Download Genetic defects causing mitochondrial respiratory

Human Reproduction, Vol. 15, (Suppl. 2), pp. 28-43, 2000
Genetic defects causing mitochondrial respiratory chain
disorders and disease
John Christodoulou1
Western Sydney Genetics Program, Royal Alexandra Hospital for Children,
Westmead, and Department of Paediatrics and Child Health, University of
Sydney, Sydney, Australia
'To whom correspondence should be addressed at: Western Sydney Genetics
Program, Royal Alexandra Hospital for Children, Westmead, NSW 2145, Australia.
E-mail: [email protected]
Genetic defects of the mitochondrial
respiratory chain show marked phenotypic
variability. Laboratory diagnosis is complicated and includes biochemical screening
tests, tissue histopathology, functional
enzyme studies, and molecular tests where
available. Normal respiratory chain function necessitates the co-ordinated expression of over 100 different gene loci, and
the interaction of two genetic systems, the
nuclear and mitochondrial genomes. Thus
genetic counselling for the mitochondrial
disorders is extremely challenging. In this
review, the classes of mitochondrial and
nuclear defects that give rise to functional
abnormalities of the mitochondrial respiratory chain are discussed, with specific
instructive examples described in some
detail.
Keywords: genetic s/mitochondria/mitochondrial disease/mitochondrial DNA/respiratory
chain
Clinical spectrum, diagnosis, and
treatment of respiratory chain disorders
The mitochondrial respiratory chain plays a
critical part in the biosynthesis of adenosine
triphosphate (ATP) by the process of oxidative
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phosphorylation, and is composed of five
enzyme complexes (Figure 1), each of which
is made up of multiple polypeptide subunits
(Shoffner and Wallace, 1995). A constant
supply of this newly formed ATP is critical
for the normal function of many key organs.
Clinically, defects of the mitochondrial
respiratory chain show marked phenotypic
variability, both between and within families,
and can interfere with the function of single
organs (including skeletal muscle, the central
nervous system, kidneys, endocrine organs
and the gastrointestinal system) or result in
life-threatening multisystem disease (DiMauro
et ai, 1998). The clinical onset may be at any
age, and the rate of progression of symptoms
can range from a rapid, fulminant lactic acidosis in the neonatal period to a slow or even
static condition at a later age, albeit usually
with impaired survival. Some mitochondrial
disorders are associated with a characteristic
set of clinical and pathological features, for
which they have been given eponymous names
(for a review see Shoffner and Wallace, 1995).
Diagnosis is a staged process, with initial
screening tests that include measuring plasma
and cerebrospinal fluid (CSF) concentrations
of lactate and pyruvate, and imaging the brain
(Munnich et al., 1996). The next phase of
investigations involves biopsy and laboratory
© European Society of Human Reproduction & Embryology
Mitochondria! respiratory chain disorders
glucose
pyruvate
intermembrane
spac
matrix
atty acids
carnitine
outer
membrane
membrane
respiratory chain
Figure 1. Schematic representation of the mitochondrial energy production pathways. The five complexes that make up the
respiratory chain are physically located at the inner mitochondrial membrane. Also shown are the tricarboxylic acid (TCA)
cycle and the fatty acid (3-oxidation spiral, both of which are the major suppliers of reduced equivalents as electron donors.
Note also the multiple copies of circular mitochondrial DNA within the mitochondrial matrix.
analysis of skeletal muscle and/or liver, using
histochemistry, electron microscopy, and functional studies, which can include polarography
or direct enzyme assays (Trounce et al, 1996).
Enzymatic studies of cultured skin fibroblasts,
isolated leukocytes, or transformed lymphoblasts can also be valuable (Rustin et al,
1994). However, as some defects are not
expressed in all tissues, including cultured
cells, selective functional assays can be problematical (Chretien et al, 1994).
Molecular analysis of specific mitochondrial (mt) DNA defects is available in many
cases, but it is important to note that certain
mtDNA molecular defects are found in some
tissues but not others, or are found at different
levels in different tissues (heteroplasmy)
(Shoffner and Wallace, 1995). While 'common' mutations have been identified in some
syndromic mitochondrial disorders (see
below), the majority of patients (including
most children), still turn out to have nuclear
or mtDNA mutations that are not identified
(Rahman et al, 1996).
The development of the rho-0 cell, an
immortal human cell line that has been
rendered mtDNA-free (King and Attardi,
1989) (though still with mitochondrial organelles), provides an extremely valuable experimental tool not only for evaluating the
biochemical and molecular pathogenicity of
putative mtDNA mutations (Attardi et al,
1995), but for distinguishing disorders with
Mendelian (nuclear) inheritance from those
due to defects of the mitochondrial genome
(Tiranti et al, 1995).
There have been many attempts at treating
the effects of mitochondrial disease, including
avoidance of agents that can precipitate lactic
acidosis, dietary modifications, treatment of
acute lactic acidosis with slow bicarbonate
infusions, administration of artificial electron
acceptors, vitamin cofactors, and other pharmacological agents (Przyrembel, 1987).
Although occasional patients show some
response to therapeutic interventions of this
type (Ogle et al, 1997; Mowat et al, 1999),
therapeutic efforts in most individuals have
generally been disappointing (Walker and
Byrne, 1995). Moreover, systematic evaluation
29
J.Christodoulou
of efficacy of these treatments is hampered by
clinical, biochemical and genetic heterogeneity, difficulty in accessing some therapeutic
agents, and difficulty assessing the response
to treatment of conditions that in any case
can show considerable fluctuation in clinical
features from week to week. It is widely felt
that the development and use of a standardized,
objective means of assessing symptoms and
response to treatment would be a step forward.
The key, meanwhile, to developing effective
treatments will lie in a better understanding
of the genetic bases of the respiratory chain
defects.
Special aspects of mitochondrial
genetics
The five enzyme complexes of the respiratory
chain are each composed of multiple polypeptides, totalling over 80 different gene products.
In addition, a number of essential proteins are
involved in regulation of mtDNA transcription
and subunit assembly. Most of these genes
(up to 80%) are encoded by the nuclear
genome, but some 37 (including specific transfer and ribosomal RNAs) are encoded by the
mitochondrial genome. Normal mitochondrial
respiratory chain function for efficient oxidative phosphorylation thus necessitates the
co-ordinated expression of >100 different
gene loci from two very different genomes
(Figure 2), with some of these showing tissuespecific expression. Thus genetic counselling
for the mitochondrial disorders is particularly
difficult and challenging.
There are a number of characteristics of the
mitochondrial genome that render its genetic
influences quite unlike that of the more familiar Mendelian inherited, and even polygenic
nuclear genomic disorders.
Maternal inheritance
In humans, mtDNA is maternally inherited,
transmitted by a female to all of her offspring
30
Human Chromosomes
100's - L000'
o
autosomes
sex chromosomes
1-22
X or Y
50-100,000 genes
2-10 copies
37 genes
proteins regulating
mtDNA replication
Figure 2. The relationship between the mitochondrial and
nuclear genomes. The protein subunits of the respiratory
chain are encoded by both nuclear and mitochondrial genes.
In addition, the mitochondrial genome encodes a full
complement of tRNAs and rRNAs, providing all the
necessary machinery to permit intramitochondrial
transcription and translation. Other (nuclear encoded) factors
are essential for the correct targeting of cytoplasmically
synthesized proteins to the mitochondria, import into the
mitochondria, and their correct assembly there. There are also
essential nuclear genes that play a role in the replication,
transcription, and translation of the mitochondrial genome.
through the ovum's cytoplasm (Giles et al.,
1980). Clinical clues to maternal inheritance
comprise involvement of multiple generations
through a common female ancestor, absence
of transmission of the defect by a male to his
offspring, and potential prevalences among
offspring higher than that expected for
autosomal dominant nuclear inheritance
(albeit with marked clinical variability
between members of the same family, see
below).
Replicative segregation
Mitochondria (and the group of mtDNA circles
they contain) are believed to behave as discrete
units within the cell (Wallace, 1995), although
there is evidence to suggest that there may
be functional intermitochondrial interactions
(Takai etal., 1997). Accordingly, during cellular replication mitochondria are randomly
segregated to daughter cells. In the case of
the female germ cells of a woman with a
mitochondrially inherited disorder, the result-
Mitochondrial respiratory chain disorders
progenitor cell
normal fully energy
competent mitochondria
mtDNA mutation develops
mitochondria replicate independently
mitochondria randomly distributed
in daughter cells
complete energy
deficiency
varying degrees of energy deficiency
fully energy
competent
Figure 3. Replicative segregation and the threshold effect. Mitochondria (and the mtDNA within them) are generally
considered to function as genetically discrete units and to replicate by a process of budding. During cell division, mitochondria
behave stochastically, i.e. they segregate in a random way among daughter cells. Thus, where a progenitor cell harbours a
pathogenic mtDNA mutation, individual daughter cells can have varying mutant loads. Once the mutant load reaches a certain
threshold, which differs from one tissue to another, respiratory chain function collapses and clinical consequences appear.
ant individual oocyte could bear mtDNA all
of normal sequence (homoplasmic normal),
all of abnormal sequence (homoplasmic for
the mutant mtDNA, which is lethal in some
instances but not in others, depending on a
number of factors), or a mixture of normal
(wild-type) and mutant mtDNA (heteroplasmy) (Wallace, 1991).
Threshold effect
During the early stages of embryogenesis there
is no mitochondrial or mtDNA replication
(Piko and Matsumoto, 1976), resulting in
random mitochondrial segregation of the fertilized oocyte's mtDNA among daughter cells
during cleavage, again producing a potential
wide range of mtDNA genotypes from virtual
homoplasmy for wild-type mtDNA to virtual
homoplasmy for the mutant mtDNA (Wallace,
1986). At a certain mutant mtDNA load, a
threshold is reached beyond which cellular
dysfunction will become evident (Figure 3)
(Shoffner et al., 1990). This threshold differs
according to the particular mutation and the
energetic needs and characteristics of individual tissues or organs, but there is generally
a predictable hierarchy of vulnerability for a
given mtDNA mutation. The organs most
likely to be affected are the central nervous
system (including the eye), type 1 skeletal
muscle, cardiac muscle, pancreatic islet tissue,
liver, and kidney (Wallace, 1992); and this is
the general pattern of abnormalities most often
encountered clinically.
High mutation rate
The mutation fixation rate of the mitochondrial
genome is 10-20 times higher than that of
the nuclear genome (Wallace, 1987). Factors
responsible for this include the fact that free
radicals generated within the mitochondrion
are highly mutagenic (Richter et al., 1988)
and that mitochondria contain limited DNA
repair mechanisms (Tritschler and Medori,
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J.Christodoulou
1993). Thus, with advancing age in postmitotic (differentiated) tissues, there is thought
to be a potentially important level of accumulation of mitochondrial mutations that can
exceed the threshold for clinical consequence.
In summary, where the metabolic disorder
is due to a defect of a mitochondrially encoded
gene, the clinical consequences of that mtDNA
mutation are a function of the mtDNA mutant
load in a given tissue, the seriousness of the
mtDNA mutation itself, and the energetic
requirements and functional reserve of the
organ or tissue in question. In addition, nuclear
genes, age-related accumulation of somatic
cell mtDNA mutations, and certain environmental insults (Shoffner and Wallace, 1995)
can all modulate the clinical phenotype.
Mutations causing mitochondrial disease
In recent years there have been great advances
in our understanding of the range of molecular
defects that can give rise to functional abnormalities in the mitochondrial respiratory chain
(Table I). Below is a description of a number
of different types of mutations. The list is not
exhaustive but is sufficient to highlight some
of the important clinical features of mitochondrial diseases.
Primary mtDNA defects
mtDNA point mutations: protein reading
frames
In all, 13 mtDNA genes encode some of the
respiratory chain subunits (seven subunits of
complex I, one subunit of complex III, three
subunits of complex IV, two subunits of complex V). Mutations have been described in
some but not all of these genes. Clinical
examples are described below.
Leber hereditary optic neuropathy (LHON)
This disorder usually has its onset in early
adulthood, and causes a progressive retinal
32
Table I. Classes of genetic defects giving rise to respiratory
chain disorders
Defects
Primary mtDNA defects
mtDNA point mutations
Protein subunits
tRNAs
rRNAs
mtDNA rearrangements
Large-scale deletions
Large-scale duplications
Small deletions
Nuclear encoded defects
Protein subunits
Protein trafficking, unfolding,
assembly
mtDNA replication,
transcription, translation
Other defects
a
Examples8
LHON, NARP
MEL AS, MERRF
Sensorineural deafness
KSS
CPEO
Recurrent myoglobinuria
NDUFS4, NDUFS8
hsp60, SURF-1
Thymidine phosphorylase
Frataxin, paraplegin
For explanations of abbreviations, see text.
microangiopathy and optic nerve degeneration, resulting in progressive blindness (Harding and Sweeney, 1994). Some individuals
also develop cardiac arrhythmias, but lactic
acidaemia (a consequence of widespread metabolic disruption) is very uncommon. For
reasons that are unclear, males are 5-10 times
more likely to be affected than females. LHON
was the first elucidated mtDNA defect
(Wallace et al., 1988), with >15 mtDNA
nucleotide substitutions now identified (1999).
A guanine to adenine mutation at nucleotide
position 11778 (notation, G11778A), which
results in the substitution of histidine for a
highly conserved arginine in a subunit NADH
dehydrogenase 4 (ND4) gene, accounts for
50-70% of cases. Unlike other mitochondrial
diseases, homoplasmy for the mutation is
common, even before the onset of symptoms.
Neuropathy, ataxia and retinitis pigmentosa
(NARP) syndrome
This condition is characterized by a pigmentary retinal dystrophy and blindness from
macular degeneration in association with
neurological dysfunction, including neuro-
Mitochondria! respiratory chain disorders
genie muscle weakness and ataxia from
olivopontocerebellar atrophy (Holt et al.,
1990). It is caused by a thymidine to guanine
mutation involving nucleotide 8993 (T8993G),
which swaps an arginine for a highly conserved leucine in ATP synthase subunit 6, and
thus places an additional positive charge group
in the proton channel of ATP synthase. This
seriously interferes with proton pumping into
the intermembranal space, thus blocking ATP
production and uncoupling oxidative phosphorylation. Death always occurs before
homoplasmy can be reached. With high levels
of inherited mutation, maternally inherited
Leigh disease can manifest instead.
Leigh disease
This is a progressive neurodegenerative disorder notable for optic atrophy, ophthalmoplegia, nystagmus, respiratory abnormalities,
ataxia, hypotonia, spasticity, and regression,
with its onset in the neonatal period or the
first few months of life, and survival spanning
only a few years (van Erven et al., 1987).
Neuropathologically, there is symmetric
gliosis, demyelination, and necrosis predominantly involving the basal ganglia and brainstem, with relative sparing of cerebral cortex.
Most cases are thought to be caused by
autosomal recessive mutations affecting nuclear-coded subunits of the mitochondrial respiratory chain, but some cases are due to the
T8993G (or T8993C) mutation also responsible for NARP. In addition, functional defects
of pyruvate dehydrogenase, or complexes I
and/or IV have been described with Mendelian
(nuclear) inheritance patterns (Robinson,
1995), including a recently described mutation
in the SURF-1 gene (resulting in isolated
complex IV deficiency, see below).
mtDNA point mutations: transfer RNA
mutations
Transfer RNAs are critical for translation of
all the mitochondrially inherited subunits of
the electron transport chain, so tRNA defects
£re usually associated with multiple functional
respiratory chain abnormalities. Muscle biopsies often show a massive accumulation of
subsarcolemmal mitochondria, which when
stained with modified Gomori trichrome give
the so-called ragged red fibre picture. Two
clinically recognized phenotypes are described below.
Myoclonic epilepsy and ragged red fibres
(MERRF) syndrome
This disorder has its onset in early childhood
through to adulthood, and is characterized by a
mitochondrial myopathy, myoclonic epilepsy,
and slowly progressive dementia, with hearing
loss and ataxia being common findings
(Moraes et al., 1989). On laboratory testing,
patients typically show functional abnormalities of complexes I and IV, and sometimes
complex III. Mutations have been identified
in several tRNAs, with the tRNA for lysine
(tRNALys) most often affected, particularly by
the mutation G8344A (Shoffner et al., 1990).
Mitochondrial encephalopathy, lactic
acidosis, and stroke-like episodes (MELAS)
Recurrent stroke-like episodes and a mitochondrial myopathy are characteristic of this
progressive
neurodegenerative
disorder
(Shoffner and Wallace, 1995). These 'metabolic strokes', which can have their onset
between 5 and 15 years of age, do not follow
a vascular distribution and are thought to be
the result of transient cerebral respiratory chain
dysfunction. Most patients to date have been
found to have mutations in the tRNA for
leucine (tRNALeu), with the mutation A3243G
being most common (Goto, 1995). Migrainelike headaches are common in these families.
Other combinations of problems can be seen,
including cardiomyopathy, deafness, ophthalmoplegia, cardiac conduction defects, diabetes
mellitus, easy fatiguability, dementia, and
33
J.Christodoulou
renal tubular dysfunction, as well as lactic
acidaemia (Schon, 1994).
mtDNA point mutations: ribosomal RNA
mutations
There have been several families reported with
maternally inherited childhood-onset sensorineural deafness, which in one family was
apparently induced by aminoglycoside antimicrobial usage. A homoplasmic mutation of
the mitochondrial genome was identified
(A1555G), which is within the gene for 12S
rRNA (Prezant et al, 1993). This mutation has
also been associated with maternally inherited
cardiomyopathy (Santorelli et al, 1999). In
addition, other mutations in the 12S rRNA
gene have been described in association with
type 2 diabetes mellitus (Tawata et al, 1998).
mtDNA rearrangements
Large-scale deletions or duplications of
mtDNA are associated with distinct phenotypes, which in almost cases do not appear to
be maternally inherited but present as sporadic
mutational events.
mtDNA rearrangements: major deletions
In these patients up to 50% (occasionally
more) of the 16.6 kb mitochondrial genome
can be deleted, which if homoplasmic in
the embryo would almost certainly be lethal
because of the profound impact this would
have on respiratory chain function (Wallace,
1991; Schon, 1994). Therefore, all individuals
are heteroplasmic for the deletion. Over 100
mtDNA deletions have been identified (MITOMAP, 1999). Most are flanked by direct
repeats, suggesting that for at least some of
these deletions slipped mispairing may be
responsible for the development of the deletion
(Schon et al, 1989; Shoffner et al, 1989).
The deletions are generally in the range 1.334
7.6 kb, localized between the end of the
D-loop and site of origin of light chain synthesis, thus spanning more than one gene (and
including tRNA and rRNA genes as well as
respiratory
chain
polypeptide
genes).
Examples of recognized clinical phenotypes
are described below.
Kearns-Sayre syndrome (KSS)
This neurodegenerative disorder usually has its
onset at <20 years, with affected individuals
typically manifesting a progressive external
ophthalmoplegia and partial ptosis, with retinitis pigmentosa (Berenberg et al, \911;
Zeviani et al, 1988). Additional features not
seen in all patients include cerebellar signs,
cardiac conduction abnormalities, and a raised
CSF protein. Less commonly seen problems
include diabetes mellitus, hypoparathyroidism,
deafness, peripheral neuropathy, seizures,
dementia, renal tubular and glomerular dysfunction, cardiomyopathy, and lactic acidosis.
Patients with an onset at >20 years usually
have a less severe and more slowly progressive
variant called chronic progressive external
ophthalmoplegia (CPEO) (Shoffner and
Wallace, 1995). Most KSS and CPEO patients
have a heteroplasmic deletion, 4.9-9 kb in size
(including the so-called 'common' deletion,
AmtDNA4977), which can be readily identified
by Southern-blot analysis or specifically
designed polymerase chain reaction (PCR)based assays (Manfredi et al, 1997). In some
patients no deletion is identified; some of these
patients have a mtDNA point mutation, while
others could harbour a nuclear DNA mutation
(Shoffner and Wallace, 1995).
Over 100 cases of KSS have been reported.
The condition is usually sporadic: as explained
above, oocyte transmission of the deletion
generally fails and the large-scale rearrangement presumably occurs as a somatic mutation
after embryogenesis has commenced (De
Vivo, 1993). A reported maternally inherited
case is thought to have involved transmission
Mitochondrial respiratory chain disorders
of a duplication (prone to subsequent deletion)
(Rotig et al, 1992). The birth of normal
children to affected women has occurred (De
Vivo, 1993).
The higher the mutational load is, the earlier
the clinical presentation, with a preference for
expression in mitotically active tissues (see
below). With lower mutational loads respiratory function is not significantly compromised
in mitotically active tissues and the disease
does not express until an age-dependent rise
in the mutational load occurs in post-mitotic
tissues. Sometimes mitotic tissue presentations
ameliorate spontaneously, as mitochondrial
selection takes place, only to give way to postmitotic tissue involvement at a later age (as has
been seen in Pearson syndrome; see below).
Pearson syndrome
This is also a progressive degenerative disorder caused by large deletions in the mitochondrial genome, but (unlike KSS) with onset
in early infancy (Rotig et al, 1991). The
clinical picture is characterized by aplastic or
sideroblastic anaemia, with vacuolated myeloid and erythroid cell lines, and ringed sideroblasts in bone marrow, watery diarrhoea,
pancreatic insufficiency, liver failure, proximal
renal tubule dysfunction, and lactic acidosis
(Pearson et al, 1979; Rotig et al., 1990). The
deletion is initially most prominent in blood
mtDNA (i.e. in mitotically active cell lines),
but in children who survive beyond infancy the
deletion disappears from blood cells, probably
because of successful clonal advantage for
non-affected cell lines (Shoffner and Wallace,
1994). There is, however, a progressive
increase in the proportion of deleted mtDNA
in muscle (unlike blood, a post-mitotic tissue),
and as the surviving child grows older the
clinical phenotype can evolve into KSS
(McShane et al., 1991). Most cases appear to
be due to de-novo deletions, although there is
a report of maternal transmission of a deletion
resulting in Pearson syndrome (Bernes et al.,
1993).
Villous atrophy and lactic acidosis
This infantile-onset disorder has features that
overlap with KSS and Pearson syndrome,
including watery diarrhoea, lactic acidosis,
growth retardation, sensorineural deafness,
retinitis pigmentosa, cerebellar ataxia, diabetes
mellitus, and renal failure, with death usually
in the second decade (Cormier-Daire et al.,
1994). Muscle samples often show ragged red
fibres, with a mitochondrial genomic deletion
3.4^.2 kb in size.
Diabetes mellitus and deafness
Affected individuals usually manifest deafness
in mid-childhood to late adolescence, with
insulin-dependent diabetes mellitus developing in the third to fourth decade, and vascular
strokes (Ballinger et al., 1992). Muscle analysis shows functional defects of complexes
I, III, and IV, but not complex II (DiMauro
and Moraes, 1993). (This laboratory pattern
of respiratory chain abnormalities is highly
suggestive of a mtDNA disorder, because all
of the four complex II subunits are encoded
by the nuclear genome.) Large heteroplasmic
deletions, up to 10.4 kb, have been found in
these families, whilst others are found to have
the A3243G mutation also seen with MELAS
(van den Ouweland et al., 1992).
mtDNA rearrangements: major
duplications
Duplications of the mitochondrial genome
are also heteroplasmic, and usually sporadic
(occasional maternally inherited cases have
been reported). Patients with a KSS or CPEO
phenotype have been described who have a
duplication of up to 10 kb in size (Poulton
et al., 1989), as have some patients with
Pearson syndrome (Rotig et al, 1991).
Duplications and deletions can co-exist, and
35
J.Christodoulou
recent studies suggest that in such individuals
it is the deletion rather than the duplication
which appears to be most pathogenic
(Manfredi et al, 1997).
mtDNA rearrangements: small deletions
subunits (Adams et al., 1997; Jaksch et al.,
1998; Lee et al, 1998), only a small number
of such genetic defects have been found. The
first to be discovered involved a subunit of
succinate dehydrogenase (Bourgeron et al.,
1995). More recently mutations of the
NDUFS4 subunit (van den Heuvel et al.,
1998), the NDUFS8 subunit (Loeffen et al,
1998) and NDUFV1 subunit (Schuelke et al,
1999) of complex I have been reported.
There have been only a few reports of a very
small deletion of the mitochondrial genome.
Shoffner and colleagues (Shoffner et al, 1995)
described a patient with a mitochondrial encephalomyopathy and cerebral calcifications,
Defects involving protein trafficking,
who was found to have a deletion of one of
unfolding or assembly
the three T:A base pairs (involving nucleotides
3271-3273) in the tRNALeu<UUR> gene. In Over the last few years there have been great
another report, the affected individual had advances made in understanding the processes
a transient neonatal lactic acidosis and that target cytoplasmically synthesized procardiac conduction defects, and subsequently teins to mitochondria, the mechanisms
developed growth failure, spasticity, nystag- involved in their translocation through the
mus and strabismus, and a further episode of mitochondrial membranes, and their sorting
lactic acidosis in association with an intercur- to and assembly in the different mitochondrial
rent illness (Seneca et al, 1996). This patient compartments (Lill et al, 1996; Neupert,
was found to have an apparently homoplasmic 1997; Ryan et al, 1997). Defects of the
1 bp deletion in the ATPase 6 gene. Keightley transportation and processing machinery in
and associates (Keightley et al., 1996) identi- some cases would be expected to result in
fied a 15 bp deletion in the cytochrome c multiple mitochondrial protein defects, involoxidase (COX) III subunit in a young woman ving not just proteins of the respiratory chain
with recurrent episodes myoglobinuria pro- but other key pathways. Such a disorder has
voked by exercise or catabolic illnesses. De been described in two siblings, involving heat
Coo et al. (1999) reported a patient with a shock protein 60 (hsp60) (Agsteribbe et al,
childhood onset encephalopathy and a 1993). Other defects involving mitochondria]
MELAS type phenotype who was found to protein trafficking and processing have also
have a 4 bp deletion in the cytochrome c gene. been proposed (Schapira et al, 1990), but the
All of these cases were sporadic (or de novo, molecular mechanisms have not been idenalthough muscle tissue was not available for tified.
analysis in the probands' mothers).
Most cases of Leigh disease (see above)
show Mendelian (nuclear) inheritance, either
autosomal recessive or X-linked, the latter
Nuclear mutations affecting oxidative
being the case in those patients who have
phosphorylation
been shown to have a defect in the El a
subunit of the pyruvate dehydrogenase complex
(Brown et al, 1989).
Defects of protein subunits
In the majority of patients with Leigh disDespite extensive attempts to identify ease an isolated defect involving COX is the
mutations in nuclear encoded respiratory chain primary functional abnormality. One of the
36
Mitochondrial respiratory chain disorders
somatic cell techniques which has been used
in the genetic study of Leigh disease is complementation analysis, where cell strains from
different patients exhibiting the same functional defect are fused. If the fused heterokaryon recovers the function under study, one can
infer that the two cell strains have mutations in
different genes (i.e. the two cell strains have
complemented each other's genetic defects,
and thus belong to different complementation
groups). On the other hand, if there is no
recovery of function, the two cell strains are
likely to have mutations in the same gene,
and would therefore be assigned to the same
complementation group. Complementation
analyses in COX-deficient Leigh disease have
shown that most patients belong to the same
complementation, suggesting they would have
a defect in the same gene (Brown and Brown,
1996; Munaro et al, 1997). Using different
approaches in a series of elegant experiments,
two
groups
simultaneously
identified
mutations in a highly conserved gene called
SURF-1 in patients belonging to this major
complementation group (Tiranti et al, 1998;
Zhu et al, 1998), thereby defining a new class
of genes causing neurodegenerative diseases
in humans. The function of the SURF-1 gene
product is not well understood, but it is
believed to play a role in assembly or maintenance of the COX complex. Thus, in some
instances individual genes involved in these
process may be specific for certain enzyme
complexes.
Defects affecting mtDNA replication,
transcription, or translation
Disorders with Mendelian inheritance in which
the defect appears to involve genes responsible
for the regulation of mtDNA replication, transcription, or translation have been reported.
At the molecular level, these disorders are
manifest by qualitative or quantitative abnormalities of mtDNA.
Multiple mtDNA deletions
Patients have been identified with a CPEO
clinical phenotype, but with an autosomal
dominant pattern of inheritance and additional
clinical features, including peripheral neuropathy, ataxia, cataracts, muscle weakness and
mild lactic acidosis, and muscle biopsies that
reveal mitochondrial proliferation (Zeviani
et al, 1989; Zeviani, 1992). Rather than a
single deletion as seen in typical CPEO, affected family members show multiple deletions
on Southern blots. Linkage studies (in two
unrelated families) have mapped genes likely
to be responsible to the 10q23.3-24.3 region
(Suomalainen et al., 1995) and the 3pl4.121.2 region (Kaukonen et al., 1996). Another
disorder, possibly autosomal recessive or Xlinked, and manifesting clinically with recurrent myoglobinuria in brothers, has been
reported with the identification of multiple
mtDNA deletions (Ohno et al., 1991).
The mitochondrial neurogastrointestinal
encephalomyopathy (MNGIE) syndrome is
a multisystem autosomal recessive disorder
mapped to chromosome 22ql3.32-qter, and
characterized by ptosis, progressive external
ophthalmoplegia, skeletal myopathy, gastrointestinal dysmotility, poor weight gain, and
leukoencephalopathy. Multiple mtDNA deletions have been observed in skeletal muscle
(Hirano et al., 1998) and are presumably also
present in other tissues. Recently diseasecausing mutations were identified in the thymidine phosphorylase gene, making this the first
gene isolated which plays a role in intergenomic interactions, perhaps by regulating the
availability of thymidine for DNA synthesis
and mtDNA maintenance (Nishino et al.,
1999).
mtDNA depletion
In this disorder mtDNA is qualitatively normal, but there is quantitative depletion of
mtDNA circles in mitochondria (Moraes et al,
37
J.Christodoulou
1991; Tritschler et al, 1992). The neonatalonset form is fatal in early infancy and is
characterized by a skeletal myopathy or liver
failure, while the later-onset form (initial presentation at ~1 year of age) appears to involve
only skeletal muscle. In both forms there are
multiple functional defects of the respiratory
chain. Mitochondrial proliferation occurs in
affected tissues in an apparent attempt at
compensation. The inheritance pattern is
autosomal recessive. In the early-onset form,
there is a reduction in mtDNA of up to 98%
compared with normal controls, whilst in the
late-onset form mtDNA depletion is less
severe, at -70-90% (Tritschler et al, 1992).
The molecular mechanism responsible for the
mtDNA depletion has not yet been identified.
Other nuclear defects affecting the
respiratory chain
Friedreich ataxia is an autosomal recessive
disorder in which many of the clinical features
are very reminiscent of a primary respiratory
chain defect (progressive ataxia, dysarthria,
peripheral neuropathy, diabetes mellitus and
cardiomyopathy), so it should not be surprising that it was recently found that affected
individuals have an unusual pattern of
abnormalities, namely functional defects of
complexes I, II and III (Rotig et al, 1997).
The most common genetic defect is an expansion of a GAA triplet repeat in the gene for
frataxin, and recent evidence suggests that the
pathogenesis of this disorder is related to a
disruption of iron-sulphur dependent enzymes,
including the respiratory chain complexes
listed above and the enzyme aconitase (Rotig
et al, 1997).
The hereditary spastic paraplegias are a
group of disorders characterized by progressive weakness and spasticity, particularly involving the lower limbs, and peripheral
neuropathy. Other features in this clinically
and genetically heterogeneous condition
38
include retinitis pigmentosa, optic atrophy,
deafness, and intellectual disability. Recently,
the gene responsible for an autosomal recessive form (SPG7) has been characterized (De
Michele et al, 1998). Called paraplegin, this
novel protein is highly homologous with a
family of yeast mitochondrial metalloproteinases. Recent work has found evidence
suggesting that defects of the human protein
might have similar consequences to yeast
mutants, i.e. a deficiency in the assembly of
functional respiratory and ATPase complexes
(Casari et al, 1998).
Some cases of KSS, CPEO and the mitochondrial disorder diabetes insipidus, diabetes
mellitus, optic atrophy, and deafness
(DIDMOAD) show autosomal dominant or
recessive inheritance (Shoffner and Wallace,
1995), but the genetic defects are unknown.
Genetic counselling for mitochondrial
disorders
Genetic counselling for the disorders of the
mitochondrial respiratory chain remains one
of the most challenging areas in the field of
human genetics. Where there appears to be
only a single member of a family affected,
no family history of consanguinity, and no
mutation is identified in either mitochondrial
or nuclear DNA, it is impossible to predict
with any confidence the likely recurrence risk
and one needs to resort to empiric risk data.
It has been suggested that the recurrence risk
for the offspring of affected females where
the mutation is unknown is in the order of
10-20%, whereas for the offspring of affected
males the risk is -1-2% (Hammans and
Morgan-Hughes, 1994).
A woman in whom a specific mtDNA
mutation has been identified would have a
high recurrence risk in her offspring but,
because the level of heteroplasmy is generally
only loosely correlated with disease severity,
it has been impossible to predict with confid-
Mitochondrial respiratory chain disorders
ence the likely severity of the disease in a fetus
identified as having the mutation (Poulton and
Marchington, 1996). However, as the body of
experience builds, such risk data ought to be
more reliably derived, as recently demonstrated for the T8993G and T8993C mutations
(White et al, 1999). In addition, with refinements in tissue manipulation techniques and
reproductive technologies, it is now possible
to analyse the mitochondrial genome of single
oocytes or preimplantation embryos to aid in
the assessment of recurrence risks for individual females (Blok et al, 1997; De Boer,
1999).
When there are difficulties in accurate genetic diagnosis such as those outlined above,
prenatal testing based on molecular methods
is generally not available. Respiratory chain
activities in cultured skin fibroblasts probably
best reflect those in cultured chorionic villus
cells or cultured amniocytes (Munnich et al,
1996). Thus, if one is relying on functional
enzyme assays of the respiratory chain in
cultured cells, in the main only in those
families where the parents are consanguineous
(presumed autosomal recessive inheritance)
and where the defect is clearly demonstrable
in cultured skin fibroblasts, will prenatal diagnosis be most reliable. As more gene defects
are identified, the usefulness of molecular
prenatal testing will improve.
Conclusions
In summary, although much is known, four
important questions remain to be answered in
the field of human mitochondrial disease: (i)
why is there so much phenotypic variability
between patients seemingly independent of
the degree of heteroplasmy?; (ii) what are the
genetic bases of the broad group of metabolic
defects for which no mitochondrial genomic
mutation has been identified?; (iii) will it
be possible to develop reliable methods of
prenatal diagnosis?; and (iv) will it be possible
to devise effective treatments?
The complicated biology of this group of
disorders makes their study particularly challenging. However, advances in molecular and
reproductive technologies, coupled with the
explosion of new biological data in humans
and other species generated by the Human
Genome Project, mean that the new millennium will see the evolution of methods that
will allow us to define more of the mitochondrial respiratory chain disorders at the
molecular level, and to evaluate their functional significance. It is in that climate that
accurate prenatal and symptomatic testing, and
possibly novel therapeutic strategies will be
developed for this devastating group of disorders.
Note added in proof
Recently, mutations in the WFS1 gene have
been reported in patients with DIDMOAD
(diabetes insipidus, diabetes mellitus, optic
atrophy and deafness) syndrome (Hardy
et al, 1999).
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