Download ECHS1 mutations in Leigh disease: a new inborn

doi:10.1093/brain/awu216
Brain 2014: 137; 2903–2908
| 2903
BRAIN
A JOURNAL OF NEUROLOGY
REPORT
ECHS1 mutations in Leigh disease: a new inborn
error of metabolism affecting valine metabolism
Heidi Peters,1 Nicole Buck,1 Ronald Wanders,2 Jos Ruiter,2 Hans Waterham,2 Janet Koster,2
Joy Yaplito-Lee,1 Sacha Ferdinandusse2,* and James Pitt1,3,*
1 Murdoch Childrens Research Institute, Victorian Clinical Genetics Services, Royal Children’s Hospital, Parkville, Vic 3052, Australia
2 University of Amsterdam, Laboratory Genetic Metabolic Diseases, Departments of Paediatrics and Laboratory Medicine, Academic Medical Centre,
Amsterdam, The Netherlands
3 Department of Paediatrics, University of Melbourne, Parkville, Vic 3052, Australia
*These authors contributed equally to this work.
Correspondence to: James Pitt,
Murdoch Childrens Research Institute,
Victorian Clinical Genetics Services,
Royal Children’s Hospital,
Parkville, Vic 3052,
Australia
E-mail: [email protected]
Two siblings with fatal Leigh disease had increased excretion of S-(2-carboxypropyl)cysteine and several other metabolites that
are features of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency, a rare defect in the valine catabolic pathway associated
with Leigh-like disease. However, this diagnosis was excluded by HIBCH sequencing and normal enzyme activity. In contrast to
HIBCH deficiency, the excretion of 3-hydroxyisobutyryl-carnitine was normal in the children, suggesting deficiency of shortchain enoyl-CoA hydratase (ECHS1 gene). This mitochondrial enzyme is active in several metabolic pathways involving fatty
acids and amino acids, including valine, and is immediately upstream of HIBCH in the valine pathway. Both children were
compound heterozygous for a c.473C 4 A (p.A158D) missense mutation and a c.414 + 3G4C splicing mutation in ECHS1.
ECHS1 activity was markedly decreased in cultured fibroblasts from both siblings, ECHS1 protein was undetectable by immunoblot analysis and transfection of patient cells with wild-type ECHS1 rescued ECHS1 activity. The highly reactive metabolites
methacrylyl-CoA and acryloyl-CoA accumulate in deficiencies of both ECHS1 and HIBCH and are probably responsible for the
brain pathology in both disorders. Deficiency of ECHS1 or HIBCH should be considered in children with Leigh disease. Urine
metabolite testing can detect and distinguish between these two disorders.
Keywords: biochemistry; metabolic disease; neurodegeneration
Introduction
Leigh disease (MIM 256000) is a devastating childhood, progressive, neurodegenerative disease characterized by bilateral, symmetric brain lesions. Patients typically present in infancy with
hypotonia, ataxia, psychomotor delay and regression. The
estimated incidence is 1:40 000 and the disorder is often fatal
(Finsterer, 2008). Atypical and later onset forms of the disease
occur and are often described as Leigh-like disease. Non-specific
biochemical findings include increased lactate in CSF and blood
and increased Krebs cycle intermediates in urine, pointing to a
deficit in energy metabolism. Deficiencies of the mitochondrial
Received December 16, 2013. Revised July 1, 2014. Accepted July 1, 2014. Advance Access publication August 14, 2014
ß The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
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respiratory chain complexes I, II and IV, caused by mutations in
either nuclear or mitochondrial DNA encoding subunits or assembly factors for these complexes, are frequent causes of Leigh disease with complex I deficiency being the most common (Tucker
et al., 2010) but many other causative genes have been identified,
most of them individually rare (Finsterer, 2008). The relative uniformity of clinical features in Leigh disease, lack of specific biochemical markers and the many causative genes present a
diagnostic challenge and non-targeted exome and genome
sequencing approaches are being increasingly used. These methods have revealed additional genes in patients with Leigh disease
with 435 genes thought to cause Leigh disease (Gerards et al.,
2013).
Here we describe a new inborn error of metabolism affecting
valine metabolism in two siblings with fatal Leigh disease. Reactive
intermediates in the valine pathway produce a characteristic metabolite signature and are likely to be responsible for the pathology of this disorder.
Materials and methods
Patients
The female proband was born at term (41 weeks gestation) by normal
vaginal delivery after an unremarkable pregnancy. Her parents were
non-consanguineous and of Greek ancestry. Her birth weight was
3270 g (25th–50th centile), length was 53 cm (90th–97th centile)
and head circumference was 33 cm (3rd–25th centile). Apgar scores
were 7 and 8 at 1 and 5 min, respectively. At birth she had generalized
hypotonia and poor suck. At 12 h of age, the baby developed apnoea
post-feeding. Septic work-up was negative. The baby continued to
have apnoeas with associated bradycardias and desaturations and
was intubated and ventilated by Day 3 of life. CSF lactate was
4.9 mM (reference range 53.0), pyruvate was 0.32 mM (0.06–0.13)
with a normal CSF lactate to pyruvate ratio. Serial serum lactate levels
were also increased, ranging from 2.8 to 4.7 mM (0.5–2.0). The activity of the pyruvate dehydrogenase complex was decreased in
cultured skin fibroblasts [0.15 nmol/(min.mg protein); controls
0.23–0.53].
MRI with magnetic resonance spectroscopy at Day 6 of life showed
a large lactate peak and lack of diffusion restriction suggestive of a
mitochondrial disorder. A repeat MRI at 2.5 months of age showed a
marked progressive generalized atrophy compared with the previous
MRI, particularly involving the supratentorial central white matter
(Supplementary Fig. 1). The corpus callosum was markedly thinned
and there was also progressive atrophy of the brainstem, basal ganglia
and upper cervical cord with symmetrical T2 hyperintensity in the posterior aspect of the putamen more conspicuous compared with the
previous MRI. Abnormal T2 hyperintensity was also seen in the supratentorial central white matter. There was symmetrical extensive diffusion restriction involving central periventricular white matter of the
frontal lobes, internal capsule, posterior body of the corpus callosum
extending to the medial temporal lobes, mid-brain and inferior cerebellar peduncles. Magnetic resonance spectroscopy showed increased
lactate in the basal ganglia. Skeletal muscle biopsy showed no obvious
abnormality on electron microscopy and mitochondrial respiratory
chain enzyme activities were normal. The patient remained ventilator-dependent with minimal spontaneous movements. She died at
H. Peters et al.
the age of 4 months due to respiratory failure after care was
withdrawn.
The second child was male and born at term (39 + 6 weeks gestation) by normal vaginal delivery after an unremarkable pregnancy. His
birth weight was 3300 g (50th–90th centile), length was 52 cm (90th
centile) and head circumference was 34.5 cm (50th centile). His Apgar
scores were 2 and 6 at 1 and 5 min, respectively. He required suction
and bag mask oxygen. This baby was also profoundly hypotonic and
had poor feeding and desaturations; however, he did not require respiratory support. At 3 months of age, he had severe global developmental delay and abnormal neurological examination with severe
generalized hypotonia and vertical nystagmus. In addition he also
had a ventricular septal defect and severe progressive hypertrophic
obstructive cardiomyopathy. MRI and spectroscopy at 7 weeks of
age showed reduced myelination of the globus pallidus and putamina
bilaterally with elevated lactate. Repeat MRI and spectroscopy at 4
months showed persistent elevated lactate, new diffusion restriction
in the globus pallidus, anterior thalami, midbrain and hippocampi
and interval parenchymal loss. Activity of the pyruvate dehydrogenase
complex was also decreased in cultured skin fibroblasts [0.04 nmol/
(min.mg protein); controls 0.23–0.53]. He died at home at the age
of 8 months as a result of cardiorespiratory failure. In both siblings,
dried blood spot acyl carnitine profiles and liver function tests were
unremarkable.
Biochemical and molecular analyses
Semiquantitative urine screening for S-(2-carboxypropyl)cysteine was
performed by flow injection tandem mass spectrometry of butyl derivatives as previously described (Pitt et al., 2002) with the addition of
a 320.2 4 119 m/z transition. Quantitation of urine cysteine, cysteamine and glycine conjugates and the separation of 3-hydroxybutyrylcarnitine and 3-hydroxyisobutyryl carnitine is described in the
Supplementary material. Urine 2-methyl-2,3-dihydroxybutyric was
measured by gas chromatography-mass spectrometry. The structures
of the metabolites are shown in Supplementary Fig. 2.
Measurement of short chain enoyl CoA hydratase (ECHS1) enzyme
activity, immunoblotting of ECHS1 protein, ECHS1 and HIBCH
sequencing, ECHS1 cDNA analysis and quantitative PCR of ECHS1
mRNA are described in the Supplementary material.
Functional expression of wild-type and
mutant ECSH1 in patient fibroblasts
We amplified the coding region of wild-type ECSH1 by PCR from
reverse-transcribed RNA isolated from control fibroblasts and subcloned this into the HindIII and BamHI sites of the eukaryotic expression vector pcDNA3 (Invitrogen). The entire fragment was sequenced
to exclude PCR-induced errors. The mutations identified in the patients
were subsequently introduced using the QuikChangeÕ site directed
mutagenesis kit of Agilent Technologies and primers 5’-CTGGGAC
CACCTCACCCAGTTTGGCGGGGGCTGTGAGC-3’ and 5’-GCTCACA
GCCCCCGCCAAACTGGGTGAGGTGGTCCCAG-3’ for mutation p.A1
58D and 5’-CTATGCCGGTGAGAAGGACCAGTTTGCACAGC-3’ and
5’-GCTGTGCAAACTGGTCCTTCTCACCGGCATAG-3’ for mutation
p.126_138del (i.e. the consequence of c.414 + 3G4C). The correct
introduction of the mutations was verified by sequencing.
For functional expression, we transfected fibroblasts from Sibling 1
with the different pcDNA3 vectors containing either the ECHS1 wildtype, the p.A158D or the p.126_138del mutants using the AMAXA
NHDF NucleofectorÕ kit and the NucleofectorÕ technology (Lonza).
ECHS1 mutations in Leigh disease
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Table 1 Biochemical findings in ECHS1 deficiency
Enzyme activities in cultured fibroblasts:
3-hydroxyisobutyryl-CoA hydrolase
Short-chain enoyl-CoA hydrolase
Urine metabolite levels:
Methacrylate metabolites (valine pathway)
S-(2-carboxypropyl)cysteine
S-(2-carboxypropyl)cysteamine
Acrylate metabolites (valine pathway)
S-(2-carboxyethyl)cysteine
S-(2-carboxyethyl)cysteamine
Tiglate metabolites (isoleucine pathway)
Tiglyl glycine
Crotonate metabolites (fatty acid pathway)
Crotonyl glycine
Other metabolites
2-methyl-2,3-dihydroxybutyric
Sibling 1
Sibling 2
Controls
Not done
59
10.3
59
7.9 1.3a (11)b
379 147 (12)
33, 36
27, 28
19–26 (3)
14–30 (3)
55.3 (19)
510 (19)
5.4, 3.6
5.8, 6.2
1.6–2.0 (3)
5.0–6.8 (3)
52.2 (19)
51.2 (19)
2.7, 2.6
0.9–4.5 (3)
57.8 (19)
1.6, 1.4
0.9–1.4 (3)
54.7 (19)
1560–2500 (4)
782, 972
525 (48)
Enzyme activities in nmol/(min.mg protein), urine levels in mmol/mmol of creatinine.
a
Mean SD.
b
Number of measurements in brackets.
Cells were harvested 2 days after transfection and ECHS1 activity was
measured in duplicate in cell homogenates.
Results
Biochemical results
Semiquantitative urine metabolite screening by tandem mass spectrometry showed increased levels of S-(2-carboxypropyl)cysteine,
which was confirmed by quantitative analysis in both siblings. This
analysis also revealed increases in several related cysteine and
cysteamine conjugates. Urine organic acid screening by gas chromatography-mass spectrometry also showed increased levels of 2methyl-2,3-dihydroxybutyrate. The urine metabolite levels are
summarized in Table 1. The levels were normal in the parents
(data not shown). These metabolites are features of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH, Fig. 1) deficiency (MIM
250620), a rare defect of the valine catabolic pathway also associated with Leigh-like disease (Brown et al., 1982; Loupatty et al.,
2007; Peters et al., 2013). However, sequencing of HIBCH did not
reveal any pathogenic mutations and HIBCH enzyme activity
(Loupatty et al., 2007) was normal in the second sibling (Table 1).
Further metabolite analysis showed that, in contrast to HIBCH
deficiency, the siblings had normal urine excretion of 3-hydroxyisobutyryl-carnitine (Fig. 2A), indicating a possible deficiency of
short-chain enoyl-CoA hydratase (ECHS1, also known as crotonase, ECHS1 gene product, Fig. 1). ECHS1 activity was therefore
measured in cultured skin fibroblasts and was below the limit of
detection of the enzyme assay in both siblings [59 nmol/(min.mg
protein), controls 379 147] (Table 1). The normal 27 kD ECHS1
band was completely absent in both siblings by immunoblot analysis (Fig. 2B).
Molecular results
No damaging mutations were identified in the exons and flanking
regions of the HIBCH gene of the first sibling. Sequence analysis
of the ECHS1 gene showed that both siblings were compound
heterozygous for a c.473C4A (p.A158D) missense mutation in
exon 4 and a c.414 + 3G4C mutation affecting the third base
of intron 3 (Supplementary Fig. 3). Testing of the parents
showed these mutations were maternally and paternally inherited,
respectively and both mutations were absent from variant databases (dbSNP and NHLBI Exome Variant Server). The p.A158D
mutation affects a highly conserved alanine residue at the entrance of the substrate binding pocket of the enzyme and is predicted to be damaging (PolyPhen-2).
ECHS1 cDNA was analysed to determine the effect of the
c.414 + 3G4C splicing mutation (Fig. 2C). This resulted in the
amplification of three products in both siblings, whereas only
one band was present in control samples (Fig. 2C). The abnormal,
smaller product was found to be missing 39 bp from the end of
exon 3, whereas exon 4 was intact. This suggested the usage of a
cryptic donor splice site in exon 3 as a result of the c.414 + 3G4C
mutation (as predicted by Human Splicing Finder), resulting in the
deletion of amino acids 126–138 from the ECHS1 protein. These
amino acids make up part of the interface between the two trimers of the hexameric structure (Protein Data Bank ID: 2HW5).
The normal sized 193 bp ECHS1 cDNA band (Fig. 2C, lane 1)
included both mutations and was sequenced to determine the
extent of aberrant splicing caused by the c.414 + 3G4C mutation.
This revealed that nucleotide position c.473 was predominantly
the mutated c.473A allele with only a small amount of the wildtype c.473C allele (Supplementary Fig. 4), indicating that the
c.414 + 3G4C mutation results in only a small amount of normally
spliced product. Based on gel quantitation (SynGene Gene Tools)
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H. Peters et al.
valine
(R)-methylmalonyl-CoA
2-ketoisovaleric
(S)-methylmalonyl-CoA
isobutyryl-CoA
propionyl-CoA
methacrylyl-CoA
acryloyl-CoA
2
5
S-(2-carboxypropyl)-L-cysteine
S-(2-carboxypropyl)cysteamine
4
4
(S)-3-hydroxyisobutyryl-CoA
3-hydroxypropionyl-CoA
(S)-3-hydroxyisobutyric
3-hydroxypropionic
1
(S)-methylmalonic semialdehyde
3
succinyl-CoA
ornithine
methionine
isoleucine
threonine
S-(2-carboxyethyl)-L-cysteine
S-2-carboxyethylcysteamine
2-methyl-2,3-dihydroxybutyric?
1
malonic semialdehyde
Figure 1 Valine catabolic pathway showing the formation of metabolites in short-chain enoyl-CoA hydratase deficiency. Enzymes are
numbered: 1 = 3-hydroxyisobutyryl CoA hydrolase; 2 = propionyl-CoA carboxylase; 3 = (R)-methylmalonyl-CoA mutase; 4 = short-chain
enoyl-CoA hydratase (ECHS1, crotonase, ECHS1 gene); 5 = isobutyryl-CoA dehydrogenase.
of the three bands produced from the exon 3-exon 4 cDNA PCR,
we estimated that 46% is wild-type, 34% has a deletion and
20% is a heteroduplex (both wild-type and deletion carrying sequences in equal amounts, based on sequencing) resulting in
56% wild-type and 44% incorrectly spliced mRNA overall.
Thus, aberrant splicing of ECHS1 caused by the c.414 + 3G4C
mutation is estimated to occur at least 80% of the time.
Quantitative PCR of ECHS1 mRNA also showed that the siblings
had 60% 26% (average average deviation, nine determinations) the levels of ECHS1 mRNA relative to controls (n = 2).
ECHS1 enzyme activity in the fibroblasts of Sibling 1 was rescued after transfection with wild-type ECSH1 [ECHS1 activity of
254 nmol/(min.mg protein) compared to 13 nmol/(min.mg protein) in mock transfected cells]. Transfection of fibroblasts of
Sibling 1 with the two identified mutant forms of ECSH1 (mutations p.A158D and p.126-138del) did not result in an increase of
ECHS1 activity [15 and 13 nmol/(min.mg protein), respectively].
Discussion
ECHS1 is a mitochondrial enzyme that converts unsaturated trans2-enoyl-CoA species to the corresponding 3(S)-hydroxyacyl-CoA
species through addition of a water molecule to the double bond
(Fong et al., 1977). It shows rather wide substrate specificity, but
has greatest activity towards crotonyl-CoA. It is a key component
of the b-oxidation spiral of short- and medium-chain fatty acid
oxidation. In addition, it is active in the isoleucine and valine
catabolic pathways. In the latter, it converts methacrylyl-CoA
to (S)-3-hydroxyisobutyryl-CoA and acryloyl-CoA to 3-hydroxypropionyl-CoA (Shimomura et al., 1994; Wanders et al., 2012).
The latter reaction is also relevant to a secondary pathway of
propionyl-CoA metabolism that feeds into the valine pathway
(Fig. 1). Methacrylyl-CoA and acryloyl-CoA are highly reactive
intermediates that can react spontaneously with a variety of
molecules including those with sulphhydryl groups such as cysteine
and cysteamine (Brown et al., 1982). Methacrylyl-CoA and acryloyl-CoA are therefore toxic to cells and can potentially disrupt
many biochemical reactions and protein structures.
In HIBCH deficiency, the accumulation of methacrylyl-CoA
results in the excretion of an unusual amino acid S-(2carboxypropyl)cysteine (Truscott et al., 1981). We added S-(2carboxypropyl)cysteine to a panel of metabolites measured as a
high-throughput urine screen for inborn errors of metabolism in
order to detect HIBCH deficiency. The increased urine S-(2carboxypropyl)cysteine in our two siblings with Leigh disease
prompted more detailed analyses, showing that the related
metabolites S-(2-carboxypropyl)cysteamine, S-(2-carboxyethyl)
cysteine and S-(2-carboxypropyl)cysteamine were increased
(Table 1). Urine 2-methyl-2,3-dihydroxybutyrate was also
increased. The metabolic origin of this last metabolite has not
been clearly established but there is evidence that it is derived
from acryloyl-CoA (Peters et al., 2013). The above metabolite
abnormalities occur in HIBCH deficiency which is associated with
Leigh disease, but this diagnosis was excluded by sequencing of
the HIBCH gene and normal HIBCH enzyme activity (Table 1). In
contrast to HIBCH deficiency, the two siblings had normal excretion of 3-hydroxyisobutyryl-carnitine, derived from 3-hydroxyisobutyryl-CoA (Fig. 2A) and this suggested a defect of ECHS1, the
enzyme immediately upstream of HIBCH in the valine pathway
(Fig. 1). This was confirmed by the findings of undetectable
ECHS1 enzyme activity (Table 1), absence of ECHS1 protein in
fibroblasts (Fig. 2B), mutations predicted to be damaging in the
ECHS1 gene and rescue of ECHS1 activity when Sibling 1’s cells
were transfected with wild-type ECHS1. The damaging nature of
the two mutations was further confirmed by demonstrating lack of
rescue of ECHS1 activity when Sibling 1’s cells were transfected
with mutated ECHS1.
There are many biochemical and clinical similarities between
ECHS1 and HIBCH deficiencies. The metabolite patterns in both
ECHS1 mutations in Leigh disease
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A
09M01763 IKONOMOU
11-Jun-2013
CHECK04 Sm (SG, 2x3)
MRM of 5 Channels ES+
304.2 > 85 (OHC4 carnitine)
1.47e6
3.34
100
2.98
%
HIBCH deficiency
2.90
0
2.50
2.55
2.60
CHECK06 Sm (SG, 2x3)
2.65
2.70
2.75
2.80
2.85
2.90
2.95
3.00
3.05
3.10
3.15
3.20
3.25
3.30
3.35
3.40
3.45
3.50
3.55
3.60
3.65
3.08
100
Sibling 1
2.89
%
3.70
3.75
3.80
MRM of 5 Channels ES+
304.2 > 85 (OHC4 carnitine)
8.16e5
2.50
0
2.50
2.55
2.60
CHECK01 Sm (SG, 2x3)
2.65
2.70
2.75
2.80
2.85
2.90
2.95
3.00
3.05
3.10
3.15
3.20
3.25
3.30
3.35
3.40
3.45
3.50
3.55
3.60
3.65
2.88
100
3.70
3.75
3.80
MRM of 5 Channels ES+
304.2 > 85 (OHC4 carnitine)
3.53e5
Control
%
3.09
3.63
0
2.50
2.55
2.60
2.65
2.70
2.75
2.80
2.85
2.90
2.95
3.00
3.05
3.10
3.15
3.20
3.25
3.30
3.35
3.40
3.45
3.50
3.55
3.60
3.65
3.70
3.75
Time
3.80
C
B
ECHS1
Figure 2 Characterization of ECHS1 deficiency. (A) UPLC-MSMS analysis of urine hydroxy-C4 carnitines as butyl derivatives
(304 4 85 m/z transition). Two peaks are formed as a result of derivatization. The black arrows indicate 3-hydroxybutyryl carnitine while
the grey arrows indicate 3-hydroxyisobutyryl carnitine. (B) Immunoblot with antibodies against ECHS1 (indicated by arrowhead) with the
lower panel showing loading controls with antibodies against b-actin. Lanes 1, 4: control fibroblasts; Lane 2: sibling 1; Lane 3: sibling 2. (C)
Gel electrophoresis of ECHS1 cDNA in fibroblast samples after reverse transcriptase-PCR and PCR of exons 3–4 showing the effect of the
c.414 + 3G4C mutation. The 193 bp fragment shows the presence of normal sized ECHS1 cDNA. Lane 1: sibling 1. The abnormal lower
band is the product of abnormal splicing while the abnormal upper band is a heteroduplex; Lane 2: control.
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| Brain 2014: 137; 2903–2908
disorders reflect the accumulation of methacrylyl-CoA and acryloyl-CoA and it is likely that the brain pathology in the two disorders is due to damage caused by these reactive intermediates.
Deficiencies of respiratory chain enzymes and the pyruvate dehydrogenase complex in HIBCH deficiency (Loupatty et al.,
2007; Ferdinandusse et al., 2013; Peters et al., 2013) also suggest
secondary protein damage. Indeed, our patients also had
decreased activity of the pyruvate dehydrogenase complex in cultured skin fibroblasts, increased lactate levels and normal lactate/
pyruvate ratios indicating that secondary enzymatic deficiencies
also occur in ECHS1 deficiency. However, they suffered a more
severe clinical course and died within the first year of life, in contrast to HIBCH deficiency with four of the five reported patients
surviving beyond 1 year of age (Loupatty et al., 2007;
Ferdinandusse et al., 2013; Peters et al., 2013).
The metabolite abnormalities in the two siblings appeared to be
confined to the valine pathway. Increased excretion of crotonylglycine and tiglylglycine are predicted consequences of ECHS1 deficiency in the fatty acid and isoleucine pathways, respectively.
However, both these metabolites were normal (Table 1). This apparent discrepancy may be due to low flux through these pathways at the times patients were sampled but further research into
the role of ECHS1 in these pathways is required.
Our findings demonstrate that ECHS1 mutations result in ECHS1
deficiency and are another cause of Leigh disease. There is considerable overlap in the clinical and biochemical features of ECHS1
and HIBCH deficiencies. However, unlike most other genetic disorders causing Leigh disease, there is a specific pattern of urine
metabolites that should facilitate the recognition of both ECHS1
and HIBCH deficiencies in future.
Acknowledgements
We thank Dr Kenneth Tan and Dr Martin Tuszynski for patient
management, Ava Mishra, Mary Eggington and Kai Mun Hong for
performing urine organic acid analysis and tandem mass spectrometry screening and Lodewijk Ijlst, Dr Avihu Boneh and Prof. Ron
Wevers for helpful advice. Dr David Thorburn and Ms Simone
Tregoning performed PDHc enzyme activity measurements.
Funding
Sections of this work were supported by the Victorian
Government’s Operational Infrastructure Support Program.
Web Resources
NHLBI Exome Variant Server: http://evs.gs.washington.edu/EVS/
dbSNP: https://www.ncbi.nlm.nih.gov/SNP/
H. Peters et al.
PolyPhen-2: http://genetics.bwh.harvard.edu/pph2/index.shtml
Online Mendelian Inheritance in Man (OMIM): http://www.ncbi.
nlm.nih.gov/omim/
Human Splicing Finder: http://www.umd.be/HSF/
Protein Data Bank: http://www.rcsb.org/pdb/home/home.do
Exon primer design: http://ihg.helmholtz-muenchen.de/cgi-bin/
primer/ExonPrimerUCSC.pl?db=hg19&acc=uc001lmu.3
Supplementary material
Supplementary material is available at Brain online.
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