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Depletion of mtDNA: Syndromes and genes
Mitochondrion 7 (2007) 6–12 www.elsevier.com/locate/mito
Review
Depletion of mtDNA: Syndromes and genes Simona Alberio, Rossana Mineri, Valeria Tiranti, Massimo Zeviani
*
Unit of Molecular Neurogenetics – Pierfranco and Luisa Mariani Center for the Study of Children’s Mitochondrial Disorders, ‘‘C. Besta’’ Neurological Institute Foundation, IRCCS, Italy Received 4 May 2006; accepted 6 October 2006 Available online 5 December 2006
Abstract Maintenance of mitochondrial DNA (mtDNA) requires the concerted activity of several nuclear-encoded factors that participate in its replication, being part of the mitochondrial replisome or ensuring the balanced supply of dNTPs to mitochondria. In the past decade, a growing number of syndromes associated with dysfunction due to tissue-specific depletion of mtDNA (MDS) have been reported. This article reviews the current knowledge of the genes responsible for these disorders, the impact of different mutations in the epidemiology of MDS and their role in the pathogenic mechanisms underlying the different clinical presentations. Ó 2006 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: mtDNA depletion; OXPHOS; Tissue specificity
1. Introduction Profound reduction of the mtDNA copy number is the molecular hallmark of the mtDNA depletion syndromes (MDS), a heterogeneous group of severe mitochondrial disorders of infancy and childhood. Three main clinical presentations of MDS are known: myopathic, encephalomyopathic and hepatocerebral (Moraes et al., 1991; Tritschler et al., 1992; Vu et al., 1998; Ducluzeau et al., 1999; reviewed by Elpeleg, 2003). These syndromes are inherited as autosomal recessive traits, while no mutations were identified in the mtDNA molecule, which suggests that MDS results from defects in nucleus-encoded factors, involved in mtDNA maintenance.
Abbreviations: MDS, mtDNA depletion syndrome; DGUOK, deoxyguanosine kinase; TK2, thymidine kinase 2; POLG, DNA polymerase subunit c1; SUCLA2, succinate-CoA ligase, ADP-forming, beta subunit; dCK, deoxycytidine kinase; SCAE, spinocerebellar ataxia with Epilepsy; SANDO, sensory ataxic neuropathy dysarthria and ophthalmoparesis. * Corresponding author. Tel.: +39 02 23942630; fax: +39 02 23942619. E-mail addresses: [email protected], [email protected] (M. Zeviani). URL: www.mitopedia.org (M. Zeviani).
Maintenance of mtDNA is controlled by an intricated and well orchestrated homeostatic network, whose effectors are the various components of the mitochondrial replisome, the still largely unknown protein set that forms the mitochondrial nucleoid, and the many enzymes and carrier proteins that provide the mitochondrion with a balanced supply of deoxyribonucleotides, that is, the ‘‘building blocks’’ of mtDNA (Bogenhagen and Clayton, 1976). In principle, abnormalities in each of these very many actors involved in mtDNA replication can cause MDS. However, mutations in proteins encoded by only five genes have so far been implicated in the pathogenesis of MDS. Namely, the mitochondrial isoform of thymidine kinase (TK2) in myopathic MDS (Saada et al., 2001); the beta subunit of the ADP-forming succinylCoA synthase (SUCLA2) in encephalomyopatic MDS (Elpeleg et al., 2005); and deoxyguanosine kinase (DGUOK) (Mandel et al., 2001), the catalytic subunit of mtDNA polymerase (Pol-cA) (Naviaux and Nguyen, 2005) and MPV17, a protein of still unknown function, in hepatocerebral MDS (Spinazzola et al., 2006). Mutations in these proteins account for only a minority of the MDS cases, confirming the existence of several additional MDS genes.
1567-7249/$ - see front matter Ó 2006 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2006.11.010
S. Alberio et al. / Mitochondrion 7 (2007) 6–12
2. Clinical and molecular genetics
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in the DGUOK gene, suggesting a strict and specific correlation between TK2 mutations and myopathic MDS.
2.1. Myopathic form 2.2. Encephalomyopathic form (OMIM#609560) Onset of symptoms usually occurs in the first year of life, with feeding difficulty, failure to thrive, hypotonia, and muscle weakness. Serum CK is often increased; this is an important clue for the diagnosis, since it is uncommon in patients with other mitochondrial myopathies. Death usually occurs early for pulmonary insufficiency and infections, but some patients have survived into their teens (Moraes et al., 1991; Tritschler et al., 1992). These clinical and biochemical manifestations are accompanied by morphological signs typical of mitochondrial myopathy such as no or ‘‘patchy’’ reactivity to cytochrome c oxidase. Proliferation of mitochondria in the form of ragged-red fibers, RRF, is not a consistent feature at onset, although RRF can appear later during the disease course. Biochemical defects of all mtDNA-encoded respiratory chain complexes are always present in muscle mitochondria. Mutations in thymidine kinase-2 (TK2, OMIM*188250) seem to be specifically associated with myopathic MDS, although a single case had also clinical and EMG (electromyogram) features referable to spinal muscular atrophy (Mancuso et al., 2002). TK2 is a mitochondrion-specific deoxyribonucleoside kinase that phosphorylates thymidine, deoxycytidine, and deoxyuridine, as well as antiviral and anticancer nucleoside analogs. In 2001, Saada et al., identified two mutations in TK2, a H90N and an I181N in four infants with severe myopathic MDS. TK2-specific activity was reduced to 13–32% in muscle mitochondria of these patients, compared to controls. Since this first observation, a total of 11 mutations of TK2 have been reported in 12 patients. The prevalence of TK2 mutations in myopathic MDS is approximately 20%. We have summarized these results in Table 1. The amino acid positions of the TK2 mutations are shown according to two different nomenclatures: the nomenclature based on the cDNA sequence (U77088) reported by Johansson and Karlsson (1997) and that based on a more recently released cDNA sequence (NM_004614.3) which appeared on April 2006 and contains an additional extension of exon 1. Contrary to the previous sequence, the latter sequence predicts the synthesis of a putative mitochondrial translocation signal encompassing the first 30 amino acid residues on the N-terminus portion. The C-terminus end of the targeting sequence contains a potential cleavage site between amino acid 26 and 27, as predicted by dedicated softwares. As for our own experience, we found two patients with TK2 mutations in a cohort of 10 individuals affected by myopathic MDS. One patient was a compound heterozygote for the already described T77M and for a new R161K mutation (Wang et al., 2005); the other patient carried two novel mutations: Y154N and R172Q, corresponding to Y81N and R99Q according to the ‘‘old’’ TK2 sequence (see Table 1). In the same group of patients, we failed to identify mutations
Elpeleg et al. (2005) described an autosomal recessive form of encephalomyopathy associated with mtDNA depletion in a consanguineous Arab family. The proband had severe psychomotor retardation with prominent muscle hypotonia, impaired hearing, and generalized seizures. Brain MRI was suggestive of Leigh syndrome with lesions in the basal ganglia. An affected cousin had muscle hypotonia, lack of voluntary movements, bilateral hearing loss, generalized seizures, and severe psychomotor retardation. Both patients showed significantly decreased activity of complexes I and IV in skeletal muscle while activities of complexes III and V were less compromised. The same tissue showed a profound depletion of mtDNA with a ratio of mtDNA to nuclear DNA that was 32% of normal values. Liver and renal tests in both patients were normal. In these two patients Elpeleg et al. (2005) identified a homozygous mutation in the SUCLA2 gene (OMIM*603921) that encodes the beta-subunit of the ADP-forming succinyl-CoA synthetase (SCS-A). SCS is a mitochondrial matrix enzyme that catalyzes the reversible synthesis of succinyl-CoA from succinate and CoA. The mutation was a complex genomic rearrangement at the 3 0 end of exon 6, composed of a 43-bp deletion and a 5-bp insertion. The deletion encompassed the last 14 bp of exon 6 plus the first 29 bp of intron 6. The mutation was not identified in 105 consecutive Arab controls. 2.3. Hepatocerebral form This is probably the most common variant of MDS (OMIM#251880). Onset is in infancy and early symptoms include persistent vomiting, failure to thrive, hypotonia, and hypoglycemia (Mazziotta et al., 1992). Histological changes in the liver biopsy include fatty degeneration, bile duct proliferation, fibrosis, and collapse of liver architecture. Liver mitochondria usually show combined deficiency of mtDNA-encoded RC complexes (Slama et al., 2005). A peculiar form of hepatocerebral MDS is Alpers–Huttenlocher syndrome (AHS; progressive infantile poliodystrophy) (OMIM#203700). Spongiotic degeneration in the cortex and deep grey structures of the brain, which evolve into global brain atrophy, account for the typical neurological findings in AHS, i.e. refractory seizures, usually multifocal, which may evolve into epilepsia partialis continua, and psychomotor regression. The latter is often episodic at first, but becomes global and progressive during the course of the disease. The liver dysfunction is usually progressive as well, evolving from microvesicular steatosis with bile duct proliferation, into overt cirrhosis and chronic liver failure. Acute liver failure may be precipitated by exposure to valproic acid for the treatment of seizures.
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Table 1 Mutations in MDS genes Gene mutations
Published patients
New INN patients
TK2 (NM_004614.3) 487–489GC->AA 763T->A 487–489GC->AA/449C->T 287C->G/287C->G 449C->T/449C->T 547C>G/760C>Ta nc 675C->G/NNNdel 449C->T/701G->A 324C->G/770T->C 542C->T/542C->T 460T->A/515G->A SUCLA2 43bpdel + 5bp ins DGUOK 204delA/204delA 313C->T/313C->T 763dupGAAT/763dupGAAT 609delGT/609delGT 425G->A/679G->A nc 749T->C/749T->C 603-604 delGA/-8T->G
1 3 1 1 3 brothers 1 1 1 1 1 1 1
Protein Accession NP_004605 H163N I254N H163N/T150M I95M/I95M T150M/T150M R225G/R296X fs191X/R225G/K244del T150M/R234K C108W/L257P A181V/A181V Y154N/R172Q
1
References Accession AAC51167 H90N I181N H90N/T77M I22M/I22M T77M/T77M R183G/R254Xa fs149X/-a R152G/K171del T77M/R161K nc nc Y81N/R99Q
Saada et al. (2001) Saada et al. (2001) Mancuso et al. (2002) Mancuso et al. (2002) Mancuso et al. (2003) Carrozzo et al. (2003) Carrozzo et al. (2003) Vila et al. (2003) Wang et al. (2005) Galbiati et al. (2006) Galbiati et al. (2006) Present study Elpeleg et al. (2005)
3 1 1 1 1 3(2 families) 1 1
del68fs80X R105X fs fs R142K/E227K D255Y L250S/L250S K201fs214X/ IVS4splicing site
Mandel et al. (2001) Taanman et al. (2002) Salviati et al. (2002) Salviati et al. (2002) Salviati et al. (2002) Tadiboyina et al. (2005) Wang et al. (2005) Present study
Ferrari et al. (2005) Ferrari et al. (2005) Ferrari et al. (2005); Nguyen et al. (2005); Horvath et al. (2006) Ferrari et al. (2005) Ferrari et al. (2005) Ferrari et al. (2005); Nguyen et al. (2005) Ferrari et al. (2005) Nguyen et al. (2006) Nguyen et al. (2006) Naviaux and Nguyen (2005) Nguyen et al. (2006); Horvath et al. (2006) Nguyen et al. (2006) Nguyen et al. (2005) Nguyen et al. (2006) Horvath et al. (2006) Horvath et al. (2006) Horvath et al. (2006) Horvath et al. (2006) Horvath et al. (2006) Horvath et al. (2006)
POLG 2243G->C/3629Ains 731C->T/2243G->C 1399G->A/2542G->A
1 1 3
W748S/Y1210X L244P/W748S A467T/G848S
1399G->A/3482 + 2T->C 1399G->A/2869G->C 1399G->A/1399G->A
1 1 2
A467T/Splice site A467T/A957P A467T/A467T
694C->G/752C->T;1760C->T 1399G->A/2246T->C 1399G->A/2554C->T 1399G->A/2617G->T 1399G->A/2740A->C
1 1 1 1 2
R232G/T251I;P587L A467T/F749S A467T/R852C A467T/E873X A467T/T914P
1399G->A/2897T->G 1399G->A/3057G->A 1399G->A/3518insGATC 1399G->A/3573G->T 926G->A/1880G->A 2209G->C/2300C->A 1399G->A/IVS15-9_12del 1399G->A/IVS21 + 1T->C 3286C->T;3708G->T/3286C->T;3708G->T
1 1 1 1 1 1 1 1 1
1399G->A;2653A->T/2637T->G;3428A->G
1
1880G->A/3287G->A 2243G->C/3428A->G-2542G->A
1 5
A467T/L966R A467T/W1020X A467T/L1173X A467T/K1191N R309H/R627Q G737R/A767N A467T/A467T/R1096C;Q1236H/ R1096C;Q1236H A467T;T885S/ Q879H;E1143G R627Q/R1096H W748S/E1143G-G848S
MPV17 149G->A/149G->A 498C->A/498C->A 148C->T/116-141del
1 1 1
R50Q/R50Q N166K/N166K R50W/fs
nc, not calculated. a The TK2 reference sequence utilized in the paper is O00142.
Horvath et al. (2006) Horvath et al. (2006) Davidzon et al. (2005); Nguyen et al. (2005) Spinazzola et al. (2006) Spinazzola et al. (2006) Spinazzola et al. (2006)
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Hepatocerebral MDS has been associated with mutations in three genes, encoding DGUOK, POLG, and MPV17, respectively. 2.3.1. DGUOK The mitochondrial deoxyguanosine kinase (dGK), (OMIM*601465) is a 2-deoxyribonucleoside kinase that mediates the phosphorylation of purine deoxyribonucleosides into the corresponding nucleotides. A total of 10 mutations in 12 patients have been reported for DGUOK. Contrary to what observed for TK2 mutations, most of the mutations found in the DGUOK gene are nonsense changes, although missense mutations have also been reported (Table 1). In our own series of five cases with hepatocerebral MDS, we found 3 DGUOK mutations in two unrelated patients. One patient had a homozygous missense mutation 749T->C resulting in a L250S amino acid change (Wang et al., 2005). The second patient was a compound heterozygote for two novel mutations, a 603–604del GA resulting in a frameshift with the creation of a premature stop codon, and a splicing site 8T->G mutation in intron 4 (Table 1). 2.3.2. POLG The mitochondrial DNA polymerase (pol c) (OMIM*174763) is essential for mitochondrial DNA replication and proofreading-based repair. It is composed of a 140-kDa catalytic subunit (pol cA) and a 55-kDa accessory subunit (pol cB), which functions as a DNA binding factor increasing the processivity of the polymerase holoenzyme. The holoenzyme works as an ab2 heterotrimer. The catalytic subunit is encoded by the POLG gene, which includes 23 exons, and is located on chromosome 15q25. Mutations in POLG are associated with an extremely heterogeneous spectrum of clinical presentations, from dominant and recessive forms of progressive external ophthalmoplegia (PEO) (Van Goethem et al., 2001; Lamantea et al., 2002; Agostino et al., 2003), sometimes with parkinsonism (Luoma et al., 2004), to juvenile spinocerebellar ataxia and epilepsy (SCAE) with or without dysarthria and lateonset ophthalmoplegia (SANDO) (Van Goethem et al., 2003, 2004; Winterthun et al., 2005), to AHS (Naviaux and Nguyen, 2005; Ferrari et al., 2005). Accumulation of multiple mtDNA deletions, rather than mtDNA depletion, in affected tissues such as muscle and brain, is the molecular hallmark of adPEO and arPEO. Multiple mtDNA deletions are also detected in the muscle tissue of patients affected with SCAE or SANDO, but this abnormality is neither precocious nor prominent in these syndromes. Depletion of liver mtDNA is more consistently associated with AHS, but again this feature may be absent in the early stages of the disease, indicating that it is neither sensitive, nor specific, for AHS (Nguyen et al., 2006). Therefore, the early phenotype in AHS, and also in SCAE and SANDO, may result from a subtler, non-replicative consequence of POLG mutations, for instance abnormalities in mtDNA proofreading and repair.
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The 1239 aa long Pol cA polypeptide is organized in three adjacent functional domains: a 50 > 30 exonucleolytic proofreading domain (amino acid 1–417), a linker domain (amino acid 418–755), and a polymerase domain (amino acid 756–1239). By reviewing POLG mutations identified in 31 AHS patients by different laboratories (Naviaux and Nguyen, 2005; Davidzon et al., 2005; Ferrari et al., 2005; Nguyen et al., 2005, 2006; Horvath et al., 2006) a total of 26 mutations were found. In several cases at least one allele carried either one of two mutations: the A467T mutation (34% of the alleles), or, more rarely, the W748S mutation (11% of the alleles). Eleven of the remaining 13 POLG mutations associated with AHS were unique to individual families. In conclusion, AHS can be considered a monogenic form of hepatocerebral MDS associated with a specific combination of POLG mutant alleles. In particular the A467T and the W748S are alternatively detected in most cases (Nguyen et al., 2006). In all cases, at least one allele is contained within the linker region (Horvath et al., 2006), which facilitates the genetic diagnosis of this condition. 2.3.3. MPV17 We recently found a new locus for hepatocerebral MDS on chromosome 2p21–23 by linkage analysis on a large multi-consanguineous Italian family. By using an integrative genomic approach (Calvo et al., 2006), we identified MPV17 (OMIM*137960) as a candidate gene for this disease. MPV17 is the human ortholog of the murine kidney disease gene Mpv17. Mutations in MPV17 have so far been found in several probands from three unrelated families (Spinazzola et al., 2006). The mutations were a 149G > A (R50Q) homozygous transition, found in our original Italian family; a 498C > A (N166K) homozygous transversion in the proband from a family of Moroccan origin; a missense change in one allele (148C > T, R50W), and a 25-bp deletion in the other allele (116–141del) in a proband from a Canadian family. The 116–141del predicts the synthesis of an aberrant and prematurely truncated polypeptide. The clinical presentation of MPV17 mutant MDS patients was characterized by severe, often fatal, hypoglycemic episodes, jaundice, and elevated levels of both lactate and hepatic enzymes in blood (Spinazzola et al., 2006). The liver showed progressive portal and lobular fibrosis evolving into terminal cirrhosis. Marked mtDNA depletion in liver, ranging from 5% to 15% of mean normal controls, was the molecular hallmark in the probands of all three families, associated with multiple defects of mtDNA-related respiratory chain (RC) complexes, particularly complex I and complex IV. Normal or mildly reduced levels of both mtDNA content and RC activities were found in muscle. We obtained experimental data indicating that, contrary to the alleged peroxisomal localization of the MPV17 gene product, MPV17p is a mitochondrial protein tightly bound to the inner mitochondrial membrane (Spinazzola et al., 2006).
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3. Pathogenic mechanisms 3.1. TK2, DGUOK, and SUCLA2 deficiency A balanced supply of deoxynucleotides is needed to warrant mtDNA replication. Thymidine kinase and deoxyguanosine kinase are part of the mitochondrion-specific salvage pathways of pyrimidine and purine nucleotides, respectively. Both pathways are essential for the replication of mtDNA since the mitochondrion is unable to synthesize dNTPs de novo. In addition, since mtDNA is replicated throughout the whole cell cycle, there is a constant need for nucleotides for mtDNA replication (Bogenhagen and Clayton, 1976; Saada, 2004). Elpeleg et al. (2005) postulated that also mutations in the SUCLA2 gene could determine a defect in the last step of the mitochondrial deoxyribonucleoside triphosphate (dNTP) salvage pathway, since SCS-A copurifies with and is tightly associated in a complex with nucleoside diphosphate kinase (NDPK) (Kadrmas et al., 1991; Kavanaugh-Black et al., 1994; Kowluru et al., 2002). NDPKs are ubiquitous protein kinases that also catalyze the exchange of terminal phosphates between tri- and di-phosphoribonucleosides and are crucial for maintaining the homeostasis of ribonucleotides and deoxyribonucleotides (Parks et al., 1973). One of the most striking features of MDS is the remarkable tissue specificity of the molecular and biochemical abnormalities. Different organs can be affected not only by mutations in different genes, but even by mutations in different part of the same gene, as we saw for pol cA. What are the mechanisms underlying this phenomenon? This important question remains largely unanswered. In case of TK2 or DGUOK mutations, the tissue-specific expression of MDS may be due to the differential expression of other genes regulating the mitochondrial dNTP pool, or to the variable effect of different gene mutations on the dNTP pools. The possibility should also be considered of a ‘‘rescue effect’’ on unaffected tissues by the activities of cytoplasmic deoxyribonucleoside kinases. For instance, Saada (2004) has hypothesized that in case of DGUOK mutations the activity of dCK, a cytosolic enzyme, could compensate for the lack of DGUOK activity in unaffected tissues; however, dCK activity is very low in brain and liver, which could explain why DGUOK deficiency leads to hepatocerebral MDS. As for TK2 deficiency, a different mechanism must be hypothesized, since cytosolic TK1 activity is very low in non-proliferating cells, including skeletal muscle. Therefore, defective supply of dTTP to mitochondria, due to mutations in TK2, cannot be compensated by the activity of TK1 in muscle. Saada et al. (2003) noted that none of the reported TK2 mutant patients is homozygous for a loss-of function mutation; at least one missense mutation is always present in these patients, suggesting that some residual enzymatic activity is retained in all cases, and is probably sufficient to rescue those tissues that have a high
basal TK2 expression. The activity of TK2 is the lowest in muscle mitochondria but it progressively increases in the heart and liver. In addition skeletal muscle has a very high energetic demand, as compared to other tissues. These two factors combined could explain the selective vulnerability of skeletal muscle in TK2-associated MDS. 3.2. POLG1 deficiency Disturbances of the nucleotide pool available for mtDNA replication, as well as abnormalities in the mitochondrial DNA polymerase, are likely to affect the rate, processivity or fidelity of replication, which could ultimately lead to instability of mtDNA. With a few exceptions (Ferrari et al., 2005; Horvath et al., 2006) the early-onset infantile encephalopathy + hepatopathy syndromes associated with POLG mutations are typical AHS patients. Almost all of them carried either the A467T or the W748S in the linker region of pol cA and in all cases at least one mutation affected the linker region (Ferrari et al., 2005; Horvath et al., 2006; see also Table 1). What is the basis of such a specific association? We do not know. And of course the mechanisms of tissue specificity remain unexplained as well. Recent studies in patients and in cell mutants may offer some clues. The A467T amino acid substitution determines a profound defect in the DNA polymerase activity of pol c, and it also impairs its processivity, by preventing the interaction of pol cA with pol cB, the accessory subunit (Chan et al., 2005). These effects of the A467T substitution are likely to cause the stalling of the enzyme during replication and, as a consequence, to promote the generation of deletions or the depletion of mtDNA. Patients homozygous for the A467T allele develop a later-onset form of AHS, or juvenile SCAE, which is also typically found in patients with homozygous W748S, or in A467T/W748S compound heterozygotes. The latter patients, however, consistently manifest a much more rapid downhill course (Tzoulis et al., 2006). Likewise, AHS, which is an extremely severe and early onset condition, is consistently associated with the presence of either the A467T or the W748S changes in one allele, in combination with a different pathogenic mutation in the second allele. These observations suggest a dominant negative effect of the A467T or W748S alleles over other pathological alleles and raise the possibility of a quaternary interaction between catalytic subunits in different heterotrimers, a phenomenon compatible with the synchronous model of mtDNA replication (Yang et al., 2002). 3.3. MPV17 deficiency The function of this gene and its role in the pathogenesis of MDS are still unknown (Spinazzola et al., 2006). The Dsym1 yeast strain shows a temperature-sensitive OXPHOS phenotype, suggesting a role for this protein in the cellular response to stress. In fibroblasts derived from Mpv17 knock-out mice the production of reactive
S. Alberio et al. / Mitochondrion 7 (2007) 6–12
oxygen species (ROS) is reduced, while its over-expression seems to induce the opposite effect, i.e. an increase level of ROS (Zwacka et al., 1994). These observations, together with the identification of MPV17 gene mutations causing hepatocerebral MDS suggest a role of Mpv17p in controlling ROS metabolism and the cellular response to stress. The availability of Mpv17 KO mouse, the first animal model for MDS syndrome, provides an invaluable tool to elucidate the function of this gene in mitochondrial homeostasis and to investigate its role in the pathogenesis of this disease. 4. Conclusion Mitochondrial DNA depletion syndromes could result from any disturbance in the mtDNA replication machinery. Imbalance of mitochondrial dNTPs pool as well as abnormalities of the mitochondrial DNA polymerase, can cause disorders characterize by a wide spectrum of clinical presentations. The recent discovery of the MPV17 gene and the future identification of additional MDS genes will probably expand and improve our knowledge on the pathogenic mechanisms underlining these disorders. Acknowledgments Supported by Fondazione Telethon-Italy (Grant No. GGP030039), Fondazione Pierfranco e Luisa Mariani, MITOCIRCLE and EUMITOCOMBAT network grants from the European Union Framework Program 6. References Agostino, A., Valletta, L., Chinnery, P.F., Ferrari, G., Carrara, F., Taylor, R.W., Schaefer, A.M., Turnbull, D.M., Tiranti, V., Zeviani, M., 2003. Mutations of ANT1, Twinkle, and POLG1 in sporadic progressive external ophthalmoplegia (PEO). Neurology 60, 1354–1356. Bogenhagen, D., Clayton, D.A., 1976. Thymidylate nucleotide supply for mitochondrial DNA synthesis in mouse L-cells. Effect of 5-fluorodeoxyuridine and methotrexate in thymidine kinase plus and thymidine kinase minus cells. J. Biol. Chem. 251, 2938–2944. Calvo, S., Jain, M., Xie, X., Sheth, S.A., Chang, B., Goldberger, O.A., Spinazzola, A., Zeviani, M., Carr, S.A., Mootha, V.K., 2006. Systematic identification of human mitochondrial disease genes through integrative genomics. Nat. Genet. 38, 576–582. Carrozzo, R., Bornstein, B., Lucioli, S., Campos, Y., de la Pena, P., Petit, N., Dionisi-Vici, C., Vilarinho, L., Rizza, T., Bertini, E., Garesse, R., Santorelli, F.M., Arenas, J., 2003. Mutation analysis in 16 patients with mtDNA depletion. Hum. Mutat. 21, 453–454. Chan, S.S., Longley, M.J., Copeland, W.C., 2005. The common A467T mutation in the human mitochondrial DNA polymerase (POLG) compromises catalytic efficiency and interaction with the accessory subunit. J. Biol. Chem. 280, 31341–31346. Davidzon, G., Mancuso, M., Ferraris, S., Quinzii, C., Hirano, M., Peters, H.L., Kirby, D., Thorburn, D.R., DiMauro, S., 2005. POLG mutations and Alpers syndrome. Ann. Neurol. 57, 921–924. Ducluzeau, P.H., Lachaux, A., Bouvier, R., Streichenberger, N., Stepien, G., Mousson, B., 1999. Depletion of mitochondrial DNA associated with infantile cholestasis and progressive liver fibrosis. J. Hepatol. 30, 149–155.
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Review
Depletion of mtDNA: Syndromes and genes Simona Alberio, Rossana Mineri, Valeria Tiranti, Massimo Zeviani
*
Unit of Molecular Neurogenetics – Pierfranco and Luisa Mariani Center for the Study of Children’s Mitochondrial Disorders, ‘‘C. Besta’’ Neurological Institute Foundation, IRCCS, Italy Received 4 May 2006; accepted 6 October 2006 Available online 5 December 2006
Abstract Maintenance of mitochondrial DNA (mtDNA) requires the concerted activity of several nuclear-encoded factors that participate in its replication, being part of the mitochondrial replisome or ensuring the balanced supply of dNTPs to mitochondria. In the past decade, a growing number of syndromes associated with dysfunction due to tissue-specific depletion of mtDNA (MDS) have been reported. This article reviews the current knowledge of the genes responsible for these disorders, the impact of different mutations in the epidemiology of MDS and their role in the pathogenic mechanisms underlying the different clinical presentations. Ó 2006 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: mtDNA depletion; OXPHOS; Tissue specificity
1. Introduction Profound reduction of the mtDNA copy number is the molecular hallmark of the mtDNA depletion syndromes (MDS), a heterogeneous group of severe mitochondrial disorders of infancy and childhood. Three main clinical presentations of MDS are known: myopathic, encephalomyopathic and hepatocerebral (Moraes et al., 1991; Tritschler et al., 1992; Vu et al., 1998; Ducluzeau et al., 1999; reviewed by Elpeleg, 2003). These syndromes are inherited as autosomal recessive traits, while no mutations were identified in the mtDNA molecule, which suggests that MDS results from defects in nucleus-encoded factors, involved in mtDNA maintenance.
Abbreviations: MDS, mtDNA depletion syndrome; DGUOK, deoxyguanosine kinase; TK2, thymidine kinase 2; POLG, DNA polymerase subunit c1; SUCLA2, succinate-CoA ligase, ADP-forming, beta subunit; dCK, deoxycytidine kinase; SCAE, spinocerebellar ataxia with Epilepsy; SANDO, sensory ataxic neuropathy dysarthria and ophthalmoparesis. * Corresponding author. Tel.: +39 02 23942630; fax: +39 02 23942619. E-mail addresses: [email protected], [email protected] (M. Zeviani). URL: www.mitopedia.org (M. Zeviani).
Maintenance of mtDNA is controlled by an intricated and well orchestrated homeostatic network, whose effectors are the various components of the mitochondrial replisome, the still largely unknown protein set that forms the mitochondrial nucleoid, and the many enzymes and carrier proteins that provide the mitochondrion with a balanced supply of deoxyribonucleotides, that is, the ‘‘building blocks’’ of mtDNA (Bogenhagen and Clayton, 1976). In principle, abnormalities in each of these very many actors involved in mtDNA replication can cause MDS. However, mutations in proteins encoded by only five genes have so far been implicated in the pathogenesis of MDS. Namely, the mitochondrial isoform of thymidine kinase (TK2) in myopathic MDS (Saada et al., 2001); the beta subunit of the ADP-forming succinylCoA synthase (SUCLA2) in encephalomyopatic MDS (Elpeleg et al., 2005); and deoxyguanosine kinase (DGUOK) (Mandel et al., 2001), the catalytic subunit of mtDNA polymerase (Pol-cA) (Naviaux and Nguyen, 2005) and MPV17, a protein of still unknown function, in hepatocerebral MDS (Spinazzola et al., 2006). Mutations in these proteins account for only a minority of the MDS cases, confirming the existence of several additional MDS genes.
1567-7249/$ - see front matter Ó 2006 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2006.11.010
S. Alberio et al. / Mitochondrion 7 (2007) 6–12
2. Clinical and molecular genetics
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in the DGUOK gene, suggesting a strict and specific correlation between TK2 mutations and myopathic MDS.
2.1. Myopathic form 2.2. Encephalomyopathic form (OMIM#609560) Onset of symptoms usually occurs in the first year of life, with feeding difficulty, failure to thrive, hypotonia, and muscle weakness. Serum CK is often increased; this is an important clue for the diagnosis, since it is uncommon in patients with other mitochondrial myopathies. Death usually occurs early for pulmonary insufficiency and infections, but some patients have survived into their teens (Moraes et al., 1991; Tritschler et al., 1992). These clinical and biochemical manifestations are accompanied by morphological signs typical of mitochondrial myopathy such as no or ‘‘patchy’’ reactivity to cytochrome c oxidase. Proliferation of mitochondria in the form of ragged-red fibers, RRF, is not a consistent feature at onset, although RRF can appear later during the disease course. Biochemical defects of all mtDNA-encoded respiratory chain complexes are always present in muscle mitochondria. Mutations in thymidine kinase-2 (TK2, OMIM*188250) seem to be specifically associated with myopathic MDS, although a single case had also clinical and EMG (electromyogram) features referable to spinal muscular atrophy (Mancuso et al., 2002). TK2 is a mitochondrion-specific deoxyribonucleoside kinase that phosphorylates thymidine, deoxycytidine, and deoxyuridine, as well as antiviral and anticancer nucleoside analogs. In 2001, Saada et al., identified two mutations in TK2, a H90N and an I181N in four infants with severe myopathic MDS. TK2-specific activity was reduced to 13–32% in muscle mitochondria of these patients, compared to controls. Since this first observation, a total of 11 mutations of TK2 have been reported in 12 patients. The prevalence of TK2 mutations in myopathic MDS is approximately 20%. We have summarized these results in Table 1. The amino acid positions of the TK2 mutations are shown according to two different nomenclatures: the nomenclature based on the cDNA sequence (U77088) reported by Johansson and Karlsson (1997) and that based on a more recently released cDNA sequence (NM_004614.3) which appeared on April 2006 and contains an additional extension of exon 1. Contrary to the previous sequence, the latter sequence predicts the synthesis of a putative mitochondrial translocation signal encompassing the first 30 amino acid residues on the N-terminus portion. The C-terminus end of the targeting sequence contains a potential cleavage site between amino acid 26 and 27, as predicted by dedicated softwares. As for our own experience, we found two patients with TK2 mutations in a cohort of 10 individuals affected by myopathic MDS. One patient was a compound heterozygote for the already described T77M and for a new R161K mutation (Wang et al., 2005); the other patient carried two novel mutations: Y154N and R172Q, corresponding to Y81N and R99Q according to the ‘‘old’’ TK2 sequence (see Table 1). In the same group of patients, we failed to identify mutations
Elpeleg et al. (2005) described an autosomal recessive form of encephalomyopathy associated with mtDNA depletion in a consanguineous Arab family. The proband had severe psychomotor retardation with prominent muscle hypotonia, impaired hearing, and generalized seizures. Brain MRI was suggestive of Leigh syndrome with lesions in the basal ganglia. An affected cousin had muscle hypotonia, lack of voluntary movements, bilateral hearing loss, generalized seizures, and severe psychomotor retardation. Both patients showed significantly decreased activity of complexes I and IV in skeletal muscle while activities of complexes III and V were less compromised. The same tissue showed a profound depletion of mtDNA with a ratio of mtDNA to nuclear DNA that was 32% of normal values. Liver and renal tests in both patients were normal. In these two patients Elpeleg et al. (2005) identified a homozygous mutation in the SUCLA2 gene (OMIM*603921) that encodes the beta-subunit of the ADP-forming succinyl-CoA synthetase (SCS-A). SCS is a mitochondrial matrix enzyme that catalyzes the reversible synthesis of succinyl-CoA from succinate and CoA. The mutation was a complex genomic rearrangement at the 3 0 end of exon 6, composed of a 43-bp deletion and a 5-bp insertion. The deletion encompassed the last 14 bp of exon 6 plus the first 29 bp of intron 6. The mutation was not identified in 105 consecutive Arab controls. 2.3. Hepatocerebral form This is probably the most common variant of MDS (OMIM#251880). Onset is in infancy and early symptoms include persistent vomiting, failure to thrive, hypotonia, and hypoglycemia (Mazziotta et al., 1992). Histological changes in the liver biopsy include fatty degeneration, bile duct proliferation, fibrosis, and collapse of liver architecture. Liver mitochondria usually show combined deficiency of mtDNA-encoded RC complexes (Slama et al., 2005). A peculiar form of hepatocerebral MDS is Alpers–Huttenlocher syndrome (AHS; progressive infantile poliodystrophy) (OMIM#203700). Spongiotic degeneration in the cortex and deep grey structures of the brain, which evolve into global brain atrophy, account for the typical neurological findings in AHS, i.e. refractory seizures, usually multifocal, which may evolve into epilepsia partialis continua, and psychomotor regression. The latter is often episodic at first, but becomes global and progressive during the course of the disease. The liver dysfunction is usually progressive as well, evolving from microvesicular steatosis with bile duct proliferation, into overt cirrhosis and chronic liver failure. Acute liver failure may be precipitated by exposure to valproic acid for the treatment of seizures.
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Table 1 Mutations in MDS genes Gene mutations
Published patients
New INN patients
TK2 (NM_004614.3) 487–489GC->AA 763T->A 487–489GC->AA/449C->T 287C->G/287C->G 449C->T/449C->T 547C>G/760C>Ta nc 675C->G/NNNdel 449C->T/701G->A 324C->G/770T->C 542C->T/542C->T 460T->A/515G->A SUCLA2 43bpdel + 5bp ins DGUOK 204delA/204delA 313C->T/313C->T 763dupGAAT/763dupGAAT 609delGT/609delGT 425G->A/679G->A nc 749T->C/749T->C 603-604 delGA/-8T->G
1 3 1 1 3 brothers 1 1 1 1 1 1 1
Protein Accession NP_004605 H163N I254N H163N/T150M I95M/I95M T150M/T150M R225G/R296X fs191X/R225G/K244del T150M/R234K C108W/L257P A181V/A181V Y154N/R172Q
1
References Accession AAC51167 H90N I181N H90N/T77M I22M/I22M T77M/T77M R183G/R254Xa fs149X/-a R152G/K171del T77M/R161K nc nc Y81N/R99Q
Saada et al. (2001) Saada et al. (2001) Mancuso et al. (2002) Mancuso et al. (2002) Mancuso et al. (2003) Carrozzo et al. (2003) Carrozzo et al. (2003) Vila et al. (2003) Wang et al. (2005) Galbiati et al. (2006) Galbiati et al. (2006) Present study Elpeleg et al. (2005)
3 1 1 1 1 3(2 families) 1 1
del68fs80X R105X fs fs R142K/E227K D255Y L250S/L250S K201fs214X/ IVS4splicing site
Mandel et al. (2001) Taanman et al. (2002) Salviati et al. (2002) Salviati et al. (2002) Salviati et al. (2002) Tadiboyina et al. (2005) Wang et al. (2005) Present study
Ferrari et al. (2005) Ferrari et al. (2005) Ferrari et al. (2005); Nguyen et al. (2005); Horvath et al. (2006) Ferrari et al. (2005) Ferrari et al. (2005) Ferrari et al. (2005); Nguyen et al. (2005) Ferrari et al. (2005) Nguyen et al. (2006) Nguyen et al. (2006) Naviaux and Nguyen (2005) Nguyen et al. (2006); Horvath et al. (2006) Nguyen et al. (2006) Nguyen et al. (2005) Nguyen et al. (2006) Horvath et al. (2006) Horvath et al. (2006) Horvath et al. (2006) Horvath et al. (2006) Horvath et al. (2006) Horvath et al. (2006)
POLG 2243G->C/3629Ains 731C->T/2243G->C 1399G->A/2542G->A
1 1 3
W748S/Y1210X L244P/W748S A467T/G848S
1399G->A/3482 + 2T->C 1399G->A/2869G->C 1399G->A/1399G->A
1 1 2
A467T/Splice site A467T/A957P A467T/A467T
694C->G/752C->T;1760C->T 1399G->A/2246T->C 1399G->A/2554C->T 1399G->A/2617G->T 1399G->A/2740A->C
1 1 1 1 2
R232G/T251I;P587L A467T/F749S A467T/R852C A467T/E873X A467T/T914P
1399G->A/2897T->G 1399G->A/3057G->A 1399G->A/3518insGATC 1399G->A/3573G->T 926G->A/1880G->A 2209G->C/2300C->A 1399G->A/IVS15-9_12del 1399G->A/IVS21 + 1T->C 3286C->T;3708G->T/3286C->T;3708G->T
1 1 1 1 1 1 1 1 1
1399G->A;2653A->T/2637T->G;3428A->G
1
1880G->A/3287G->A 2243G->C/3428A->G-2542G->A
1 5
A467T/L966R A467T/W1020X A467T/L1173X A467T/K1191N R309H/R627Q G737R/A767N A467T/A467T/R1096C;Q1236H/ R1096C;Q1236H A467T;T885S/ Q879H;E1143G R627Q/R1096H W748S/E1143G-G848S
MPV17 149G->A/149G->A 498C->A/498C->A 148C->T/116-141del
1 1 1
R50Q/R50Q N166K/N166K R50W/fs
nc, not calculated. a The TK2 reference sequence utilized in the paper is O00142.
Horvath et al. (2006) Horvath et al. (2006) Davidzon et al. (2005); Nguyen et al. (2005) Spinazzola et al. (2006) Spinazzola et al. (2006) Spinazzola et al. (2006)
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Hepatocerebral MDS has been associated with mutations in three genes, encoding DGUOK, POLG, and MPV17, respectively. 2.3.1. DGUOK The mitochondrial deoxyguanosine kinase (dGK), (OMIM*601465) is a 2-deoxyribonucleoside kinase that mediates the phosphorylation of purine deoxyribonucleosides into the corresponding nucleotides. A total of 10 mutations in 12 patients have been reported for DGUOK. Contrary to what observed for TK2 mutations, most of the mutations found in the DGUOK gene are nonsense changes, although missense mutations have also been reported (Table 1). In our own series of five cases with hepatocerebral MDS, we found 3 DGUOK mutations in two unrelated patients. One patient had a homozygous missense mutation 749T->C resulting in a L250S amino acid change (Wang et al., 2005). The second patient was a compound heterozygote for two novel mutations, a 603–604del GA resulting in a frameshift with the creation of a premature stop codon, and a splicing site 8T->G mutation in intron 4 (Table 1). 2.3.2. POLG The mitochondrial DNA polymerase (pol c) (OMIM*174763) is essential for mitochondrial DNA replication and proofreading-based repair. It is composed of a 140-kDa catalytic subunit (pol cA) and a 55-kDa accessory subunit (pol cB), which functions as a DNA binding factor increasing the processivity of the polymerase holoenzyme. The holoenzyme works as an ab2 heterotrimer. The catalytic subunit is encoded by the POLG gene, which includes 23 exons, and is located on chromosome 15q25. Mutations in POLG are associated with an extremely heterogeneous spectrum of clinical presentations, from dominant and recessive forms of progressive external ophthalmoplegia (PEO) (Van Goethem et al., 2001; Lamantea et al., 2002; Agostino et al., 2003), sometimes with parkinsonism (Luoma et al., 2004), to juvenile spinocerebellar ataxia and epilepsy (SCAE) with or without dysarthria and lateonset ophthalmoplegia (SANDO) (Van Goethem et al., 2003, 2004; Winterthun et al., 2005), to AHS (Naviaux and Nguyen, 2005; Ferrari et al., 2005). Accumulation of multiple mtDNA deletions, rather than mtDNA depletion, in affected tissues such as muscle and brain, is the molecular hallmark of adPEO and arPEO. Multiple mtDNA deletions are also detected in the muscle tissue of patients affected with SCAE or SANDO, but this abnormality is neither precocious nor prominent in these syndromes. Depletion of liver mtDNA is more consistently associated with AHS, but again this feature may be absent in the early stages of the disease, indicating that it is neither sensitive, nor specific, for AHS (Nguyen et al., 2006). Therefore, the early phenotype in AHS, and also in SCAE and SANDO, may result from a subtler, non-replicative consequence of POLG mutations, for instance abnormalities in mtDNA proofreading and repair.
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The 1239 aa long Pol cA polypeptide is organized in three adjacent functional domains: a 50 > 30 exonucleolytic proofreading domain (amino acid 1–417), a linker domain (amino acid 418–755), and a polymerase domain (amino acid 756–1239). By reviewing POLG mutations identified in 31 AHS patients by different laboratories (Naviaux and Nguyen, 2005; Davidzon et al., 2005; Ferrari et al., 2005; Nguyen et al., 2005, 2006; Horvath et al., 2006) a total of 26 mutations were found. In several cases at least one allele carried either one of two mutations: the A467T mutation (34% of the alleles), or, more rarely, the W748S mutation (11% of the alleles). Eleven of the remaining 13 POLG mutations associated with AHS were unique to individual families. In conclusion, AHS can be considered a monogenic form of hepatocerebral MDS associated with a specific combination of POLG mutant alleles. In particular the A467T and the W748S are alternatively detected in most cases (Nguyen et al., 2006). In all cases, at least one allele is contained within the linker region (Horvath et al., 2006), which facilitates the genetic diagnosis of this condition. 2.3.3. MPV17 We recently found a new locus for hepatocerebral MDS on chromosome 2p21–23 by linkage analysis on a large multi-consanguineous Italian family. By using an integrative genomic approach (Calvo et al., 2006), we identified MPV17 (OMIM*137960) as a candidate gene for this disease. MPV17 is the human ortholog of the murine kidney disease gene Mpv17. Mutations in MPV17 have so far been found in several probands from three unrelated families (Spinazzola et al., 2006). The mutations were a 149G > A (R50Q) homozygous transition, found in our original Italian family; a 498C > A (N166K) homozygous transversion in the proband from a family of Moroccan origin; a missense change in one allele (148C > T, R50W), and a 25-bp deletion in the other allele (116–141del) in a proband from a Canadian family. The 116–141del predicts the synthesis of an aberrant and prematurely truncated polypeptide. The clinical presentation of MPV17 mutant MDS patients was characterized by severe, often fatal, hypoglycemic episodes, jaundice, and elevated levels of both lactate and hepatic enzymes in blood (Spinazzola et al., 2006). The liver showed progressive portal and lobular fibrosis evolving into terminal cirrhosis. Marked mtDNA depletion in liver, ranging from 5% to 15% of mean normal controls, was the molecular hallmark in the probands of all three families, associated with multiple defects of mtDNA-related respiratory chain (RC) complexes, particularly complex I and complex IV. Normal or mildly reduced levels of both mtDNA content and RC activities were found in muscle. We obtained experimental data indicating that, contrary to the alleged peroxisomal localization of the MPV17 gene product, MPV17p is a mitochondrial protein tightly bound to the inner mitochondrial membrane (Spinazzola et al., 2006).
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3. Pathogenic mechanisms 3.1. TK2, DGUOK, and SUCLA2 deficiency A balanced supply of deoxynucleotides is needed to warrant mtDNA replication. Thymidine kinase and deoxyguanosine kinase are part of the mitochondrion-specific salvage pathways of pyrimidine and purine nucleotides, respectively. Both pathways are essential for the replication of mtDNA since the mitochondrion is unable to synthesize dNTPs de novo. In addition, since mtDNA is replicated throughout the whole cell cycle, there is a constant need for nucleotides for mtDNA replication (Bogenhagen and Clayton, 1976; Saada, 2004). Elpeleg et al. (2005) postulated that also mutations in the SUCLA2 gene could determine a defect in the last step of the mitochondrial deoxyribonucleoside triphosphate (dNTP) salvage pathway, since SCS-A copurifies with and is tightly associated in a complex with nucleoside diphosphate kinase (NDPK) (Kadrmas et al., 1991; Kavanaugh-Black et al., 1994; Kowluru et al., 2002). NDPKs are ubiquitous protein kinases that also catalyze the exchange of terminal phosphates between tri- and di-phosphoribonucleosides and are crucial for maintaining the homeostasis of ribonucleotides and deoxyribonucleotides (Parks et al., 1973). One of the most striking features of MDS is the remarkable tissue specificity of the molecular and biochemical abnormalities. Different organs can be affected not only by mutations in different genes, but even by mutations in different part of the same gene, as we saw for pol cA. What are the mechanisms underlying this phenomenon? This important question remains largely unanswered. In case of TK2 or DGUOK mutations, the tissue-specific expression of MDS may be due to the differential expression of other genes regulating the mitochondrial dNTP pool, or to the variable effect of different gene mutations on the dNTP pools. The possibility should also be considered of a ‘‘rescue effect’’ on unaffected tissues by the activities of cytoplasmic deoxyribonucleoside kinases. For instance, Saada (2004) has hypothesized that in case of DGUOK mutations the activity of dCK, a cytosolic enzyme, could compensate for the lack of DGUOK activity in unaffected tissues; however, dCK activity is very low in brain and liver, which could explain why DGUOK deficiency leads to hepatocerebral MDS. As for TK2 deficiency, a different mechanism must be hypothesized, since cytosolic TK1 activity is very low in non-proliferating cells, including skeletal muscle. Therefore, defective supply of dTTP to mitochondria, due to mutations in TK2, cannot be compensated by the activity of TK1 in muscle. Saada et al. (2003) noted that none of the reported TK2 mutant patients is homozygous for a loss-of function mutation; at least one missense mutation is always present in these patients, suggesting that some residual enzymatic activity is retained in all cases, and is probably sufficient to rescue those tissues that have a high
basal TK2 expression. The activity of TK2 is the lowest in muscle mitochondria but it progressively increases in the heart and liver. In addition skeletal muscle has a very high energetic demand, as compared to other tissues. These two factors combined could explain the selective vulnerability of skeletal muscle in TK2-associated MDS. 3.2. POLG1 deficiency Disturbances of the nucleotide pool available for mtDNA replication, as well as abnormalities in the mitochondrial DNA polymerase, are likely to affect the rate, processivity or fidelity of replication, which could ultimately lead to instability of mtDNA. With a few exceptions (Ferrari et al., 2005; Horvath et al., 2006) the early-onset infantile encephalopathy + hepatopathy syndromes associated with POLG mutations are typical AHS patients. Almost all of them carried either the A467T or the W748S in the linker region of pol cA and in all cases at least one mutation affected the linker region (Ferrari et al., 2005; Horvath et al., 2006; see also Table 1). What is the basis of such a specific association? We do not know. And of course the mechanisms of tissue specificity remain unexplained as well. Recent studies in patients and in cell mutants may offer some clues. The A467T amino acid substitution determines a profound defect in the DNA polymerase activity of pol c, and it also impairs its processivity, by preventing the interaction of pol cA with pol cB, the accessory subunit (Chan et al., 2005). These effects of the A467T substitution are likely to cause the stalling of the enzyme during replication and, as a consequence, to promote the generation of deletions or the depletion of mtDNA. Patients homozygous for the A467T allele develop a later-onset form of AHS, or juvenile SCAE, which is also typically found in patients with homozygous W748S, or in A467T/W748S compound heterozygotes. The latter patients, however, consistently manifest a much more rapid downhill course (Tzoulis et al., 2006). Likewise, AHS, which is an extremely severe and early onset condition, is consistently associated with the presence of either the A467T or the W748S changes in one allele, in combination with a different pathogenic mutation in the second allele. These observations suggest a dominant negative effect of the A467T or W748S alleles over other pathological alleles and raise the possibility of a quaternary interaction between catalytic subunits in different heterotrimers, a phenomenon compatible with the synchronous model of mtDNA replication (Yang et al., 2002). 3.3. MPV17 deficiency The function of this gene and its role in the pathogenesis of MDS are still unknown (Spinazzola et al., 2006). The Dsym1 yeast strain shows a temperature-sensitive OXPHOS phenotype, suggesting a role for this protein in the cellular response to stress. In fibroblasts derived from Mpv17 knock-out mice the production of reactive
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oxygen species (ROS) is reduced, while its over-expression seems to induce the opposite effect, i.e. an increase level of ROS (Zwacka et al., 1994). These observations, together with the identification of MPV17 gene mutations causing hepatocerebral MDS suggest a role of Mpv17p in controlling ROS metabolism and the cellular response to stress. The availability of Mpv17 KO mouse, the first animal model for MDS syndrome, provides an invaluable tool to elucidate the function of this gene in mitochondrial homeostasis and to investigate its role in the pathogenesis of this disease. 4. Conclusion Mitochondrial DNA depletion syndromes could result from any disturbance in the mtDNA replication machinery. Imbalance of mitochondrial dNTPs pool as well as abnormalities of the mitochondrial DNA polymerase, can cause disorders characterize by a wide spectrum of clinical presentations. The recent discovery of the MPV17 gene and the future identification of additional MDS genes will probably expand and improve our knowledge on the pathogenic mechanisms underlining these disorders. Acknowledgments Supported by Fondazione Telethon-Italy (Grant No. GGP030039), Fondazione Pierfranco e Luisa Mariani, MITOCIRCLE and EUMITOCOMBAT network grants from the European Union Framework Program 6. References Agostino, A., Valletta, L., Chinnery, P.F., Ferrari, G., Carrara, F., Taylor, R.W., Schaefer, A.M., Turnbull, D.M., Tiranti, V., Zeviani, M., 2003. Mutations of ANT1, Twinkle, and POLG1 in sporadic progressive external ophthalmoplegia (PEO). Neurology 60, 1354–1356. Bogenhagen, D., Clayton, D.A., 1976. Thymidylate nucleotide supply for mitochondrial DNA synthesis in mouse L-cells. Effect of 5-fluorodeoxyuridine and methotrexate in thymidine kinase plus and thymidine kinase minus cells. J. Biol. Chem. 251, 2938–2944. Calvo, S., Jain, M., Xie, X., Sheth, S.A., Chang, B., Goldberger, O.A., Spinazzola, A., Zeviani, M., Carr, S.A., Mootha, V.K., 2006. Systematic identification of human mitochondrial disease genes through integrative genomics. Nat. Genet. 38, 576–582. Carrozzo, R., Bornstein, B., Lucioli, S., Campos, Y., de la Pena, P., Petit, N., Dionisi-Vici, C., Vilarinho, L., Rizza, T., Bertini, E., Garesse, R., Santorelli, F.M., Arenas, J., 2003. Mutation analysis in 16 patients with mtDNA depletion. Hum. Mutat. 21, 453–454. Chan, S.S., Longley, M.J., Copeland, W.C., 2005. The common A467T mutation in the human mitochondrial DNA polymerase (POLG) compromises catalytic efficiency and interaction with the accessory subunit. J. Biol. Chem. 280, 31341–31346. Davidzon, G., Mancuso, M., Ferraris, S., Quinzii, C., Hirano, M., Peters, H.L., Kirby, D., Thorburn, D.R., DiMauro, S., 2005. POLG mutations and Alpers syndrome. Ann. Neurol. 57, 921–924. Ducluzeau, P.H., Lachaux, A., Bouvier, R., Streichenberger, N., Stepien, G., Mousson, B., 1999. Depletion of mitochondrial DNA associated with infantile cholestasis and progressive liver fibrosis. J. Hepatol. 30, 149–155.
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