Download Assembly factors as a new class of disease

doi:10.1093/brain/awr261
Brain 2012: 135; 12–22
| 12
BRAIN
A JOURNAL OF NEUROLOGY
REVIEW ARTICLE
Assembly factors as a new class of disease genes
for mitochondrial complex I deficiency: cause,
pathology and treatment options
Jessica Nouws, Leo G. J. Nijtmans, Jan A. Smeitink and Rutger O. Vogel
Nijmegen Centre for Mitochondrial Disorders at the Department of Paediatrics, Radboud University Nijmegen Medical Centre, 6500 HB, Nijmegen,
The Netherlands
Correspondence to: Leo G. J. Nijtmans,
Department of Paediatrics,
Radboud University Nijmegen Medical Centre,
Geert Grooteplein 10,
PO BOX 9101, 6500 HB,
Nijmegen,
The Netherlands
E-mail: [email protected]
Complex I deficiency is the most frequent cause of oxidative phosphorylation disorders. The disease features a large diversity of
clinical symptoms often leading to progressive encephalomyopathies with a fatal outcome. There is currently no cure, and
although disease-causing mutations have been found in the genes encoding complex I subunits, half of the cases remain
unexplained. However, in the past 5 years a new class of complex I disease genes has emerged with the finding of specific
assembly factors. So far nine such genes have been described and it is believed that in the near future more will be found.
In this review, we will address whether the functions of these chaperones point towards a general molecular mechanism of
disease and whether this enables us to design a treatment for complex I deficiency.
Keywords: complex I deficiency; assembly factors; treatment
Abbreviations: NADH = nicotinamide adenine dinucleotide; OXPHOS = oxidative phosphorylation
Introduction
Mitochondrial complex I [nicotinamide adenine dinucleotide
(NADH) : ubiquinone oxidoreductase; EC 1.6.5.3] is vital to the
energy supply in the body. It is the first enzyme in oxidative phosphorylation (OXPHOS), the process in which breakdown of
organic nutrients is coupled to ATP production. Disturbances in
complex I function can result in complex I deficiency (OMIM
252 010), which is the most common biochemical defect of the
OXPHOS system. It is estimated that OXPHOS disorders occur
once in every 5000 newborns (Skladal et al., 2003), of which
complex I deficiency underlies 25–35% (Loeffen et al., 2000;
Bugiani et al., 2004; Scaglia et al., 2004; Thorburn, 2004). It
typically features a severe multisystem decline with a poor prognosis, and currently lacks a cure. Although complex I dysfunction
initially attracted attention in the context of inherited metabolic
disorders, it is now increasingly implicated in diabetes and neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s
disease (Mootha et al., 2003; Eckert et al., 2010; Winklhofer and
Haass, 2010). This makes a better understanding of complex I
deficiency of relevance for the general population.
Complex I is the product of seven mitochondrial and 38 nuclear
genes (Carroll et al., 2006). During the past two decades only
33% of the complex I deficiencies could be explained by
Received May 13, 2011. Revised July 4, 2011. Accepted July 11, 2011. Advance Access publication October 27, 2011
ß The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
A new class of complex I disease genes
mutations in these genes (Calvo et al., 2010). To explain most of
the remaining cases it was inferred that complex I assembly factors
must exist analogous to the 20 or more found essential to complex IV assembly. Indeed, in 2002, the first complex I assembly
factor was found (Janssen et al., 2002), and in 2005 the first
factor was implicated in complex I deficiency (Ogilvie et al.,
2005). Since then genetic, bioinformatic and proteomic analyses
have led to the identification of no less than nine disease-causing
assembly factors in only half a decade. Seven of these were found
in the past 3 years, promising great growth in the field for the
coming years.
In this review, we will address whether these individual discoveries
share similarities in terms of function or disease pathology.
Furthermore, we will investigate the putative pathological distinction
of complex I deficiency caused by mutations in assembly factors
versus structural subunits, and highlight therapeutic approaches
that may prove particularly effective for assembly disturbances.
Complex I function and
deficiency
Complex I is embedded in the mitochondrial inner membrane
where it oxidizes the NADH produced during glycolysis, fatty
Brain 2012: 135; 12–22
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acid oxidation and the Krebs cycle. In more detail, electrons extracted from NADH are funnelled to the electron carrier ubiquinone via a chain of iron-sulphur clusters, resulting in conformational
changes that allow proton translocation across the mitochondrial
inner membrane (Efremov et al., 2010). This proton translocation,
also performed by complexes III and IV, results in a mitochondrial
membrane potential used by complex V (ATP synthase) to generate ATP from ADP and inorganic phosphate (Fig. 1). Disturbances
in the function of complex I can have various physiological consequences, such as disturbed ATP production, oxygen consumption and calcium homeostasis. In addition, it can cause
accumulation of reactive oxygen species (Distelmaier et al.,
2009) and lactic acid (Robinson, 2006), and induce apoptosis
(Perier et al., 2005).
Although there is no obvious genotype–phenotype correlation
(Distelmaier et al., 2009), several disease phenotypes for complex
I deficiency can be distinguished based on inheritance.
Mitochondria have their own circular genome, which in humans
measures 16 569 bp. In 1988, it first became clear that mitochondrial disorders can be caused by a point mutation or deletion in
the mitochondrial genome and that the cell can harbour a mixture
of healthy and mutated mitochondrial DNA, called heteroplasmy
(Holt et al., 1988; Wallace et al., 1988). The thousands of copies
of mitochondrial DNA are inherited via the mother and randomly
Figure 1 The molecular basis and pathology of complex I deficiency. Left: Complex I is part of a system of five enzyme complexes
(CI–CV) embedded in the mitochondrial inner membrane, which together perform oxidative phosphorylation. In this system, complex I
oxidizes nicotinamide adenine dinucleotide (NADH) to transfer electrons to acceptor ubiquinone via a series of iron-sulphur (FeS) clusters.
In turn, this leads to proton translocation across the mitochondrial inner membrane to maintain a proton gradient required for complex V
to produce ATP. Complex I is assembled from nuclear subunits translated by cytosolic ribosomes (Ribo) and mitochondrial subunits
translated by the mitochondrial ribosome (MitoRibo). Disturbances in the assembly and/or stability of the complex can result in reduced
conversion of NADH to NAD + , reduced proton translocation, increased release of reactive oxygen species (ROS), and can ultimately lead
to reduced ATP production and the accumulation of lactate. Deficiency of complex I can lead to various cellular abnormalities and
apoptosis, which translates into a heterogeneous disease phenotype (right). Affected organs are primarily those with energy-demanding
tissue, such as the nervous system, muscle, liver and kidneys. Complex I deficiency is generally an early onset, progressive multisystem
disorder featuring severe neurological and muscular problems.
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| Brain 2012: 135; 12–22
distributed to daughter cells during development in a process
called segregation. These characteristics of mitochondrial DNA explain why disorders caused by mutations in the mitochondrial DNA
generally feature a high prevalence of mitochondrial symptoms
within one family and a high variability in clinical course. In contrast, mitochondrial deficiencies caused by nuclear DNA mutations
generally have a much more severe phenotype with early onset.
Mammalian complex I has seven mitochondrial genes (ND1–6
and ND4L) and 38 nuclear genes (Carroll et al., 2006). Mutations
in mitochondrial genes are associated with maternally inherited
complex I deficiency. The most prevalent pathologies associated
with mutations in mitochondrial complex I genes are Leber’s hereditary optic neuropathy (OMIM 535 000), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes
(OMIM 540 000) and Leigh syndrome (OMIM 256 000)
(Mitchell et al., 2006). Leber’s hereditary optic neuropathy
involves degeneration of retinal ganglion cells and their axons
resulting in acute loss of vision. Age of onset is generally between
the ages of 15 and 30 years, with males being affected five times
more than females (Harding et al., 1995; Yu-Wai-Man et al.,
2009). Mitochondrial myopathy, encephalopathy, lactic acidosis
and stroke-like episodes is a clinically and genetically heterogeneous phenotype of a mostly maternally inherited neurological
syndrome. It is characterized by epilepsy or dementia, often with
additional neurological symptoms such as migraine or psychiatric
problems, lactic acidosis (present in 94% of patients; Hirano and
Pavlakis, 1994), and stroke-like episodes (hemianopia, hemiparesis, aphasia and hemineglect, etc.), associated with additional
symptoms such as epileptic seizures, ataxia, migraine-like headaches, impaired vision or hearing, or disturbances in consciousness
(Finsterer, 2009). Leigh syndrome is a devastating neurodegenerative disease (OMIM 256 000; Loeffen et al., 2000). Most of the
cases typically present within the first 2 years of life with psychomotor retardation in combination with signs of brainstem or extrapyramidal dysfunction and lactic acidaemia, usually in a period of
increased stress, such as infection (Rahman et al., 1996; Finsterer,
2008). Other symptoms include neurological symptoms (ophthalmoparesis, optic atrophy, ataxia, dysphagia, cranial nerve palsies,
muscle weakness, hypotonia, dystonia, respiratory impairment,
lethargy and polyneuropathy) whether or not in combination
with dysmorphic features, endocrine abnormalities, hypertrichosis,
short stature, cardiac abnormalities (hypertrophic or dilated cardiomyopathy) or gastrointestinal problems (diarrhoea), diabetes,
vomiting, anaemia or renal problems. Death usually occurs
within the first years of life as a consequence of respiratory failure
caused by ongoing brainstem dysfunction, which can be accompanied by increasing muscle weakness (Finsterer, 2008).
Mutations in nuclear genes show Mendelian inheritance. The
majority (26%) of patients with mutations in nuclear complex I
genes present with Leigh syndrome. Other phenotypes associated
with mutations in nuclear complex I genes include neonatal cardiomyopathy with lactic acidosis, leucoencephalopathy and other
undefined progressive or stable encephalomyopathies. Neonatal
cardiomyopathy with lactic acidosis involves a relatively common
early onset, progressive hypertrophic cardiomyopathy leading to
death usually within the first year of life (Loeffen et al., 2000;
Bugiani et al., 2004). Leucoencephalopathy has been described
J. Nouws et al.
with a progressive occurrence of large cavitations in subcortical
white matter, accompanied by ventricular enlargement and
macrocephaly. The neurological condition comprises involuntary
movements and signs of brainstem involvement, developmental
regression, failure to thrive and spasticity (Bugiani et al., 2004;
Hoefs et al., 2010).
Complex I assembly factors:
a novel cause for disease
Sequencing of the seven mitochondrial DNA-encoded and 38 nuclear DNA-encoded complex I genes could provide a genetic explanation for 550% of the patients. This made clear that the
cause of many disease cases must be sought outside the structure
of complex I. Prime suspects were complex I assembly factors,
analogous to those identified for complex IV in the yeast model
organism (Fontanesi et al., 2006). Assembly factors aid assembly
of a complex without being part of the final structure. Although
the lack of complex I in yeast initially hampered their identification, pioneering work in the fungus Neurospora crassa delivered
the first two complex I assembly factors, called CIA30 and CIA84
(Kuffner et al., 1998). The human orthologue of CIA30,
NDUFAF1, proved essential to human complex I assembly and
mutations in the gene were linked to complex I deficiency
(Vogel et al., 2005; Dunning et al., 2007).
Lacking additional genetic pointers to go on, homozygosity
mapping in consanguineous families was key to finding human
complex I assembly factors. Some new factors were found
simply by sequencing all candidate genes in homozygous regions
(e.g. C20orf7 and NDUFAF4) (Sugiana et al., 2008; Saada et al.,
2008). Additional clues for identifying new complex I disease
genes have come from recent developments in high-throughput
analysis of gene and protein function.
One example is proteomics. A liquid chromatography–mass
spectrometry-based method to profile protein interactions with
complex I assembly intermediates delivered NDUFAF3 (Wessels
et al., 2009), and simultaneous homozygosity mapping followed
by sequencing implicated the NDUFAF3 gene in complex I deficiency (Saada et al., 2009). Additional proteomic strategies, such
as blue native-polyacrylamide gel electrophoresis and tandem
affinity purification, illustrated binding of NDUFAF3 with previously identified assembly factor NDUFAF4 from early to late
stages of complex I assembly (Saada et al., 2009). Tandem affinity
purification was combined with mass spectrometric analysis to find
proteins binding to NDUFAF1, which provided the ECSIT and
ACAD9 assembly factors (Vogel et al., 2007c; Nouws et al.,
2010). Both factors were previously implicated in other cellular
processes than OXPHOS (Kopp et al., 1999; Zhang et al., 2002;
Xiao et al., 2003). ACAD9 mutations turn out to be an important
cause for complex I deficiency, as recently three research groups
identified pathogenic mutations in the ACAD9 gene in 10 patients
(Haack et al., 2010; Nouws et al., 2010; Gerards et al., 2011).
The examples above show the strength of combining homozygosity mapping with proteomic approaches to verify or direct identification of new assembly factors.
A new class of complex I disease genes
Another example is bioinformatics. Benefiting from the fact that
yeast does not have complex I and other organisms do, subtractive genome analysis provided assembly factor NDUFAF2 (Ogilvie
et al., 2005). This protein is a paralogue of complex I subunit
NDUFA12 and is associated with a late-stage assembly intermediate. The NDUFAF2 gene proved an important source of disease-causing mutations associated with complex I deficiency
(Ogilvie et al., 2005; Barghuti et al., 2008; Hoefs et al., 2009;
Janssen et al., 2009; Calvo et al., 2010; Herzer et al., 2010).
Innovations in the computation of large data sets allowed
for more expansive, genome-wide bioinformatic approaches.
Pagliarini et al. (2008) combined in-depth protein mass spectrometry, microscopy and machine learning to generate a compendium
of 1098 mitochondrial genes, which was further used in conjunction with phylogenetic profiling to develop a list of 19 putative
complex I disease-causing genes. A pathogenic mutation in
C8orf38 was found after subsequent homozygosity mapping.
High-throughput pooled sequencing in a complex I-deficient patient cohort identified mutations in both FOXRED1 and NUBPL
(Calvo et al., 2010). NUBPL, also known as huInd1, plays a role
in iron-sulphur cluster incorporation during early complex I assembly (Sheftel et al., 2009). A second patient harbouring mutations
in FOXRED1 was reported shortly after (Fassone et al., 2010), but
the exact function of the protein remains unclear.
The role of assembly factors
during complex I assembly
Human complex I assembly has primarily been studied by investigating the accumulation of assembly intermediates either in patient cell lines or after inhibition of mitochondrial translation, and
by tracing 35S or epitope labelled complex I subunits during the
assembly process (Antonicka et al., 2003; Ugalde et al., 2004;
Lazarou et al., 2007; Vogel et al., 2007a; Perales-Clemente
et al., 2010). This has led to the identification of multiple assembly
intermediates. For example, tracing the NDUFS3 subunit during
assembly revealed at least six assembly intermediates (Vogel
et al., 2007b). In addition, at least five entry points were recently
identified for mitochondrial DNA-encoded subunits (PeralesClemente et al., 2010). Based on the temporal order of appearance and the composition of reported assembly intermediates a
basic assembly scheme can be devised (Fig. 2, subcomplex numbers based on Vogel et al., 2007b). Briefly summarized, a complex
of several core nuclear DNA-encoded subunits is formed and tethered to the mitochondrial membrane during early assembly (Fig. 2,
subcomplexes 1, 2 and 3). In parallel, hydrophobic membrane
complexes are formed and incorporated into the maturing complex (Fig. 2, at subcomplexes 4 and 5). Subsequent expansion of
the complex includes addition of subunits from the dehydrogenase
module (Fig. 2, subcomplex 6), resulting in fully assembled complex I (Fig. 2, complex 7).
Altogether, nine assembly factors have been implicated in complex I deficiency in a short period of time (Fig. 2). Currently, the
precise mechanism of action of most complex I assembly factors is
unclear. An exception is NUBPL, which is required for the
Brain 2012: 135; 12–22
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incorporation of iron-sulphur clusters (Sheftel et al., 2009), present
in six subunits (NDUFV1, NDUFV2, NDUFS1, NDUFS2, NDUFS7
and NDUFS8). Despite the lack of mechanistic data, the stages in
which the other assembly factors operate can be derived from the
accumulation of assembly intermediates upon their mutation or
knockdown, and by examining their protein–protein interactions
by affinity purification. The proposed involvement of each factor
during assembly is summarized in the scheme in Fig. 2. 35S labelling of mitochondrial protein showed that mutations in C20orf7
lead to the accumulation of early membrane subcomplexes, suggesting that C20orf7 acts in early assembly (Sugiana et al., 2008).
However, as C20orf7 mutations have also been implicated in
combined complex I/complex IV deficiency, its role may not be
exclusive to complex I assembly (Saada et al., 2011). NDUFAF1
and ACAD9 have been found in conjunction with assembly factor
ECSIT, and NDUFAF1 was reported to associate with a number of
complex I subunits (Dunning et al., 2007; Vogel et al., 2007c;
Nouws et al., 2010). Knockdown of any of these assembly factors
results in accumulation of subcomplexes 4 and 5 (Fig. 2), hinting
towards a role in the incorporation of membrane subunits. Epitope
tagging of assembly factors NDUFAF3 and NDUFAF4 revealed
their association with each other and with assembly stages 3, 4,
5 and 6, indicating a role from early to late complex I assembly
(Saada et al., 2009). NDUFAF2 is typically observed in an accumulated near holocomplex upon mutation of subunits NDUFV1 or
NDUFS4 (Ogilvie et al., 2005, Vogel et al., 2007a), indicating
that it is involved in late assembly or complex I stability.
Putative associations of assembly factors C8orf38 and FOXRED1
with complex I subunits have not been studied. An additional
protein, apoptosis inducing factor, has been implicated in complex
I deficiency in mouse studies (Vahsen et al., 2004; Joza et al.,
2005), but as mutations in human patients resulted in a combined
complex III/IV deficiency and mitochondrial DNA depletion, its
requirement for complex I assembly is as yet, ambiguous
(Ghezzi et al., 2010).
In summary, most identified assembly factors are involved in
early assembly, more specifically the incorporation of hydrophobic
membrane subunits (NDUFAF1—possibly in combination with
ACAD9 and ECSIT, NDUFAF3 and NDUFAF4 and C20orf7).
Apparently this is a process that requires careful coordination,
akin to the incorporation of prosthetic groups during complex IV
assembly (Fontanesi et al., 2006).
The pathology of assembly
factors
Do mutations in complex I assembly factors result in a unique
pathology? At first glance the symptoms listed in Table 1 are
not distinct from what is observed for previously reported complex
I deficiencies. It should be noted that making a distinction based
on symptoms is complicated by possible differences in their classification between different case reports. In addition, the same
mutation can lead to different clinical outcomes. For example, reported consanguineous siblings carrying ACAD9 and NDUFAF3
mutations may have had a similar age of onset and symptoms,
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J. Nouws et al.
Figure 2 The dawn of complex I assembly factors. The histogram (top) gives a historical overview of the cumulative number of unique
mutations associated with complex I deficiency in nuclear DNA-encoded subunit genes (nDNA subunits), mitochondrial DNA-encoded
subunit genes (mtDNA subunits) and assembly factors. Mitochondrial DNA mutations were included when the mutations were scored
510 by use of the Mitchell scoring table (Mitchell et al., 2006). The more recently published mutations were included in the chart when
these were proven to be pathogenic by the creation of cybrids or they had to fulfil three of the four following criteria: (i) absence of the
mutation in 5100 controls; (ii) presence of heteroplasmy; (iii) a highly conserved amino acid was changed; and (iv) the mutation
segregated with disease. The mutations and the corresponding references are listed in Supplementary Table 1. Bottom: The proposed
involvement of each disease-implicated assembly factor in the assembly process is indicated with arrows. Numbers of subcomplexes refer
to those described in Vogel et al. (2007b).
but siblings with the same NDUFAF4 mutation had a considerable
difference in the development of symptoms. Also, mutations in the
same gene can lead to different clinical outcomes. For example,
one patient carrying a mutation in C20orf7 died after 7 days of
high blood lactate level. Two siblings harbouring another mutation
in C20orf7 are both in their 20s and diagnosed with Leigh syndrome. After puberty the progression of the disease stopped.
A notable observation is the vanishing white matter reported for
almost all patients with NDUFAF2 mutations. This brain anomaly
has previously been reported for a mutation in a structural complex I subunit that is incorporated late in the assembly pathway
(Pagniez-Mammeri et al., 2010), the stage in which NDUFAF2 is
also active. Other gene–pathology relationships are less transparent. ACAD9 and NDUFAF1 were shown to be co-dependent and
to be active in the same assembly stages. Mutations in either
factor are associated with a high incidence of exercise intolerance
and cardiomyopathy. In contrast, two other proteins known to
interact in the assembly pathway are NDUFAF3 and NDUFAF4,
Reported mutations
N
N
Homozygous c.103delA (DT67)
Homozygous c.103delA (DT68)
Homozygous c.221G 4 A,
exon skipping (DT16)
Calvo, 2010
ND
Homozygous c.749G 4 T;
Gly250Val (5)
Saada, 2009
NDUFAF4
ND
less
ND
Homozygous c.194T 4 C;
Leu65Pro (F511)
Homozygous c.194T 4 C;
Leu65Pro (F334)
Homozygous c.194T 4 C;
Leu65Pro (F528)
ND
ND
Homozygous c.749G 4 T;
Gly250Val (4)
Homozygous c.296A 4 G;
Gln99Arg (sibling)
ND
Homozygous c.749G 4 T;
Gly250Val (3)
ND
ND
Homozygous c.749G 4 T;
Gly250Val (2)
Pagliarini, 2008 Homozygous c.296A 4 G;
Gln99Arg
C8orf38
ND
Homozygous c.592C 4 T;
Leu159Phe (IV-7)
Homozygous c.749G 4 T;
Gly250Val (1)
ND
Homozygous c.592C 4 T;
Leu159Phe (IV-11)
Gerards, 2010
Saada, 2011
ND
Homozygous c.719T 4 C;
Leu229Pro
ND
ND
Sugiana, 2008
C20orf7
N
Homozygous c.9G 4 A:Trp3X
Herzer, 2010
N
Deletion of full sequence
Janssen, 2009
N
Homozygous c.1A 4 T:Met1Leu
(Patient 2)
Homozygous c.114C 4 G;
p.Tyr38X
N
Homozygous c.1A 4 T:Met1Leu
(Patient 1)
Barghuti, 2008
Hoefs, 2009
N
Homozygous c.182C 4 T;
Arg45X 1
N
N
F
F
M
M
F
M
F
F
F
M
M
M
M
M
M
F
M
F
F
M
F
F
M
43%
67%
14%
14%
25%
32%
32%
26%
41%
45%
23%
53%
ND
520%#
36%
14%
6%
36%
25%
26%
48%
36%
25%
38%
12%
24%
36%
40%#
Term
Term
Term
ND
ND
ND
ND
ND
ND
36
ND
ND
35
ND
ND
ND
Term
38
Term
ND
ND
Term
ND
Birth
Birth
Birth
7m
10m
ND
ND
ND
ND
12m
3y
3y
1d
4m
3m
6m
14m
Birth
3m
8m
20m
12m
11m
% Res
Pregnancy Age of
Protein Sex %
CI activity duration
onset
detected
Res CI
activity in in muscle (weeks)
fibroblasts
Ogilvie, 2005
NDUFAF2
Dunning, 2007 CH: c.1001A 4 C;Thr207Pro,
c.1140 A 4 G; Lys253Arg
NDUFAF1
Publication
5d
N7Y
18m
N22m
34m
N2,5y
N6y
N4,5y
N7y
5y10m
N29y
N23y
7d
15m
14m
17m
27m
14m
12m
21m
2y
13y7m
N20y
Age of
death
Brain
abnormalities
Neurological
symptoms
Failure to
thrive
Symptoms
Table 1 Clinical phenotypes of patients with mutations in complex I assembly factors
Elevated
alanine level
2
Exercise
intolerance
Motor
development delay
(continued)
Lethargy
Developmental
delay
Other
Hepatomology
Renal
problems
Hyperthrophic
cardiomyopathy
Other organ
failure
Respiratory
disturbance
Cyanotic
Apneic
Tachhypnea
Respiratory
problems
Elevated CSF
lactate level
Elevated blood
lactate level
Metabolite
levels
Optic atrophy
Hearing loss
Ataxia
Spasticity
Hypotonia
Dystonia
Seizures
Microcephaly
Abnormal eye
movements
ND
Homozygous c.365G 4 C;
Arg122Pro (II-1)
CH: c.2T 4 C;Met1Thr,
ND
c.365G 4 C; Arg122Pro (III-1)
ND
ND
ND
ND
CH: c.130T 4 A; Phe44Ile;
c.797G 4 A; Arg266Gln (I:B)
CH: c.797G 4 A; Arg266Gln;
c.1249C 4 T; Arg417Cys (II)
CH: c.976G 4 A; Ala326Pro;
c.1594C 4 T: Arg532Trp (III)
F
CH: c.380G 4 A; Arg127Gln,
ND
c.1405C 4 T; Arg469Trp (CV)
CH: c.130T 4 A; Phe44Ile;
c.797G 4 A; Arg266Gln (I:A)
F
ND
Homozygous c.1594C 4 T;
Arg532Trp (VII-11)
F
F
M
F
M
ND
Homozygous c.1594C 4 T;
Arg532Trp (VII-8)
F
ND
F
CH: c.187G 4 T; Glu63X,
N
c.1237G 4 A;Glu413Lys (II-1)
Homozygous c.1594C 4 T;
Arg532Trp (VII-6)
F
M
M
Y
ND
ND
M
M
38%
32%
50%
40%
38%
58%
53%
70%
9%
19%
33%
18%
39%
26%
13%
14%
9%
61%
7%
9%
37%
26%
40%
32%
ND
ND
ND
39 w + 6d
ND
ND
ND
ND
35
Term
Term
31
Term
Term
Term
34–38
34–38
34–38
N40y
N38y
N31y
6m
N18y
N10y
N22y
N8y
6m
4m
3m
3m
3m
Birth
Birth
Birth
24h
2y
12y
N5Y
46d
Early CH N27y
4y
4y
4y
4m
8m
Birth
Birth
2y
3m
3w
1–3d
1–3d
1–3d
Age of
death
Exercise
intolerance
Motor
development delay
Developmental
delay
Other
Hepatomology
Renal
problems
Hyperthrophic
cardiomyopathy
Other organ
failure
Respiratory
disturbance
Cyanotic
Apneic
Tachhypnea
Respiratory
problems
Elevated
alanine level
Elevated CSF
lactate level
Elevated blood
lactate level
Metabolite
levels
Optic atrophy
Hearing loss
Ataxia
Spasticity
Hypotonia
Dystonia
Seizures
Microcephaly
Abnormal eye
movements
Brain
abnormalities
Neurological
symptoms
Failure to
thrive
Symptoms
This table encompasses all published case reports. Boxes in black indicate the symptom was reported in the original article; white boxes indicate the symptom was absent or not tested in the original article. CI = complex 1; CH = compound
heterozygous. ND = not determined; Residual CI activity in fibroblasts = complex I activity normalized for COX activity, percentage of lowest control value (values in bold are normalized to citrate synthase); Yellow box = percentage of control mean.
Residual CI activity in muscle = complex I activity normalized for citrate synthase activity, percentage of lowest control value; # = determined by in gel activity; d = days; w = weeks; m = months; y = years. Brain abnormalities = on MRI or CT and/or
diagnosed brain dysfunction. N = not dead, age at report date. 1 = Hemizygous for c.182C 4 T mutation, probably the other allele was deleted. Elevated alanine level = in blood; except for 2 = in urine. The patient number, as mentioned in the case
report, is given in brackets.
Haack, 2010
Gerards, 2011
Nouws, 2010
Homozygous c.1553G 4 A;
Arg518His (I-1)
Homozygous
c.1054C 4 T;Arg352Trp
Fassone, 2010
ACAD9
CH: c.694C 4 T; Gln232X,
c.1289A 4 G; Asn430Ser
ND
F
ND
Homozygous c.229G 4 C;
Gly77Arg (I-3)
CH:Del exon 1-4 and
c.166G 4 A; Gly56Arg,
c.815-27T 4 C
M
ND
Homozygous c.229G 4 C;
Gly77Arg (I-2)
F
ND
F
Pregnancy Age of
% Res
Protein Sex %
onset
CI activity duration
detected
Res CI
activity in in muscle (weeks)
fibroblasts
Homozygous c.229G 4 C;
Gly77Arg (I-1)
Reported mutations
Calvo, 2010
FOXRED1
Calvo, 2010
NUBPL
Saada, 2008
NDUFAF3
Publication
Table 1. Continued
Lethargy
A new class of complex I disease genes
Brain 2012: 135; 12–22
| 19
Figure 3 Age at onset and survival of complex I-deficient patients harbouring mutations in nuclear-encoded subunits and assembly
factors. (A) Kaplan–Meier graph showing age of onset as percentage of patients affected, for patients harbouring mutations in
nuclear-encoded subunits and assembly factors (subunits n = 82, assembly factors n = 45). (B) Kaplan–Meier survival analysis for
patients harbouring mutations in nuclear-encoded subunits and assembly factors (subunits n = 89; assembly factors, n = 49). The
mutations and corresponding references are listed in Supplementary Table 2. AF = assembly factors.
yet apart from high lactate levels the symptoms deviate. Despite
NUBPL’s seemingly crucial role in complex assembly, the patient
carrying a mutation in the gene is 8 years old and suffering from
encephalopathy but no other organs are affected.
In summary, given the small number of mutations found it is
not yet possible to relate an individual assembly factor gene to a
specific clinical phenotype, nor to make a clear distinction from
mutations in nuclear subunit genes. However, when we analyse
mutations in assembly factor genes as a separate class we noted
that disorders caused by mutations in assembly factors (versus
nuclear complex I subunit genes) generally result in a significantly
later age of death, which becomes prominent after 12 months of
life (Fig. 3). Although the age of onset is reported to differ between complex I deficiency caused by mutation in nuclear versus
mitochondrial complex I genes (Swalwell et al., 2011), we found
no apparent difference in the age of onset between complex I
deficiency caused by mutations in complex I nuclear subunit
genes versus complex I assembly genes (Fig. 3).
Clinical perspectives
Current therapeutic strategies for complex I deficiency can relieve
symptoms, but are unable to cure or even delay the progress of
the disease. General approaches include administration of vitamins
and cofactors, such as different forms of ubiquinone (Q10), even
though beneficial effects are unclear because proper clinical trials
are scarce (Chinnery et al., 2006; Glover et al., 2010; Kerr, 2010).
Fortunately several encouraging new approaches are being developed, some of which may prove particularly useful for the
treatment of complex I deficiency caused by disturbances in the
assembly process. The assembly process can be viewed as a multistep reaction that involves input of subunits, catalysis by assembly
factors and output of a holoenzyme. Mutations in assembly factors generally slow down or halt the reaction even though the
subunits are without error. With this notion in mind, to restore
assembly, one could either improve the function of any residual
mutated assembly factor or increase the flow of substrates into the
20
| Brain 2012: 135; 12–22
assembly ‘reaction’. Of note, the success of such strategies will
depend on the relative contribution of the assembly factor to the
assembly process and the functional impact of the mutation.
Stabilization of assembly factors can be achieved by targeted
administration of cofactors. For example, a high dosage
(300 mg/day) of riboflavin increased complex I activity and improved the clinical condition of patients with the ACAD9 mutation
(Gerards et al., 2011). Furthermore, riboflavin was reported to
stabilize the FAD group in complex I assembly protein, apoptosis
inducing factor (Ghezzi et al., 2010). Likewise, this strategy has
been reported to work for destabilized subunits, as the effects of a
mutation of NDUFV1 could be alleviated by riboflavin supplementation (Benit et al., 2001).
Increasing the amount of substrate (the subunits) to partly restore assembly is another promising strategy. Activation of PPAR
(peroxisome proliferator-activated receptor) and PGC-1 transcriptional pathways led to increased mitochondrial biogenesis
and improved mitochondrial physiology in cellular models for
OXPHOS deficiency (Srivastava et al., 2007). More importantly,
PGC-1 overexpression led to a significant improvement in endurance and healthy life span of a mouse knockout of complex IV
assembly factor COX10 (Wenz et al., 2008). Administration of
PPAR panagonist bezafibrate to the diet of these mice had a similar effect. As another example, overexpression of transcriptional
activator NF-Y could partially rescue complex IV deficiency caused
by mutation in assembly factor SURF1 by increasing complex IV
expression (Fontanesi et al., 2008). Metabolic sensor kinases such
as metabolic sensor kinase mTOR can also induce mitochondrial
biogenesis (Cunningham et al., 2007). Recently, D’Antona et al.
(2010) showed that mTOR stimulation with branched chain amino
acids elevated mitochondrial biogenesis in middle-aged mice and
up-regulated the reactive oxygen species defence mechanism.
The quality control system, which includes molecular chaperones
such as heat shock proteins and the ubiquitin proteasome, plays a
key role in certain neurodegenerative diseases and cancer.
Prevention of aggregation of misfolded proteins via small molecular regulators, chemical chaperones or neutraceuticals provides
therapeutic options for treatment of Alzheimer’s disease and
Parkinson’s disease (Wisniewski and Sadowski, 2008; Ali et al.,
2010; Rose et al., 2011). Likewise, a similar approach to prevent
aggregation of assembly intermediates may prove a useful tool to
treat mitochondrial diseases caused by mutations in assembly factors. A direct link between protein aggregation and disease pathology has not yet been established for complex I deficiency.
However, disturbances in mitochondrial protein quality control
have been implicated in respiratory deficiency. For example,
mutations in the m-AAA protease component AFG3L2 lead to
respiratory deficiency and spastic paraplegia (Di Bella et al., 2010).
Conclusion
The rapid emergence of complex I assembly factors has had several implications. It provided a genetic solution for part of the thus
far unexplained disease cases, aiding diagnosis and prevention.
The recent identification of multiple assembly factors working
in similar assembly stages has helped to direct future research
J. Nouws et al.
into the molecular basis of disease. By analogy to complex IV
for which 420 assembly factors aid assembly of a 13 subunit
complex, 10–20 new complex I assembly factors are expected to
be found in the next couple of years. This search is greatly facilitated by implementation of technical novelties, such as
high-throughput bioinformatic screening and whole genome
sequencing.
Although highly desirable, the clinical heterogeneity of complex
I deficiency may preclude a general therapeutic approach. Most
likely tailored combinations of approaches will provide the most
effective personalized medicine. Several therapeutic options are
being developed to alleviate disturbed complex I biogenesis.
A prerequisite to further develop these approaches is detailed
mechanistic knowledge of the molecular basis of disease.
Therefore, it is of the utmost importance to accompany the association of new mutations with disease by detailed functional analysis of the involved assembly factor.
Acknowledgements
The authors would like to thank Thatjana Gardeitchik for her help
with statistical analysis.
Funding
Netherlands Organization for Scientific Research (863.10.018 to
R.O.V.).
Supplementary material
Supplementary material is available at Brain online.
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