Download Mutations in SUCLA2: a tandem ride back to the Krebs cycle

doi:10.1093/brain/awm023
Brain (2007), 130, 606 ^ 609
SCIENTIF IC COMMENTARY
Mutations in SUCLA2: a tandem ride back
to the Krebs cycle
Mitochondria are small membrane-bound intracellular
organelles that are the principal source of adenosine
triphosphate (ATP), the high-energy phosphate molecule
required by all human cells. ATP is produced by the
mitochondrial respiratory chain which is linked to oxidative
phosphorylation. Thirteen essential respiratory chain polypeptides are synthesized from small circles of DNA present
within each mitochondrion (the mitochondrial genome,
mtDNA). Qualitative defects of mtDNA are a major cause
of human disease: both point mutations and deletions of
mtDNA cause a biochemical defect which often affects
skeletal muscle and the nervous system, presenting with
neurological features (Zeviani and Di Donato, 2004).
MtDNA is inherited exclusively down the maternal line,
and pathogenic mtDNA mutations are either maternally
inherited or sporadic, affecting at least 1 in 5000
(Schaefer et al., 2004). More recently, autosomal recessive
mitochondrial disorders have gained increasing prominence
(Table 1). Mutations have been described in nuclear genes
coding for respiratory chain proteins or assembly factors,
and nuclear-encoded intra-mitochondrial translation factors
have recently entered the lime-light (Coenen et al., 2004;
Valente et al., 2007). However, a new class of genetic
disease has emerged as an important cause of human
pathology, ranking along-side the primary mtDNA
mutations in terms of their frequency. These diseases are
also due to nuclear gene mutations, and all affect proteins
that maintain a healthy mitochondrial genome.
Genetic linkage in families with autosomal dominant
chronic progressive external ophthalmoplegia (ad-PEO) led
the way to pathogenic mutations in three nuclear genes:
POLG which codes for the intra-mitochondrial DNA
polymerase (pol g), PEO1 which codes for the mtDNA
helicase Twinkle, and SLC25A4 which codes for the
adenine nucleotide translocase ANT1 (Kaukonen et al.,
1996; Spelbrink et al., 2001; Van Goethem et al., 2001).
Subsequent candidate gene sequencing identified the first
mutation in POLG2 which codes for the pol g accessory
subunit (Longley et al., 2006). In all of these dominant
disorders, the clinical phenotype is due to the formation of
multiple deletions of the mtDNA molecule which develop
during life. POLG appears to be a major disease gene, with
a plethora of clinical presentations [recently described in
Brain (Horvath et al., 2006; Tzoulis et al., 2006)], some of
which are autosomal recessive (Naviaux and Nguyen, 2004;
Ferrari et al., 2005), and cause a ‘quantitative’ defect of
mtDNA: mtDNA depletion.
MtDNA depletion or the loss of mtDNA copies has
been linked to five additional genes: TK2, DGUOK,
TP, MPV17 and SUCLA2. Each gene is associated with
an emerging clinical phenotype, although there is some
overlap. Mutations in POLG, DGUOK and MPV17 cause
an encephalopathy and liver failure in early childhood
(Mandel et al., 2001; Naviaux and Nguyen, 2004; Spinazzola
et al., 2006), with POLG being the principal cause of the
Alpers–Huttenlocher syndrome (Nguyen et al., 2006).
Mutations in TK2 typically present with a progressive
myopathy in childhood (Saada et al., 2003), or spinal
muscular atrophy without liver involvement (Oskoui et al.,
2006), and mutations in TP cause thymidine phosphorylase
(TP) deficiency and mitochondrial neurogastro-intestinal
encephalomyopathy (MNGIE) (Nishino et al., 1999).
TP, TK2 and DGUOK code for proteins involved in
the essential processes regulating the concentration of
nucleotide building-blocks of mtDNA (dNTPs) within
mitochondria. Being unable to synthesize their own
nucleotides, mitochondria are critically dependent on
cytosolic dNTPs, which are partly regulated by TP,
and also on the recycling of nucleotides within the
mitochondrial matrix by thymidine kinase (TK) and
dexoyguanosine kinase (DGUOK) (Marti et al., 2003).
Although the function of the inner mitochondrial
membrane protein MPV17 remains unclear (Spinazzola
et al., 2006), a common theme is emerging, with
mtDNA depletion arising through the dysregulation of
intra-mitochondrial nucleotide pools. But what about
SUCLA2, which codes for the b subunit of the mitochondrial matrix enzyme succinyl-CoA synthase (SCS-A)? SCS-A
forms part of the tricarboxylic acid (TCA) cycle first
described by Hans Krebs in 1937. At first sight this seems
an unlikely member of the pack, but two articles in this
volume of Brain add to the single previously described
family with mutated SUCLA2, expanding the phenotype
into adult life, and firmly establishing SUCLA2 as gene
involved in mtDNA maintenance.
Homozygous mutations in SUCLA2 were first identified
in two cousins from a consanguineous Israeli-Muslim
family with a presumed recessive mitochondrial
encephalomyopathy (Elpeleg et al., 2005). Both had
a biochemical defect affecting three respiratory chain
ß 2007 The Author(s)
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Scientific Commentary
Brain (2007), 130, 606 ^ 609
607
Table 1 Nuclear genes causing mitochondrial disease
Nuclear genetic disorders of the mitochondrial respiratory chain, mutations in structural subunits:
Leigh syndrome (complex I deficiencyçmutations in NDUFS1, NDUFS4, NDUFS7, NDUFS8, NDUFV1. Complex II deficiency, SDHA)
Cardiomyopathy and encephalopathy (complex I deficiency, mutations in NDUFS2)
Optic atrophy and ataxia (complex II deficiencyçmutations in SDHA)
Hypokalaemia and lactic acidosis (complex III, mutations in UQCRB)
Nuclear genetic disorders of the mitochondrial respiratory chain, mutations in assembly factors:
Leigh syndrome (mutations in SURF I and the mRNA binding protein LRPPRC)
Hepatopathy and ketoacidosis (mutations in SCO1)
Cardiomyopathy and encephalopathy (mutations in SCO2)
Leucodystrophy and renal tubulopathy (mutations in COX10)
Hypertrophic cardiomyopathy (mutations in COX15)
Encephalopathy, liver failure, renal tubulopathy (with complex III deficiency, mutations in BCS1L)
Encephalopathy (with complex V deficiency, mutations in ATP12)
Nuclear genetic disorders of the mitochondrial respiratory chain, mutations in translation factors:
Leigh syndrome, Liver failure and lactic acidosis (mutations in EFG1)
Lactic acidosis, developmental failure and dysmophism (mutations in MRPS16)
Myopathy and sideroblastic anemia (mutations in PUS1)
Leukodystrophy and polymicrogyria (mutations in EFTu)
Nuclear genetic disorders associated with multiple mtDNA deletions or mtDNA depletion:
Autosomal progressive external ophthalmoplegia (mutations in POLG, POLG2, PEO1 and SLC25A4)
Mitochondrial neurogastrointestinal encephalomyopathy (thymidine phosphorylase deficiencyçmutations in TP)
Alpers^Huttenlocher syndrome (mutations in POLG and MPV)
Infantile myopathy/spinal muscular atrophy (mutations in TK2)
Encephalomyopathy and liver failure (mutations in DGUOK )
Hypotonia, movement disorder and/or Leigh syndrome with methylmalonic aciduria (mutations in SUCLA2)
Others
Co-enzyme Q10 deficiency (mutations in COQ2)
Barth syndrome (mutations in TAZ)
Cardiomyopathy and lactic acidosis (Mitochondrial phosphate carrier deficiency, mutations in SLC25A3)
complexes containing mtDNA encoded subunits (I, III and
IV), associated with mtDNA depletion. Microsatellite
markers spaced every 10 cM (10 million, or mega-bases,
Mb) across the whole genome revealed regions of shared
homozygosity spanning 20 Mb, where the cousins had
identical pairs of alleles. Most mitochondrial proteins are
synthesized in the cytoplasm with an additional peptide
sequence at the N-(amino)-terminal, which targets the
protein to mitochondria. Of the 113 predicted genes within
the homozygous region, only three coded for proteins with
a mitochondrial target predicted in silico. One of these was
SUCLA2, which contained a complex rearrangement (both
a deletion and an insertion) of the gene. The affected
cousins were homozygous for the mutation, which is
predicted to alter the way that the coding (exonic) and
non-coding (intronic) regions of the gene are spliced to
make the mature messenger RNA coding for SCS-A.
SCS-A consists of two different protein subunits
(a heterodimer). The a subunit is shared with SCS-G,
which uses GTP as a substrate, with the nucleotide
specificity being determined by the b subunit (Johnson
et al., 1998). Both SCS-G and SCS-A are widely expressed
in the liver, kidney and heart, but SCS-A is the dominant
enzyme in the central nervous system (Lambeth et al.,
2004), possibly explaining the tissue-selectivity seen in the
original family (Elpeleg et al., 2005).
Twelve patients are described in the first paper
(page 853), all with ancestors originating from the Faroe
islands, with five linked geneologically (Ostergaard et al.,
2007). Clinically the disorder presented at birth or early
infancy with hypotonia and motor delay, followed by
failure to thrive leading to gastrostomy feeding. All subjects
developed a hyperkinetic movement disorder and severe
sensorineural deafness requiring cochlear implantation in
some. Seven patients died at between 8 months and
21 years of age, usually from respiratory infections, with
a 10-year-old being the oldest of five surviving children.
Fourteen cases were described in the second paper
(page 862) (Carrozzo et al., 2007), including three from
southern Italy, with one being of Rumanian extraction. The
clinical course in both cohorts was strikingly similar,
although careful scrutiny reveals some overlap, with the
same 10 patients being described in both papers (Table 2).
This makes a total number of 16 new cases described in this
edition of Brain.
Brain imaging revealed abnormalities in the putamen and
caudate nuclei, global atrophy in the more advanced cases
and dysmyelination in some. The extent and severity
differed both between subjects and between the two studies,
probably reflecting the age of each subject and the different
imaging modalities used. Proton magnetic resonance
spectroscopy in one case showed a high lactate level in
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Brain (2007), 130, 606 ^ 609
Scientific Commentary
Table 2 Correspondence between the patients with
SUCLA2 mutations described by Ostergaard et al., 2007 and
Carrozzo et al., 2007
Carrozzo et al., 2007
Table 1, patient number
Ostergaard et al., 2007
Table 1, patient initials
1
2
3
4
5
6
7
8
9
10
11
12
13
14
^
^
^
^
^
^
BC
EH
AH
JR
TO
DJ
FT
BN
TD
RJ
LD
BD
^ ¼ not described in that paper.
the basal ganglia, providing a clue to the underlying
metabolic disturbance. This was reflected by high lactate
levels in plasma and CSF in some. Rather surprisingly,
epilepsy is not a prominent feature of this encephalopathy.
In both studies, all of the patients had abnormal respiratory
chain activity in skeletal muscle, with multiple complex
defects and depletion of mtDNA (ranging between 15 and
41% of control values across both cohorts). Given the
tendency to carry out less invasive procedures on infants, it
is important to note that similar investigations on cultured
fibroblasts were normal in some patients.
The presumed recessive inheritance and ethic homogeneity led one group (Ostergaard et al., 2007) to map the
whole human genome with single nucleotide polymorphisms (SNPs) using 10 000 SNPs on a microarray to
look for regions of shared homozygosity. This lead to a
1.4 Mb locus by a classical ‘reverse genetic’ approach,
refining 25 000 genes to a short list of 10 which included
SUCLA2. The other group (Carrozzo et al., 2007) took a
‘forward genetic’ approach which led them, in tandem,
to the same gene. They noted high levels of methylmalonic
acid in plasma and urine in all patients, which pointed to
enzymes in the relevant metabolic pathway and the
underlying genetic defect (see figure 1 Carrozzo et al.,
2007 or figure 5 in Ostergaard et al., 2007). Both groups
identified the same homozygous SUCLA2 mutations in all
of the Faroese patients (IVS4 þ 1G4A and c.534 þ 1G4A
are actually the same mutation, described by different
nomenclature). This mutation was predicted to alter exon
splicing and introduce a premature stop codon, further
shortening the mRNA transcript. Analysis of the cDNA
(formed in vitro from the processed mRNA) revealed exon
skipping, although it is intriguing that the two groups
identified different transcripts in cultured skin fibroblasts
from the same underlying mutation. Further work at the
protein level will clarify the principal molecular mechanism,
which could vary from tissue to tissue. Both groups
determined the carrier frequency in Faroese control
subjects, and report a reassuringly similar value (2 vs 3%
of the population, which is not significantly different,
Fisher’s exact P ¼ 0.27), explaining the high prevalence of
the disease in this geographically isolated population (1 in
2500). The patients from southern Italy harboured different
mutations, both predicted to alter the protein sequence.
SCS-A activity was measured in one patient and was low,
and the amount of the SUCLA2 encoded b subunit was
reduced in muscle from the Italian and Faroese patients
(Carrozzo et al., 2007).
The description of additional cases with mutations
affecting the coding sequence and a different splice site
confirms the role of SUCLA2 as an important human
disease gene, and identifies another cause of methylmalonic
acidaemia. The methylmalonic acid accumulates because a
build up of substrate limits its conversion to succinyl-CoA
via methylmalonyl-CoA (see figure 1 Carrozzo et al., 2007
or figure 5 in Ostergaard et al., 2007). So far, the clinical
presentation seems consistent, despite the different underlying molecular mechanisms, and measuring urine organic
acids provides a non-invasive out-patient-based screening
test for the condition. In the future, cases are likely to be
detected by tandem mass spectrometry in neonates based
on the profile of abnormal carnitine esters seen in their
patients (Carrozzo et al., 2007). However, developing an
effective treatment depends on our understanding of the
disease mechanism—and the fundamental question still
remains: how does a Krebs cycle enzyme cause mtDNA
depletion? SCS-A is tightly associated with nucleoside
diphosphate kinase (NDPK) (Kowluru et al., 2002), which
catalyses the exchange of phosphates between tri- and
di-phosphoribonucleosides, and is critically involved in the
salvage dNTPs during mtDNA synthesis (Bradshaw and
Samuels, 2005). Precisely how the two enzymes
interact is not clear, but the fundamental role of NDPK
in rate-limiting mtDNA replication, with its limited
expression in brain (Milon et al., 1997), strongly
implicates this interaction as being fundamentally important. This is encouraging, because new treatments are under
development for related disorders of intra-mitochondrial
nucleoside metabolism, including bone marrow transplantation (Hirano et al., 2006; Lara et al., 2006). Measuring
nucleosides levels within different cellular compartments
will hopefully clarify the role of NDPK in patients with
SUCLA2 mutations. Unfortunately, although some cases
of methylmalonic aciduria are vitamin B12 responsive,
anecdotally this treatment did not help the Faroese group
(Ostergaard et al., 2007).
The SUCLA2 story provides further support for ongoing
genetic-mapping studies of rare recessive disorders,
encouraging us to think ‘outside the box’ when searching
Scientific Commentary
for molecular mechanisms of disease. Moreover, mutations
in SUCLA2 provide key evidence that SCS-A is ‘moonlighting’—beyond its conventional role described by
Hans Krebs. Other intra-mitochondrial proteins have
similar secondary functions (Jeffery, 1999), which may
actually be more important for human disease than their
primary affiliation.
Patrick F. Chinnery
Mitochondrial Research Group and Institute of
Human Genetics
Newcastle University
The Medical School
Framlington Place
Newcastle upon Tyne
NE2 4HH, UK
E-mail: [email protected]
Acknowledgements
PFC is a Wellcome Trust Senior Clinical Research Fellow
who also receives funding from the United Mitochondrial
Diseases Foundation, a research grant from the United
States Army, and the EU FP program EUmitocombat and
MITOCIRCLE. Funding to pay the Open Access publication charges for this article was provided by The Wellcome
Trust.
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