybridization for detecting the 4977 bp mtDNA deletion while using only a low number of PCR cycles that minimize damage to tissue architecture. By using this method, we have demonstrated that the mutation does not occur uniformly among the cells of a given tissue/organ. This technique will be useful studying the distribution/localization of mtDNA mutations in individual cells of tissues and when combined with enzyme histochemical procedures in adjacent sections will enable the correlation between mtDNA mutations and bioenergy defects in single cells. Hum Mutat 10:489–495, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of mutant DNA polymerase gamma in patients with mitochondrial DNA depletion

We studied six unrelated children with depletion of mitochondrial DNA (mtDNA). They presented with Leigh syndrome, infantile hepatocerebral mtDNA depletion syndrome, or Alpers-Huttenlocher syndrome. Several genes have been implicated in mtDNA depletion. Screening of candidate genes indicated that all six patients were compound heterozygous for missense mutations in the gene for the catalytic subunit of DNA polymerase gamma (POLG). Three of the identified mutations, c.3328C>T (p.H1110Y), c.3401A>G (p.H1134R), and c.3406G>A (p.E1136K), have not been reported earlier. To investigate the functional consequences of the mutations, we carried out a series of biochemical assays in cultured fibroblasts. These studies revealed that fibroblast cultures from the patients with infantile hepatocerebral mtDNA depletion syndrome progressively lost their mtDNA during culturing, whereas fibroblast cultures from patients presenting with Leigh syndrome or Alpers-Huttenlocher syndrome had reduced but stable levels of mtDNA. DNA polymerase gamma activity was below the normal range in all patient cultures, except for one; however, this culture showed low levels of the heterodimeric enzyme and poor DNA polymerase gamma processivity. Parental fibroblast cultures had normal catalytic efficiency of DNA polymerase gamma, consistent with the observation that all carriers are asymptomatic. Thus, we report the first patient with Leigh syndrome caused by POLG mutations. The cell culture experiments established the pathogenicity of the identified POLG mutations and helped to define the molecular mechanisms responsible for mtDNA depletion in the patients' tissues. The assays may facilitate the identification of those patients in whom screening for POLG mutations would be most appropriate.     

慢性进行性眼外肌瘫痪和Kearns-Sayre综合征的线粒体DNA突变分析

目的 探讨慢性进行性眼外肌瘫痪(chronic progressive external ophthalmoplegia，CPEO)和Kearns-Sayre综合征(Kearns—Sayre syndrome，KSS)的线粒体DINA(mitochondrial DNA，mtDNA)突变特点。方法 用Southern印迹方法检测7例CPEO和4例KSS患者的肌肉组织mtDNA，并进一步用聚合酶链反应产物直接测序来明确缺失的具体范围；用聚合酶链反应-限制性内切酶分析法检测有无mtDNA A3243G点突变。结果 发现5例患者(2例CPEO和3例KSS)存在mtDNA的大片段缺失；1例KSS患者存在A3243G点突变。5例大片段缺失的大小及缺失范围各不相同，从3．0～8．0kb不等，缺失型mtDNA占总mtDNA的比例为37．6％～87．0％。聚合酶链反应产物测序表明这5例缺失类型均未见文献报道。结论 与CPEO和KSS患者相关的最常见的mtDNA突变为大片段缺失，A3243G点突变也可在少数患者中检测到。     

The mitochondrial protein frataxin prevents nuclear damage

The mitochondrial protein frataxin helps maintain appropriate iron levels in the mitochondria of yeast and humans. A deficiency of this protein in humans causes Friedreich's ataxia, while its complete absence in yeast (Delta yfh1 mutant) results in loss of mitochondrial DNA, apparently due to radicals generated by excess iron. We found that the absence of frataxin in yeast also leads to nuclear damage, as evidenced by inducibility of a nuclear DNA damage reporter, increased chromosomal instability including recombination and mutation, and greater sensitivity to DNA-damaging agents, as well as slow growth. Addition of a human frataxin mutant did not prevent nuclear damage, although it partially complemented the Delta yfh1 mutant in preventing mitochondrial DNA loss. The effects in Delta yfh1 mutants result from reactive oxygen species (ROS), since (i) Delta yfh1 cells produce more hydrogen peroxide, (ii) the effects are alleviated by a radical scavenger and (iii) the glutathione peroxidase gene prevents an increase in mutation rates. Thus, the frataxin protein is concluded to have a protective role for the nucleus as well as the mitochondria.     

Novel POLG mutations in progressive external ophthalmoplegia mimicking mitochondrial neurogastrointestinal encephalomyopathy

Autosomal recessive progressive external ophthalmoplegia (PEO) is one clinical disorder associated with multiple mitochondrial DNA deletions and can be caused by missense mutations in POLG, the gene encoding the mitochondrial DNA polymerase gamma. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is another autosomal recessive disorder associated with PEO and multiple deletions of mitochondrial DNA in skeletal muscle. In several patients this disorder is caused by loss of function mutations in the gene encoding thymidine phosphorylase (TP). We report a recessive family with features of MNGIE but no leukoencephalopathy in which two patients carry three missense mutations in POLG, of which two are novel mutations (N846S and P587L). The third mutation was previously reported as a recessive POLG mutation (T251I). This finding indicates the need for POLG sequencing in patients with features of MNGIE without TP mutations.     

Respiratory chain complex I deficiency caused by mitochondrial DNA mutations

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.     

Mitochondrial DNA Polymerase γ Mutations and Their Implications in mtDNA Alterations in Colorectal Cancer

Mitochondrial DNA was found to be highly mutated in colorectal cancer cells. One of the key molecules involved in the maintenance of the mitochondrial genome is the nuclear‐encoded polymerase gamma. The aim of our study was to determine if there is a link between polymorphisms within the polymerase gamma gene (POLG) and somatic mutations within the mitochondrial genome in cancer cells. We investigated POLG sequence variability in 50 colorectal cancer patients whose complete mitochondrial genome sequences were determined. Relative mtDNA copy number was also determined. We identified 251 sequence variants in the POLG gene. Most of them were germline‐specific (～92%). Twenty‐one somatic changes in POLG were found in 10 colorectal cancer patients. We have found no association between the occurrence of mtDNA somatic mutations and the somatically occurring variants in POLG. MtDNA content was reduced in patients carrying somatic variants in POLG or germline nucleotide variants located in the region encoding the POLG polymerase domain, but the difference did not reach statistical significance. Our findings suggest that somatic mtDNA mutations occurring in colorectal cancer are not a consequence of somatic mutations in POLG. Nevertheless, POLG nucleotide variants may lead to a decrease in mtDNA content, and consequently result in mitochondrial dysfunction.     

Two pathogenic point mutations exist in the authentic mitochondrial genome, not in the nuclear pseudogene

Technical advancements in molecular genetics have shown various mitochondrial DNA (mtDNA) abnormalities in patients with mitochondrial myopathies. Recently, it has been revealed that, in these patients, the nuclear DNA carries sequences similar to those of the mtDNA (nuclear pseudogene) and it has several point mutations previously reported to be pathogenic. We verified the existence of the T3250C and T3291C mutations, which we have found in patients with mitochondrial myopathy, in the authentic mitochondrial genome. A long polymerase chain reaction provides a powerful tool for avoiding nuclear pseudogene amplification and for ruling out ambiguity in the detection of the mutation for diagnosis. Received: August 2, 2000 / Accepted: August 30, 2000     

Depletion of the other genome-mitochondrial DNA depletion syndromes in humans

We present the current knowledge on the genetic and phenotypic aspects of mitochondrial DNA depletion syndromes. The human mitochondrial DNA encodes 13 of the 82 structural proteins of the mitochondrial electron transport chain. The replication and maintenance of the mtDNA require a large number of nuclear encoded enzymes and balanced nucleotide pools. Mitochondrial nucleotide synthesis is of major importance because of the constant need for nucleotides for mtDNA maintenance even in quiescent cells. As de novo enzymes are not present in the mitochondria, synthesis is accomplished via the salvage pathway. Defective mtDNA synthesis and maintenance manifest by multiple deletions or by depletion of the mitochondrial genome. Patients with multiple deletions typically present with progressive external ophthalmoplegia, ptosis and, exercise intolerance after the first decade of life. mtDNA depletion is usually an infantile disease characterized by severe muscle weakness, hepatic failure, or renal tubulopathy with fatal outcome. Linkage analysis in families with multiple mtDNA deletions reveal mutations in proteins that participate in mtDNA replication, the mitochondrial DNA polymerase gene, and the Twinkle gene, a putative mitochondrial helicase and in factors which play a role in mitochondrial nucleotide metabolism, the adenine nucleotide translocator, and the thymidine phosphorylase gene. We have recently identified mutations in an additional two essential proteins in the nucleotide salvage pathway, the mitochondrial deoxyribonucleoside kinases. The phenotype was distinctive for each gene, with hepatic failure and encephalopathy associated with mutations in the deoxyguanosine kinase gene and isolated devastating myopathy as the sole manifestation of thymidine kinase 2 deficiency. The tissue selectivity of these disorders and especially the exclusive muscle involvement in thymidine kinase 2 mutations is puzzling. The normal sequence of the remaining mtDNA copies in spite of a serious mitochondrial nucleotide imbalance is also unexpected. We propose several tissue-specific protective mechanisms and a time window, likely encompassing fetal life and even early infancy, during which nuclear nucleotide synthesis provides mitochondrial needs in all organs. We also speculate on future genes to be discovered in other phenotypes of mtDNA depletion.     

线粒体肌病患者线粒体DNA的突变分析

目的分析线粒体肌病患者线粒体DNA的突变情况，为疾病诊断提供依据。方法用常规HE、酶组化染色和电镜检查等病理形态学方法对3例线粒体肌病疑似患者进行诊断，并用聚合酶链反应-单链构象多态和DNA测序等方法对患者线粒体DNA中全部22个tRNA基因进行突变筛查。结果3例患者均被确诊为线粒体肌病，其中例1tRNA—VaI基因发生A1627G纯合突变，例2tRNA—Val基因发生A1627G／A杂合突变，例3tRNA—Trp基因发生T5554C突变、tRNA—Arg基因发生A10412C／A杂合突变。结论线粒体DNA中的tRNA基因突变是线粒体肌病的重要病因之一。