Lactic acidemia and mitochondrial disease

Molecular Genetics and Metabolism 89 (2006) 3–13 www.elsevier.com/locate/ymgme

Lactic acidemia and mitochondrial disease Brian H. Robinson ¤ Metabolism Research Programme, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ont., Canada M5G 1X8 Departments of Biochemistry and Paediatrics, University of Toronto, 1 King’s College Circle, Toronto, Ont., Canada M5S 1A8 Received 28 March 2006; received in revised form 25 May 2006; accepted 26 May 2006 Available online 18 July 2006

Abstract Lactic acidemia is present in the majority of patients with mitochondrial oxidative defects as well as in disorders of gluconeogenesis. An understanding of the dynamics of lactic acid metabolism in the human body and the inXuences on lactate/pyruvate ratios exerted by changes in cellular redox state allows for the development of diagnostic algorithms based on clinical and biochemical phenotypes. Mitochondrial disorders can be due to defects in nuclear genes directly aVecting the respiratory chain assembly or function, mtDNA genes aVecting the respiratory chain or nuclear genes inXuencing mtDNA structure and viability. In this review, we look at the classiWcation of mitochondrial disease from the perspective of not just the genetic and biochemical etiology but also from the perspective of the clinical phenotypic expression. © 2006 Elsevier Inc. All rights reserved. Keywords: Lactic acidemia; Mitochondrial disease; Mitochondrial DNA; Leigh disease

Introduction Lactic acidemia is known to be a presenting feature of many inborn errors of metabolism involving defective mitochondrial metabolism yet the appearance of increased lactic acid levels in the blood is not a constant feature of all the mitochondrial oxidative defects. It is a common presenting feature in some, intermittent in others and for some, it is not a presenting feature at all. Why is this? The overall metabolic Xuxes in the human body that involve lactic acid are in a delicate balance, with the end result that on a daily basis blood lactate levels are rarely elevated beyond the normal range, in most laboratories between 0.8 and 2.0 mM [1]. The only circumstances in which a healthy person displays an elevated blood lactate is one in which anaerobic exercise has been performed, anything from running for a bus or up a Xight of stairs, to more prolonged exercise of the fast-twitch muscles, such as might be involved in playing a game of squash or performing serial 400 m sprints [2]. Games of tennis and squash played vigorously routinely *

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result in an end of game lactate of 6–10 mM, while 3 £ 400 m sprints done in a 10–15 min period can result in a lactate of 20 mM [2]. The majority of proven mitochondrial oxidative defects present with a raised blood or CSF lactate and this is often accompanied by a raised lactate to pyruvate ratio signifying a change in cellular redox state. So what happens metabolically in a patient with mitochondrial disease that determines whether or not they display a chronically elevated lactate? The normal source of lactic acid in the body is from tissues that are glycolytically active by virtue of their structure or location. Red cells have no mitochondria, white cells have few, while skin cells and kidney medulla have a mode of metabolism that has them preferring to derive at least part of their energy from glycolytic lactate production. We can calculate that the cultured skin Wbroblast derives a quarter of its energy from glycolysis and three quarters from oxidation, so that six times as much pyruvate goes to lactate as is oxidized. Redox state and lactate/pyruvate ratios We have shown that oxidative defects can be at least partially assorted by the observed change in redox state in

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B.H. Robinson / Molecular Genetics and Metabolism 89 (2006) 3–13

cultured skin Wbroblasts. Because the respiratory chain is not aVected, most PDH defects display a normal redox state and thus a normal or even low lactate to pyruvate ratio. This is also true for pyruvate carboxylase deWciency. Many of the defects aVecting complex I and complex IV, however, do have an easily demonstrable increase in the ratio of lactate to pyruvate both in skin Wbroblasts and in the blood of the patient [3–5]. However, there are less severely aVected patients with respiratory chain defects that have normal lactate to pyruvate ratios measured in Wbroblasts. These cases are often the least likely to exhibit a raised blood lactate. Why does this happen? Skin Wbroblasts with a 50% deWcit of cytochrome oxidase (COX) activity show no change in redox state, whereas cells with residual levels of COX of 35%, 25% and 10% of normal activity show progressive increases in cellular redox state. Presumably for electron Xow in the respiratory chain, COX does not become rate limiting until activity falls below 40%. Likewise in complex V deWciency, a functional defect depressing the potential rate of ATP synthesis to 50–60% of normal also does not change redox states in skin Wbroblasts [6]. On the other hand, the complex I mtDNA defect G13513A, when it presents as Leigh disease, usually displays increased blood lactate, has a signiWcant depression of respiratory chain activity and has a change in redox state detectable in Wbroblasts, while most patients with mtDNA mutations in ND1, ND4 and ND6, leading to Leber hereditary optic neuropathy (LHON), have none of these features [7]. About 20% of complex I defects, which are mostly nuclear in origin, were reported by Smeitink et al. [8] as having a normal level of blood lactate. In general, the more severe the defect, the more likely it is to display an increased lactate in body Xuids [4].

Lactic acid Xux The cumulative total of lactate produced per day glycolytically is between 70 and 110 g for the human body, of which 33.5 g each comes from red cells and skin with another 20 g or so from skeletal muscle and the brain and a small amount from intestinal mucosa [9] (Fig. 1). Observed removal rates after exercise have been measured as high as 250–330 g per 24 h. This demonstrates that the capacity for homeostatic maintenance of lactate is dependent on rates of synthesis by glycolytic tissues and removal, largely by gluconeogenic tissues but with some oxidative removal by muscle [10]. The lactate generated by 3 £ 400 m sprints, about 52 g produced in a matter of minutes takes 3–5 h to be removed by the liver [2]. The continual Xux is regulated very carefully, so that when excess lactate in the circulation is encountered by the liver, pyruvate carboxylase becomes more active. Likewise, when muscle encounters increased lactate under aerobic conditions the resulting increase in pyruvate activates the pyruvate dehydrogenase complex by inhibition of PDH kinase [11]. These two enzymes are the key regulation sites and it is worth bearing in mind that the shutdown of pyruvate metabolism by defects in the mitochondrial respiratory chain will use the same regulation points. Gluconeogenic defects: lactic acidemia with fasting The pathway of gluconeogenesis in the liver has two major roles as indicated in the consideration of lactic acid Xux. First, the constant generation of lactate by non-oxidative tissues requires that lactate be resynthesized into

Fig. 1. The daily Xux of glucose and lactic acid in the human body. The average 70 kg man ingests 300 g of carbohydrate per 24 h. After processing by the GI tract and liver, 250 g of this is released as glucose plus another 75 g generated by the Cori cycle for a total liver output of 325 g. One hundred and Wfty grams of glucose is utilized by the brain and 100 g used by other tissues to include heart, skeletal muscle and kidney. Various tissues process glucose to lactic acid, particularly skin and blood, accounting for a net production of 75 g lactic acid per day. Small amounts of lactate are produced by brain and skeletal muscle (25–30 g) but an equivalent amount is re-used oxidatively by heart and kidney cortex. Thus, 75 g of lactic acid is returned to the liver for processing via gluconeogenesis to glucose.

B.H. Robinson / Molecular Genetics and Metabolism 89 (2006) 3–13

glucose in the liver and this happens at a Xux rate of about 70 g per day for an adult human. Second, in the fasting state, amino-acids are quantitatively released from muscle, broken down into useful precursors and made into lactate at a rate approaching 200 g per day. DeWciency of fuctose-1,6-bisphosphatase (OMIM 229600) is a rare defect whose compromise aVects the Xux in the pathway of gluconeogenesis in a fashion which produces marked lactic acidemia only when fasting occurs [12]. This can become life-threatening when the liver glycogen stores become exhausted because at this point the blood glucose falls to less than 0.5 mM. This combination of lactic acidemia and severe hypoglycemia is even more pronounced in Glycogen storage disease, type I (OMIM 23220). The lactic acidemia here is present constantly as both glycogenolysis and gluconeogenesis are eVectively compromised by deWciency of glucose-6-phosphatase (GSD-Type 1a), glucose-6-phosphate translocase (GSDType 1b), or the endoplasmic reticulum phosphate translocase. (GSD-Type 1c). The Wnal reaction of gluconeogenesis appears to be at least in part compartmentalised in the endoplasmic reticulum. so that hydrolysis of glucose-6-phophate requires G6P translocation into the compartment and glucose and phosphate translocation out of the compartment for the reaction to be fully functional [13]. Pyruvate carboxylase deWciency (see below) exhibits a similar increase in lactic acidemia in fasting with a tendency towards hypoglycemia, but the eVect is not as pronounced. PDH deWciency: the importance of carbohydrate fuelled power generation The deWciency of the pyruvate dehydrogenase complex is now well documented and mutations have been described in the genes (PDHA1, PDHB, DLAT, DLD, PDX1 and PDP1) encoding the E1, E1, E2, E3, E3BP and PDP1 proteins [1,14–18]. There is a gradation of phenotype with decreasing PDH complex activity [1]. The PDH complex activity can be documented accurately in cultured skin Wbroblasts, and with some exceptions, this activity in males is reasonably representative of somatic residual activity. Thus in a series of PDH deWciency patients with E1 defects, we showed a gradation of symptoms. The highest residual activity is about 70%. The phenotypes vary from fatal infantile lactic acidosis at the lower end to more ataxia cases only at the higher end with Leigh syndrome and cerebral atrophy cases taking up the middle. All of these patients have deWned mutations in the X-linked PDHA1 gene encoding PDH-E1 subunits (OMIM 312170). We estimate, based on PDH measurements in the human brain that the PDH capacity of the brain is 180–210 g per 24 h. Since the human brain utilizes 150 g of glucose per day, the Xow through pyruvate dehydrogenase is set at 67–79% of total capacity [1]. Measurements in mammalian brain have shown that the phosphorylation state of PDH-E1 is such that the complex is 60–70% active under fed conditions, which agrees with the required activity. The genetic defects

5

in E1 are such that even partial defects with up to 60% activity are capable of showing symptoms [1,11]. When this residual activity is close to the actual required capacity, the symptoms are intermittent, as seen with many ataxic patients. In more severe defects, the eVects are chronic and lead to progressive neurodegeneration [17]. In the extreme, the problems lead to overwhelming fatal infantile lactic acidosis [1]. There are equal numbers of male and female patients with PDHA1 defects, but with somewhat diVerent phenotypes. What holds for E1 males does not hold for E1 females because the X-inactivation pattern inXuences the outcome [19]. The E1 deWcient females can have similar phenotypes to E1 deWcient males, but there are more females with psychomotor retardation and enlarged ventricles and a category of males with carbohydrate sensitive ataxia. In females who have one normal allele and one mutant allele, the loss of cells bearing non-inactivated mutant X chromosomes and normal survival of the cells with X-inactivated mutant, there is a progressive thinning of the cerebral cortex which creates symmetrically enlarged ventricles [1]. PDH-E2 (OMIM 246348) and E3-BP (OMIM 246900) deWcient patients are similar to E1 defects depending again on residual activity. E3 protein defects are unusual because of the eVect that E3 deWciency has on 2-oxoglutarate, branched chain keto-acid dehydrogenase and glycine cleavage enzyme activities, all of which require lipoamide dehydrogenase [1,20]. While the original defects in the E3 enzyme activity documented were usually quite severe, some more recent cases have emerged with milder phenotypes. These children have diVerent degrees of developmental delay and they appear to survive [21]. The residual activity of PDH complex in some cases is only just on the deWcient range, with the 2-OGDH being substantially more compromised. Finally, the Wrst genetically proven case of PDP1 deWciency (OMIM 608782) reported by our group recently was surprising in that the presenting symptoms were relatively mild despite an almost complete absence of enzyme activity [22]. The two aVected siblings homozygous for a mutation that eVectively removed a key valine residue were aged 10 and 12 years, with slightly delayed psychomotor development and minimally elevated lactate (3 mM) which rose rapidly on exercise, precipitating exercise intolerance. Cultured skin Wbroblasts showed a native PDH complex activity of 25% of total compared to the usual 70–75%, but this could be restored to normal by pre-incubation with dichloroacetate, an inhibitor of PDH kinase. The 3 bp deletion in the PDP1 gene produced a PDP1 with »5% of normal activity when produced as a recombinant protein. This eVective decrease in phosphatase activity functionally aVects the behaviour of cultured skin Wbroblasts so that they produce more lactic acid and carry out less oxidation of pyruvate. The lactate production in Wbroblasts goes from the 133 nmol min¡1 mg¡1 seen in controls to 300 nmol min¡1 mg¡1 which is the amount expected from a

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B.H. Robinson / Molecular Genetics and Metabolism 89 (2006) 3–13

drop in pyruvate oxidation rate from 0.45 nmol min¡1 mg¡1 to the 0.24 observed in the PDP1 deWcient patients [22]. There are two PDH phosphatases in mammals, the PDP1 being the most important in brain, skeletal muscle and heart, and unlike PDP2 it is responsive to changes in Ca2+ concentration [23]. When PDP1 is absent, the PDP2 can partially deputize and keep the PDH from remaining completely phosphorylated by the PDH kinases but not enough to escape problems caused either by exercise, where Ca2+ stimulates PDH in muscle, or in neuronal stimulation where PDH is also stimulated by Ca2+ [22]. Pyruvate carboxylase: feeding the cycles Pyruvate carboxylase (PC) deWciency (OMIM 266150) with three almost distinct phenotypes is illustrative of the roles played by pyruvate carboxylase in diVerent metabolic pathways in diVerent tissues. The A-form of the deWciency was originally described as a disturbance of lactic acid metabolism, the patients having a tendency towards hypoglycemia accompanied by lacticacidemia, sometimes with increased levels of ketone bodies [24,25]. The explanation for this lies in the unusual situation that pyruvate (in the form of alanine or lactate) arrives at the liver, especially in the fasting state, ready to be converted to glucose as part of the Cori cycle. Because little PC is available two things happen. First there is a lack of oxaloacetate, the product of pyruvate carboxylation, and second there is excessive oxidation through PDH at a time when fatty acids are being oxidized. The excess of acetyl CoA generated in the liver then drives ketone body formation. Children with PC type A deWciency suVer from psychomotor retardation and while they may survive to maturity they function, albeit at a lower than average level, needing special care and schooling. Two speciWc Amerindian missense mutations, A610T and M743I were identiWed as the major cause of type A PC deWciency in North America [26,27]. The type B PC deWciency Wrst described in France had a more severe phenotype attached to it. Very few infants with this form survive past three months of age. This is because in most of these individuals there is no detectable PC protein and this produces a much more severe phenotype with citrullinemia, hyperammonemia and lactic academia all developing in the immediate post-natal period [24]. The biochemical phenotype is driven by lack of oxaloacetate as provider of 4 carbon replenishment for the citric acid cycle, gluconeogenesis and for provision of aspartate as the second nitrogen donor in the urea cycle. In both type A and B, phenotypes activity is low in all tissues [28]. In the type C phenotype known as the benign phenotype, the only abnormality is the occurrence of episodes of lactic academia [29,30]. The psychomotor development is normal. This has led to the hypothesis that the reason for this is that the two diVerent transcripts that are produced encoding PC diVer in the Wrst two exons. A mutation aVecting the Wrst two exons of the liver form would leave the brain form being expressed normally in the face of a liver

deWciency. Detailed analysis of the transcripts in one patient produced an unusual combination of a liver promoter mutation but in the same allele as a missense mutation, the other transcript being completely normal. Without analysis of liver-speciWc transcripts and resulting protein level it is not easy to judge if this scenario is a viable description of the genetic defect. Nuclear versus mitochondrial DNA (mtDNA) encoded defects The 13 open reading frames of mitochondrial DNA (mtDNA) that encode protein components of the mitochondrial respiratory chain are vital building block templates that assume a lot more signiWcance in disease than was at Wrst thought. MtDNA as a source of mitochondrial disease problems has undergone revision several times since the discovery of mitochondrial pathology [31,32]. In the context of mtDNA versus nuclear DNA as a source of genetic causative lesions, much of what has been learned revolves around the role of mtDNA versus nuclear DNA encoded components of the respiratory chain and associated assembly factors. Percentage heteroplasmy of mtDNA, function of the mutated protein, degree of assembly of mature complexes and Xux generating capacity of the complex all enter into the downstream biochemical and phenotypic characteristics of each defect. In Fig. 2, the known genetic defects have been arranged by presenting clinical phenotype versus observed biochemical phenotype. This can serve as a basis for classiWcation by phenotype. Fig. 3 shows the breakdown of some of the defects by assignment to the metabolic pathways of energy metabolism. Documented protein coding defects of mtDNA have become more numerous since the original 11778 mutation described for Lebers hereditary optic neuropathy (LHON) and disorders of comparatively more severe phenotype have been described for all complexes of both nuclear and mtDNA genetic origin [33–35]. Well-documented heteroplasmic mutations in ND3, ND5 and ND6 leading to complex I deWciency (OMIM#252010) have now been shown to be causative for Leigh syndrome [7,36–38]. Mutations in cytochrome oxidase genes COX I and COX II likewise have been documented in association with quite severe neurodegeneration. Defects in cytochrome b encoded by the MTCYB gene leading to complex III deWciency (OMIM#124000) appear to be of a milder phenotype, while mutations in ATP6 producing complex V deWciency (OMIM¤516060) are likely to produce basal ganglia lesions when present at high heteroplasmy, but at low titres ataxia and retinitis pigmentosa (RP) are more likely to be presenting features [39]. Comparison of the protein coding defects of mtDNA with those documented for nuclear DNA are instructive in that there are some diVerences but also many similarities. There are nine diVerent nuclear-encoded subunits of complex I in which disease-causing mutations have been

B.H. Robinson / Molecular Genetics and Metabolism 89 (2006) 3–13

7

Patient with Suspected Mitochondrial disease ±Lactic Acid Elevated in Blood or CSF Neuromuscular

Radiology

MRI Muscle or Skin Biopsy

Leigh or Leigh -like L/P Elevated

PDH

Complex I

Nuclear

MtDNA

NDUFS1 NDUFS3 NDUFS4 NDUFS7 NDUFS8 NDUFV1

10158T>C 10191T>C 11696A>G 11778G>A 12706T>C 13045A>T 13084A>T 13513G>A 14484T>C 14459G>A

Complex II

L/P Normal

Ophthalmology

MRI: Cerebral atrophy,leukodystrophy,spongiform changes ± Hepatopthy

Nuclear

Nuclear

PDHA1 DLAT DLD

PDHA1 PDHB PDX1 DLD NDUFS1 NDUFS4 NDUFV1

Cardiac

Neurology Ataxia,PEO

Optic Neuropathy Optic Atrophy Retinal Degeneration

Ventricular Hypertrophy

MMyopathy

Conduction defect

MtDNA

Exercise Intolerance Nuclear PDHA1

tRNA Leu tRNA Val

MtDNA

MtDNA

Nuclear

3460G>A 4160T>C 14484T>C 14459G>A

tRNA Leu

NDUFS2 NDUFS4 NDUFS8 NDUFV1 NDUFV2

tRNA Ile tRNA Gly

SDHA

MtDNA Complex III

BCS1

MTCYTB

UQCRB

Complex IV

Complex V

Others

SURF-1

MtDNA

COX15

COXI

LRPPRC

COXII

ATP12

MtDNA MtDNA 8993T>G 8993T>C 9176T>C 9185T>C

SCO1 SCO 2 COX10

MtDNA MtDNA

tRNA Lys T8993G T8993C

Nuclear

Nuclear PC Type A PC Type B

SUCLA2 DGUOK

8993T>G 8993T>C 9185T>C

Nuclear OPA1

TK2 KIF1B MFN2 FRDA

PREO1 POLG ANT1 TAZ

Fig. 2. Gene defects associated with mitochondrial disease. The diagnostic algorithm begins with a patient who presents with suspected mitochondrial disease, many of which are known present with an elevated level of blood or CSF lactate. The subsequent investigations may depend on the results of assays performed on skin Wbroblasts or muscle biopsy for the PDH complex and the mitochondrial respiratory chain, listed vertically on the left hand side of the table. Clinical investigation will produce a hierarchy of presenting symptoms, listed horizontally. Fitted into the matrix created by the horizontal and vertical axis, the known gene defects are listed as being due to nuclear or mtDNA encoded genes. The Leigh disease category are subdivided into normal or elevated lactate to pyruvate ratio in blood or Wbroblasts which is often a useful discriminator. An additional category of “others” is listed vertically below complex V so that defects aVecting mitochondrial energy delivery not primarily associated with the respiratory chain can be included in clinical presentation context. See text for further details.

linked to a decrease in complex I activity. The most common presenting features of these cases is either Leigh syndrome (e.g., NDUFS7), Leigh syndrome with cardiomyopathy (e.g., NDUFS2), or cerebral atrophy/leukodystrophy (e.g., NDUFV1). Otherwise, there is often a combination of basal ganglia necrotisation and leukodystrophy or fatal infantile lactic acidosis. A small number of complex IV deWciency (OMIM#220110) cases have also been documented as being due to mtDNA mutations in COX I, COX II or COX III [40–43]. These can resemble the nuclear-encoded defects now well documented in SURF1, LRPPRC (Leigh syndrome) and COX 10 (leukodystrophy) or COX 15 (Leigh or leukodystrophy), but in general the presentations are milder and of later onset. The potentially severe defects are tempered because of heteroplasmy, a recurring theme in mtDNA-encoded defects.

An illustration of variable phenotype is demonstrated by the rare deWciencies of complexes II (OMIM#252011) and III (OMIM#124000). Mutations in SDHA, the Xavoprotein subunit of SDH, cause Leigh syndrome, while mutations in SDHB, SDHC and SDHD in a dominant mode are the cause of predisposition to paraganglioma and pheochromocytoma [44–48]. BCS1, an assembly gene for complex III causes a severe deWciency in complex III and a clinical phenotype of Leigh syndrome, while a deletion in subunit VII (UQCRB) caused hypoglycemia with lactic acidosis [49–51]. MtDNA-encoded mutations in cytochrome b, however, produce defects resulting primarily in exercise intolerance [52]. The mutations in the two protein coding genes involved in mtDNA and complex V, ATP6 and ATP8, produce a variable picture of diVerential onset and diVerential severity. Two mutations, 8993T > G and 8993T > C, produce Leigh syndrome as the

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B.H. Robinson / Molecular Genetics and Metabolism 89 (2006) 3–13

Nuclear V

Glucose

IV

ATP12 SURF-1 COX10 COX15 LRPPRC

MtDNA ATP6

8851T>C 8993T>G 8993T>C 9176T>C 9185T>C

COXI COXII COXIII

BCS1

III

UQCRB

MTCYTB

SDHA

PC

Oxaloacetate

Pyruvate

NAD NADH Acetyl CoA

LDH

PDHa

PDK1 PDK2

Lactate

PDP1

PDK3

PDP2

PDK4

SDHB

II

SDHC SDHD

I

NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS6 NDUFS7 NDUFS8 NDUFV1 NDUFV2

ND1 ND3

ND4 ND5 ND6

3460G>A 4160T>C 10158T>C 10191T>C 11696A>G 11778G>A 12706T>C 13045A>T 13084A>T 13513G>A 14484T>C 14459G>A

PDH i Fig. 3. The relationship of the major oxidative complexes to the known gene defects of energy metabolism. All cells utilize glucose and produce pyruvate by glycolysis. The pyruvic acid is either then reduced by NADH to lactate, if the mode of metabolism is preferentially glycolytic or if there is a problem reoxidizing NADH. Alternatively, pyruvate may be metabolized to oxaloacetate by pyruvate carboxylase (PC) or to acetylCoA by the pyruvate deydrogenase complex (PDH). The PDH complex exists in two forms, an active form PDHa and an inactive form PDHi. The phosphorylation of PDHa to produce PDHi is catalyzed by one of four PDH kinases (PDK1, 2, 3 and 4) and the return to the active form is catalyzed by speciWc PDH phosphatases (PDP1 and PDP2). Re-oxidation of NADH generated by PDH and the other citric acid cycle enzymes is carried out by the respiratory chain assembly, consisting of four electron transport complexes, complex I (NADH-ubiquinone reductase), complex II, (succinate-ubiquinone reductase), complex III (ubiquinol-cytochrome c reductase) and complex IV (cytochrome c oxidase). The synthesis of ATP is catalyzed by complex V (oligomycin-sensitive ATP synthase). Nuclear or mtDNA gene defects associated with these complexes are listed on the right hand side of the diagram. The mitochondrial DNA mutations are listed as actual mutations for complexes I and V alongside the individual MtDNA genes responsible. See text for further details.

most severe phenotype, with intermediate phenotypes of ataxia and retinitis pigmentosa and a mild phenotype of retinitis pigmentosa only, all depending on the percentage heteroplasmy [6,53,54]. In the protein coding defects of mtDNA the heteroplasmy does not have wide variations across tissues, with some exceptions. 9176T > C and 8851T > C also produce a Leigh syndrome presentation but are rare, as is the 9185T > C mutation which again produces a Leigh/cerebellar phenotype of onset at 7–9 years of age at high heteroplasmy with an ataxia (CMTlike) at lower heteroplasmy [55–57]. Only one example of a nuclear-encoded complex V defect exists, that described by De Meirleir et al. in ATP12, involved in complex assembly, which produced an early onset Leigh syndrome with cataracts and dysgenesis of the corpus callosum [58]. This joins PDH deWciency and complex I deWciency as a mitochondrial disease associated with agenesis or dysgenesis of the corpus callosum. Houstek et al. [59] analysed a patient with hypertrophic cardiomyopathy and mitochondrial complex V deWciency and showed it to be a nuclear-encoded defect. No genetic defect was deWned.

tRNA defects: variability in the extreme No genotype/phenotype analysis has been as perplexing as attempts to put rhyme and reason to certain tRNA defects in mtDNA, giving rise to variable phenotypes [39]. There does appear to be at least some domination of phenotype aYliation that depends on the amino acid speciWcity of the tRNA. While much has been written on this subject, the etiology of the variations can be reduced to Wve possible major causes [60,61]. 1. The complex most aVected by the amino acid speciWc to the tRNA [62]. 2. The percentage heteroplasmy and its variation in tissues. 3. The localization of the mutation within the tRNA. 4. The position of the tRNA in relation to the processing of mtDNA transcripts. 5. The stability of the mutated tRNA and role of tRNA secondary modiWcation. tRNA defects in general have been linked with the appearance of ragged red Wbres in muscle stains with

B.H. Robinson / Molecular Genetics and Metabolism 89 (2006) 3–13

Gomori trichrome stain. That mitochondria should proliferate in muscle and other tissues under conditions of low energy production is not surprising. The mechanism for this ampliWcation is thought to be a co-operation between transcription factors PGC-1 and PPAR in induction of the respiratory factors nRF1 and nRF2 [63,64]. These in turn drive the replication of mtDNA and both the synthesis of mtDNA and nuclear-encoded mitochondrial proteins. The prototypical tRNA defects, the 3243A > G mutation in the tRNALeu(UUR) responsible for MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episodes) and the 8344A > G mutation in the tRNALys responsible for MERRF (myoclonic epilepsy with ragged red Wbres) are instructional in that they both are tRNA defects with ragged red Wbres yet the clinical presentations are diVerent [65,66]. Why is this? Should not a defect in one of the tRNA molecules necessary for protein synthesis produce similar eVects? This answer to this is complicated. The observations are that (a) the severity of the defect in both cases in any particular patient is proportional to the percentage of mutant mitochondrial DNA present in muscle (and presumably brain), (b) the ragged red Wbres that appear in MELAS are COX positive (i.e., they have positive stain for cytochrome oxidase), while in MERRF the Wbres are largely COX negative [39]. Measurement of electron transport components in the respiratory chain of mitochondria place the severity of the MELAS defect more in complex I while MERRF seems more to aVect complex IV. It is interesting to note that certain mtDNA defects exclusive to complex I, for example in the 13513 and 13514 defects in ND5, can either produce stroke-like episodes typical of MELAS or the more typical presentation of Leigh syndrome, or both, depending largely on severity [7,67]. This suggests that interface with the membrane of complex I may be crucial to producing the right conditions for MELAS to occur since it does not happen in nuclear-encoded versions of complex I deWciency [8,68–70]. The reason for the focus of deWcient activity residing in complex I for tRNALeu(UUR) could be that the mtDNA-encoded complex I subunits have the highest average leucine content (21.4%) compared to 12.9% for COX I, II and III, 17.2% for ATP6 and ATP8 and 16.9% for cytochrome b. More convincing is the observed accumulation of partially processed transcripts containing 16SrRNAtRNA-ND1, which seems to be accompanied by a decrease in mature tRNALeu(UUR) and ND1 transcripts [71–73]. This would lead to a deWcit of complex I by a more direct route of limitation of ND1 protein. In the case of MERRF there is also a decrease in the level of functional tRNALys transcripts and observed truncated versions of COX I and II [62]. In the case of the lysine content of COX this does not seem to be a viable part of the mechanism since the lysine content of COX I, II and III is not any greater than other complexes. tRNAPhe and tRNAGly defects causing MERRF and hypertrophic cardiomyopathy, respectively, also have reduced COX I and this subunit does have a high glycine content [74]. The association of tRNAIle mutations

9

with hypertrophic cardiomyopathy though may have an element of this too, since the isoleucine content of cytochrome b is the highest (10.4%) compared to mtDNAencoded subunits of other complexes [75–77]. The secondary modiWcation of tRNA sequences may also have a major eVect on the mutated tRNAs functional capacity. It has been shown that the wobble position uridine in the tRNALeu(UUR) is modiWed to taurinomethyluridine to enable it to function as an anticodon for both UUG and UUA [78]. Recently, it has been demonstrated that when the point mutations 3243A > G, 3244G > A, 3258T > C, 3271T > C and 3291T > C are present, the modiWcation is lacking and that this has the eVect of severely impeding decoding of UUG but not UUA codons [79]. A similar eVect on the secondary modiWcation of the tRNA was also seen for tRNALys in the 8344A > G leading to MERRF [80]. While the full picture of tRNA speciWcity is probably not yet understood, a combination of these critical factors will likely prove to be important. MtDNA deletion and depletion Deletion of segments of mtDNA were recognized as a causative of myopathy very early on [31,32], then it became apparent that in some disorders, mtDNA could also become severely depleted. In both scenarios, again, ragged red Wbres (RRF) are commonly seen as muscle tries to compensate for lack of energy by encouraging mitochondrial proliferation. The common deletion spanning 8432–13,460 of mtDNA which appears to be generated because of Xanking perfect repeat sequences is seen in Kearn-Sayre syndrome (OMIM#530000), Pearson syndrome (OMIM# 55700) and progressive external ophthalmoplegia [32]. Duplications also happen, but these are comparatively rare and seem to be generated by fusion of two deleted mtDNAs. The duplications present as myopathy with diabetes, deafness and sometimes nephropathy [81–83]. The major eVect of ring deletion is that tRNAs are eVectively removed in the case of the common deletion, those for Gly, Arg, His, Ser and LeuUCN together with COX III and various ND complex I transcripts. Thus the RRFs seen in deletions are COX negative. Multiple deletions are generated by a series of rare autosomal dominant nuclear defects. These include mutants of DNA polymerase (POLG) (OMIM#157640), twinkle (OMIM#605286), a DNA helicase (PEO1) and the muscle and brain speciWc adenine nucleotide transporter (ANT)1 (OMIM#609560 and OMIM#251880). All of these patients present with PEO usually as adults [84–86]. The observation is that in muscle mtDNA has multiple deletions of varying sizes. This could be generated by a free radical mechanism but it is more likely to be a consequence of either the stalling of DNA replication or the DNA repair process [87]. In another disease, spinocerebellar ataxia caused by AR mutations in the twinkle gene, there appear to be no deletions of mtDNA occurring so the mechanism may not be universal [88,89]. Mitochondrial depletion

10

B.H. Robinson / Molecular Genetics and Metabolism 89 (2006) 3–13

Table 1 The occurence of lactic acidemia in severity assorted by defect Defect

Complex I

Normal lactate <2.0 mM

Some nuclear mtDNA SDHC SDHD LHON mutations

Moderate lactate Nuclear defects 2–5 mM mtDNA ND5,ND6

Severe lactate >5 mM

Nuclear defects mtDNA ND3,ND5

Complex II Complex III Complex IV

SDHA

Complex V

PC

PDH

tRNA mutation

MtDNA depletion

Some Cyt b Some LRPPRC ATP6 Ataxia Type C Mild PDHA1 Low heteroplasmy PDHE3 tRNALeu tRNALys MTCYTB UQCRB

LRPPRC COX 10 COX 15 COX I COX II

BCS1

SURF1

syndrome has been recognized as a defect where mtDNA copy number decreases producing COX deWciency and proliferation [90]. All causes so far of mtDNA depletion have been attributed to interference with mtDNA replication via depletion of deoxynucleotide pools or depression of DNA polymerase. In the hepato-cerebral form the mitochondrial cause has been found to be homozygous mutations in the DNA polymerase gene. The mutation detected G848S/ W748S in this gene was the same for all four patients reported and clinical presentation was of progressive liver failure with spongiform encephalopathic changes. Another form of hepatocerebral mtDNA depletion has been shown to be due to mutation in the deoxyguanosine kinase gene (DGUOK). The myopathic form of the disease, however, is due to mutation in the thymidine kinase (TK) gene [91]. The succinyl CoA thiokinase gene (ADP speciWc) SUCLA2, has been shown to be responsible for another form of depletion, not apparently as a result of the resulting defect in the enzyme but due to the fact that it is in complex with the nucleotide diphosphate kinase in the mitochondria [92]. Patients with this form of the defect had a presentation of Leigh syndrome. The biochemical phenotype of depletion syndrome manifests as a COX deWciency only with rare expression of the defect in Wbroblasts. This actually emphasizes the nature of the defects leading to mtDNA depletion. The fact that most of them are corrected in culture suggests that a plentiful supply of purine and pyrimidine bases in the culture medium is a way of overcoming these defects in rat deoxynucleotide maintenance, presumably by a form of mass action eVect. The presence of lactic acidemia is a relative constant in the mtDNA depletion syndromes, lactate levels being in the 2–5 mM range for both muscle and hepatocerebral forms. These elevated levels suggest that altered Xuxes in appearance and removal are out of balance between tissues resulting in an increased ambient level. Table 1 summarises the known correlation of chronic lactic acidemia with various gene defects. In the table, the blood lactate measured at rest is correlated with the type of defect. In defects where the disease is progressive, there may be a concomitant increase in the blood lactate, for instance in cases of mtDNA depletion where hepatopathy gradually compromises lactate removal by the liver. In other cases, lactic acidosis can be

ATP 6 leigh ATP12

TypeA PDHA1, PDHB DLAT DLD PDP1

High heteroplasmy DGUOK tRNALeu tRNALys TK2 SUCLA2 POLG

TypeB Severe PDHA1

precipitated by infection, stress or trauma. A distinct cohort of patients present as severe infantile lactic acidosis. These are typically severe PDHA1 and DLD cases, severe nuclear or mtDNA encoded defects of complex I, severe type B pyruvate carboxylase deWciency and SURF1 defects associated with cytochrome oxidase deWciency. This adds up to about 10% of the cases with severely elevated lactate, 20% may have a normal lactate while the remaining 70% has a resting lactate of 2–5 mM. This latter group encompasses the typical presentation for most of the documented defects of mitochondrial oxidative metabolism. Final mention should be made of some defects listed in Fig. 2 that produce symptoms similar to oxidative defects but in fact are caused by defects in mitochondrial fusion. These include the MFN2 (OMIM#609260) and OPA1 (OMIM#165500) defects which produce ataxia and optic atrophy respectively [93,94]. Mitochondrial movement within the cell is compromised by KIF1B defects, again producing an ataxic syndrome, Charcot-Marie-Tooth-2A1(O MIM#609261) [95]. These defects are usually dominant and do not typically result in a raised lactate. Acknowledgment This work was supported by grants from the Canadian Institute of Health Research. References [1] B. Robinson, Lactic Acidemia: Disorders of Pyruvate Carboxylase and Pyruvate Dehydrogenase, McGraw-Hill, New York, Toronto, 2001. [2] M.S. Hermansen, L Pruett EDR, Vagi O, Waldum H, Wesselaas T. lactate Removal at rest and during exercise, Bekhauser Verlag, Basel, 1975. [3] B.H. Robinson, L. De Meirleir, M. Glerum, G. Sherwood, L. Becker, Clinical presentation of mitochondrial respiratory chain defects in NADH-coenzyme Q reductase and cytochrome oxidase: clues to pathogenesis of Leigh disease, J. Pediatr. 110 (1987) 216– 222. [4] B.H. Robinson, D.M. Glerum, W. Chow, R. Petrova-Benedict, R. Lightowlers, R. Capaldi, The use of skin Wbroblast cultures in the detection of respiratory chain defects in patients with lacticacidemia, Pediatr. Res. 28 (1990) 549–555. [5] B.H. Robinson, MtDNA and nuclear mutations aVecting oxidative phosphorylation: correlating severity of clinical defect with

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