Download 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
4
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
6
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
8
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.
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