Download document/47414 - UvA-DARE

UvA-DARE (Digital Academic Repository)
Branched chain amino acids : facts and defects
Loupatty, F.J.
Link to publication
Citation for published version (APA):
Loupatty, F. J. (2007). Branched chain amino acids : facts and defects
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),
other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating
your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask
the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,
The Netherlands. You will be contacted as soon as possible.
UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)
Download date: 16 Jun 2017
Branched Chain Amino Acids
Facts and Defects
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam op
gezag van de Rector Magnificus prof.
dr. J.W. Zwemmer ten overstaan van
een door het college voor promoties ingestelde commissie, in het openbaar te
verdedigen in de Aula der Universiteit op
dinsdag 29 mei 2007, te 12.00 uur door
Ference John Loupatty
geboren te Amersfoort
Promotiecommissie
Promotor
Prof. dr. R.J.A. Wanders
Co-promotor
Dr. M. Duran
Overige leden
Prof. dr. J.M.F.G. Aerts
Prof. dr. G. Brown
Prof. dr. W.H. Lamers
Prof. dr. A. Sturk
Prof. dr. F.A. Wijburg
Dr. H.R. Waterham
Faculteit der Geneeskunde
The work described in this thesis was carried out at the laboratory Genetic Metabolic Diseases, Departements
of Clinical Chemistry and Paediatrics, Emma Children’s Hospital, Academic Medical Center, University of
Amsterdam, Amsterdam, The Netherlands.
Contents
Scope of this Thesis
5
Branched Chain Amino Acids: Facts and Defects
7
3-Methylglutaconic Aciduria Type I is Caused by Mutations in AUH
19
Direct Non-isotopic Assay of 3-Methylglutaconyl-CoA Hydratase
23
Identification of a Novel Enoyl-CoA Hydratase Encoded by ECHDC2
27
Clinical, Biochemical and Molecular Findings in Three Patients with 3-Hydroxyisobutyric Aciduria
35
Mutations in the Gene Encoding 3-Hydroxyisobutyryl-CoA Hydrolase Results in Progressive Infantile Neurodegeneration 41
Summary
46
Vertakte keten aminozuren: Feiten en Fouten
47
Dankwoord
48
Am J Hum Genet. 2002; 71(6): 1463-6
Clin Chem. 2004; 50(8): 1447-50
Mol Genet Metab. 2006; 87(3): 243-8
Am J Hum Genet. 2007; 80(1):195-9
Chapter 1
Scope of this Thesis
Ference J. Loupatty
In recent years tremendous progress has been made with respect to the enzymology of the catabolic
pathway of the branched chain amino acids and defects therein. First, the introduction of tandem
mass-spectrometry for the analysis of plasma acylcarnitines has facilitated the identification
of patients with a defect in the catabolic pathways of branched chain amino acids to a great
extent. Second, a number of new enzymes have been identified, including 2-methylbutyryl-CoA
dehydrogenase, 2-methyl-3-hydroxybutyryl-CoA dehydrogenase and 3-hydroxyisobutyryl-CoA
hydrolase. Finally, several branched chain organic acidurias have been resolved at the molecular
level. These developments explain why the number of patients with an inborn error of branched
chain amino acid catabolism has increased over the last decade. An update of the current state of
knowledge regarding the clinical, biochemical and molecular aspects of branched chain organic
acidurias, which includes data presented in the consecutive chapters, is described briefly in chapter
2.
Nonetheless, at the start of this project the metabolic and molecular basis of several inborn errors
of branched chain amino acid metabolism, including 3-methylglutaconyl-CoA hydratase deficiency,
3-hydroxyisobutyric aciduria and 3-hydroxyisobutyryl-CoA hydrolase deficiency, were still elusive.
Therefore, it was the purpose of this study to examine and elucidate the underlying defect in these
disorders and to devise new and improved means to identify patients with a branched chain organic
aciduria. The results are presented in chapters 3 to 7. In chapter 3 the resolution of the molecular
basis of 3-methylglutaconyl-CoA hydratase deficiency is presented. The need to differentiate between
3-methylglutaconic aciduria type I and other forms of 3-methylglutaconic aciduria which are all
characterized by normal 3-methylglutaconyl-CoA hydratase activity necessitated the development
of a specific assay to measure this enzyme activity. The results are described in chapter 4. In our quest
to resolve the molecular basis of 3-methylglutaconic aciduria type I another gene was encountered
which encodes a protein with similar enzymatic properties as 3-methylglutaconyl-CoA hydratase.
This novel enoyl-CoA hydratase was characterized and compared to 3-methylglutaconyl-CoA
hydratase (chapter 5). In an effort to elucidate the abnormal metabolite profiles detected in patients
suffering from 3-hydroxyisobutyric aciduria, the molecular and biochemical characterization of the
human 3-hydroxyisobutyrate dehydrogenase was undertaken (chapter 6). After its initial report in
1982 we have resolved the metabolic and molecular basis of 3-hydroxyisobutyryl-CoA hydrolase
deficiency. In addition, through acylcarnitine profiling a second patient with 3-hydroxyisobutyrylCoA hydrolase deficiency has been identified of which the results are presented in chapter 7.
From the Departments of Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam,
The Netherlands (F.J.L.)
Chapter 2
Branched Chain Amino Acids: Facts and Defects
Ference J. Loupatty
General aspects of BCAAs catabolism
cells, because amino acids are little stored in free form
in mammalian species. BCAAs are metabolized by
skeletal muscle, liver, kidney, heart, brain and adipose
tissue as an alternative energy source, or used for the
biosynthesis of lipids.
In humans, the BCAAs are metabolized in the
mitochondria via the concerted action of a series of
enzymes to simple organic acid intermediates that enter
general metabolism (figure 1). The catabolic pathways
of the BCAAs can be divided into two sequential series
of reactions, referred to as the common pathway and the
distal pathway. The process begins with the transport
of the BCAAs via a Na+-dependent L-amino acid
The three branched chain amino acids (BCAA) leucine,
isoleucine and valine are neutral aliphatic amino acids,
each with a methyl branch in the side chain. As the
human body is unable to synthesize these amino acids
de novo, the BCAAs are essential nutrients. Although
they are present in all protein-containing foods, the
most prominent sources are dairy products, red meat,
whey and egg protein. Most diets provide adequate
amounts of these amino acids which is approximately
25-65 mg per kg body weight per day.1,2 The BCAAs
are indispensable amino acids as stimulants of protein
synthesis and as building blocks of proteins.1,3 BCAAs
obtained in excess are immediately catabolized in
Figure 1. Catabolic pathways for the
branched chain amino acids valine,
isoleucine and leucine. The structures and names of the intermediate
are denoted whereas the enzymes
that catalyze the reactions are indicated by their E.C. numbers.
CH3
HO
O
O
S
CoA
D-methylmalonyl-CoA
CH3
HO
O
O
S
O
CoA
L-methylmalonyl-CoA
S
HO
CoA
O
Succinyl-CoA
From the Departments of Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam,
The Netherlands (F.J.L.)
specific transport protein across the plasma membrane
into the cells. Once in the cytoplasm, the BCAAs can
enter the mitochondria in two ways. First, the BCAAs
can be transaminated in the cytosol by the reversible
action of a cytosolic branched chain aminotransferase.
The resulting branched chain 2-oxo acids are then
transported across the mitochondrial innermembrane
via a specific transporter protein into the matrix space.
Alternatively, the BCAAs can enter the mitochondria
via a neutral amino acid carrier protein, and are then
converted to their corresponding branched chain 2oxo acids by a mitochondrial aminotransferase. Once
inside the mitochondria, the branched chain 2-oxo
acids are oxidatively decarboxylated by the branched
chain 2-oxo acid dehydrogenase (BCKADH) complex
to form branched chain acyl-CoA thioesters with one
carbon atom less than the parent amino- or 2-oxoacids.
This reaction is irreversible and considered the ratelimiting step in the catabolic pathway of BCAAs. After
these common steps the degradative pathway for each
of the BCAAs diverges, in a so-called distal pathway,
generating different end products. Moreover, the distal
pathways are completely different for the three BCAAs,
and comprise enzymes specific for each amino acid.
Remarkably, the catabolic pathways of leucine, valine
and isoleucine present many analogies to fatty acid
oxidation.
Isoleucine degradation
The major pathway of isoleucine degradation
proceeds via metabolites having the S-stereochemical
configuration to the common intermediates acetylCoA and propionyl-CoA. This S-pathway of isoleucine
degradation is depicted in figure 2. First, isoleucine is
transaminated to 2-oxo-3-methylvaleric acid, followed
by oxidative decarboxylation to 2-methylbutyryl-CoA.
Next, like any 2-methyl branched chain fatty acid, 2methylbutyryl-CoA undergoes further breakdown
by oxidation via a four-step pathway involving
dehydrogenation to tiglyl-CoA, hydration to 2methyl-3-hydroxybutyryl-CoA, dehydrogenation to 2methylacetoacetyl-CoA, and finally, thiolytic cleavage
to produce acetyl-CoA plus propionyl-CoA. This fourstep sequence constitutes a complete cycle of fatty acid
β-oxidation. It is generally accepted that the enzyme
crotonase which is involved in the β-oxidation of fatty
acids is responsible for the hydratase activity in both
the isoleucine and valine oxidation. Human crotonase
deficiency has not been reported so far. As isoleucine
has an asymmetrical carbon atom it can be converted
to its diastereomer, alloisoleucine. The degradation of
alloisoleucine proceeds through the minor R-pathway
(not discussed here), which results in different end
products as compared to the major S-pathway for
Figure 2. Catabolic pathway of Isoleucine.
The structures and the names of the
intermediates are shown in the center with
the names of the enzyme on the left and the
metabolites that may be elevated due to a
deficiency in one of these enzymes shown
on the right. (BCKADH complex: branched
chain ketoacid dehydrogenase complex;
SBCAD: short/branched chain acyl-CoA
dehydrogenase; SCHMAD: short chain
hydroxymethylacyl-CoA dehydrogenase)
Figure 3. Catabolic pathway of leucine.
The structures and the names of the
intermediates are shown in the center
with the names of the enzyme on the
left and the metabolites that may be
elevated due to a deficiency in one of
these enzymes shown on the right.
(BCKADH complex: branched chain
ketoacid dehydrogenase complex; IVD:
isovaleryl-CoA dehydrogenase; MCC: 3methylcrotonyl-CoA carboxylase; 3MGH:
3-methylglutaconyl-CoA hydratase)
isoleucine. Three inborn errors of the distal pathway
of isoleucine degradation, i.e. 2-methylbutyrylCoA
dehydrogenase
deficiency,
2-methyl-3hydroxybutyryl-CoA dehydrogenase deficiency and 2methylacetoacetyl-CoA thiolase deficiency, have now
been resolved at both the enzymatic and molecular
level and will be discussed below.
Four inborn errors of the distal pathway of isoleucine
degradation, including: (1) isovaleric acidemia, (2)
isolated 3-methylcrotonyl-CoA carboxylase deficiency,
(3) 3-methylglutaconyl-CoA hydratase deficiency and
(4) 3-hydroxy-3-methylglutaryl-CoA lyase deficiency,
have been identified and will be discussed below.
Leucine degradation
Valine degradation
The catabolic pathway of leucine is depicted in figure
3. First, leucine is transaminated to 2-oxoisocapronic
acid, which is subsequently converted into isovalerylCoA. In contrast to 2-methylbutyryl-CoA, isovalerylCoA cannot undergo fatty acid β-oxidation as
the 3-methyl group blocks β-oxidation. Instead,
isovaleryl-CoA undergoes a four-step process,
involving dehydrogenation to 3-methylcrotonyl-CoA,
carboxylation to 3-methylglutaconyl-CoA, hydration to
3-hydroxy-3-methylglutaryl-CoA, and finally a thioester
hydrolysis forming acetyl-CoA and acetoacetic acid.
Leucine degradation is different from the degradation of
the other branched chain amino acids, in that it includes
a carboxylation step, which requires a biotin cofactor.
The metabolic and molecular basis of valine
degradation has always been poorly understood. Now,
with the recent resolution of 3-hydroxyisobutyryl-CoA
hydrolase deficiency (see chapter 7)4 and isobutyrylCoA dehydrogenase deficiency some light has been
shed on this enigmatic pathway. The catabolic route
of valine is exceptional, because it is believed that part
of the pathway between 3-hydroxyisobutyryl-CoA and
propionyl-CoA proceeds via free acids, thus requiring a
specific hydrolase (figure 4). This is in marked contrast
to the degradation pathways of the other branched
chain amino acids, i.e. leucine and isoleucine, in which
the intermediates distal to the 2-oxoacids are all CoA
thioesters. Indeed, the hydrolysis of an activated
Figure 4. Catabolic pathway of valine.
The structures and the names of the
intermediates are shown in the center
with the names of the enzyme on the
left and the metabolites that may be elevated due to a deficiency in on of these
enzymes shown one the right. (BCKADH
complex: branched chain ketoacid dehydrogenase complex; ACAD8: isobutyryl-CoA dehydrogenase; HIBCH: 3-hydroxyisobutyryl-CoA hydrolase; HIBADH:
3-hydroxyisobutyrate
dehydrogenase;
MMSDH: methylmalonic semialdehyde
dehydrogenase
acyl group in the heart of a catabolic pathway is not
only uncommon, but also energetically unfavourable
especially when subsequent steps of the pathway again
involve CoA thioester intermediates. The resolution of
the aforementioned disorder has provided conclusive
evidence which indicates that in humans, part of the
catabolic pathway of valine does indeed proceed via free
acids. This opens the door to a better understanding of
the catabolic pathway of valine and defects therein.
Inborn errors of
branched chain amino acids metabolism
Maple Syrup Urine Disease (MSUD; MIM 248600)
MSUD is caused by a defect in the branched chain 2oxo acid dehydrogenase (BCKADH) complex which
catalyses the second step in branched chain amino acid
metabolism.5-10 This multi enzyme system is composed
of three components: (1) a decarboxylase that requires
thiamine as a cofactor (E1α and E1β), (2) a dihydrolipoyl
acyltransferase (E2), and (3) a dihydrolipoamide
dehydrogenase (E3). A deficiency of each of these
10
catalytic subunits can cause MSUD. The diagnosis can
be readily made by amino acid analysis and organic
acid analysis. The BCAAs are considerably elevated
in body fluids which include blood, cerebrospinal
fluid and urine, and the presence of alloisoleucine is
pathognomonic for MSUD. In addition, a defect in
BCKADH will result in the increased excretion of 2oxo-3-methylvaleric acid, 2-oxoisocaproic acid and
2-oxoisovaleric acid. These organic ketoacids give
the urine its intensive and characteristic maple-syrup
like odour, hence the name of the disease. Since its
initial report by Menkes and associates11 five distinct
clinical and biochemical phenotypes – classic,
intermediate, intermittent, thiamine-responsive and
dihydrolipoamide dehydrogenase deficient MSUD –
have been recognized.5,6,8,9,12 However, categorization of
the variant cases is often complicated since no uniform
criteria are present.
Classic MSUD with a neonatal onset of
encephalopathy represents a severe, but most common
form of this disorder. Affected newborns appear normal
at birth, and symptoms often develop between four to
seven days of age. Lethargy and poor feeding are usually
the first signs followed by weight loss and alternating
hypertonia and hypotonia. Ketosis and the maple
syrup-like odour now become apparent. The majority
of these patients die within the early months of life from
recurrent metabolic crises, neurological deterioration
and coma if left untreated. The surviving patients
often manifest severe neurological damage which
includes mental retardation. In classic MSUD BCAAs
levels, in particular leucine, are markedly elevated and
more than 50 % of the branched chain 2-oxo acids
are derived from leucine. BCKADH activities are less
than 2% of normal. The intermediate and intermittent
forms of MSUD are mild clinical phenotypes which are
associated with higher BCKADH activities, i.e. 3-30
% and 5-20 % of normal, respectively.13 Most patients
with the intermediate form were diagnosed between
the age of 5 months and seven years when evaluated
for developmental delay or seizures. Episodes of
ketoacidosis and acute encephalopathy were rare in
these patients. Persistent elevated excretion of BCAAs
and 2-oxo acids – 75% derived from isoleucine – were
noted. In contrast, patients with the intermittent
form of MSUD demonstrated normal BCAAs levels
when asymptomatic. Moreover, the hallmark of the
intermittent type is its late onset and the episodes of
acute behavioural change and unsteady gait, often
associated with increased protein consumption or
intercurrent illness, that may progress to seizures and
coma. Some patients have died during these episodes.
A number of patients with thiamine-responsive
MSUD have been reported in which the metabolic
abnormalities were ameliorated with large doses of
thiamine (vitamin B1). In general, these patients do
not present with an acute neonatal illness, and their
early clinical course resembles that of the intermediate
form of MSUD. Dihydrolipoamide dehydrogenase
(E3) is not exclusive for the BCKADH, but is also a
component of α-ketoglutarate dehydrogenase and
pyruvate dehydrogenase. A defect in E3 will result in
combined ketoacid dehydrogenase deficiency and the
clinical manifestations are similar to the intermediate
form of MSUD. However, the organic acid profile of E3
deficiency demonstrates abnormalities of both lactic
acidosis and MSUD. Plasma BCAAs are moderately
increased as compared to classic MSUD.
Dietary restriction of BCAAs has proven a valuable
strategy in the treatment of MSUD. However, life-long
treatment is an absolute necessity as potentially fatal
episodes of metabolic decompensation may occur
in MSUD as a result of environmental factors which
include infection, fever or any other recurrent illness
at any age. Thiamine therapy is advisable in every novel
patient to determine thiamine-responsiveness. Marked
accumulation of BCAAs and their corresponding
ketoacids will cause an acute deterioration of cerebral
functions if left untreated. This life-threatening
situation requires aggressive and prompt treatment
which may include peritoneal dialysis or hemodialysis.5
11
These aggressive approaches are warranted with
plasma leucine in excess of 800 µmol/L. Orthotopic
liver transplantation has been performed in several
patients with MSUD and turned out to be an effective
method in controlling plasma BCAA levels.14,15 More
importantly, these patients remained metabolically and
neurologically stable on an unrestricted diet for more
than two years.
Inborn Errors of Leucine Degradation
Isovaleric acidemia (MIM 243500) Isovaleric acidemia
is caused by a deficiency of the enzyme isovalerylCoA dehydrogenase. This flavoenzyme catalyses the
conversion of isovaleryl-CoA to 3-methylcrotonylCoA and transfers electrons to the electron transfer
flavoprotein. Since its first report by Tanaka and coworkers16 approximately one hundred cases have
been reported17-19, but many more are known todate.
Initially, two distinct forms of isovaleric acidemia were
recognized. The acute neonatal form which causes
massive metabolic acidosis, encephalopathy and will
lead to rapid death, and the chronic form which is
characterized by episodes of severe ketoacidotic attacks
with asymptomatic intervals. More recently, a third
phenotype has been identified in which patients manifest
mild biochemical abnormalities or are asymptomatic.20
The phenotypic abnormalities of isovaleric acidemia
are due to the accumulation of isovaleric acid which
is toxic to the central nervous system. Interestingly,
during episodes of acute metabolic decompensation
the concentration of isovaleric acid can reach levels as
high as several hundred times normal values, but due
to its rapid conjugation to other compounds isovaleric
acid itself is not the hallmark of this disorder. In fact,
isovalerylcarnitine in conjunction with extremely low
carnitine, and isovalerylglycine are the distinctive
metabolites elevated in this disorder in plasma and
urine, respectively.21-23 The majority of patients with
isovaleric acidemia today are diagnosed through
newborn screening by use of acylcarnitine profiling
in dried blood spots which reveals increased amounts
of C5-acylcarnitine, i.e. isovalerylcarnitine. The gene
encoding isovaleryl-CoA dehydrogenase is mapped
to chromosome 15q14-q15 and consists of 12 exons
that span 15 kb of genomic DNA. Although, several
disease-causing mutations have been documented
in the numerous patients with isovaleric acidemia,
no genotype-phenotype correlation has been found.
The reduction of leucine intake and supplementation
with either glycine or carnitine have proven useful
strategies in the management of this disorder. Indeed,
administration of glycine reduces isovaleric acidemia
in neonates by conjugating isovaleric acid, with urinary
excretion of the conjugate.24,25
Isolated 3-methylcrotonyl-CoA carboxylase deficiency
(MIM 210200 and MIM 210210) 3-Methylcrotonyl-
CoA carboxylase is a heteromeric mitochondrial
enzyme comprising biotin-containing α subunits
and smaller β subunits, encoded by the MCCA and
MCCB gene, respectively. The enzyme catalyses
the fourth step in leucine catabolism converting 3methylcrotonyl-CoA to 3-methylglutaconyl-CoA in
a reversible ATP-dependent reaction using biotin as
source of the carboxyl-group. 3-Methylcrotonyl-CoA
carboxylase deficiency is caused either by mutations in
the structural genes MCCA and MCCB, or by deficient
activities of the enzymes involved in the metabolism of
its cofactor biotin. In addition to 3-methylcrotonyl-CoA
carboxylase, three other biotin-dependent carboxylases
exist in human – pyruvate carboxylase, acetyl-CoA
carboxylase and propionyl-CoA carboxylase – and
a deficit in biotin will result in multiple-carboxylase
deficiency. Isolated 3-methylcrotonyl-CoA carboxylase
deficiency is inherited as an autosomal recessive
trait and the molecular basis of this disorder was
resolved in 2001 by two independent groups.26,27 The
characteristic abnormal urinary metabolites in isolated
3-methylcrotonyl-CoA carboxylase deficiency are 3hydroxyisovaleric acid and 3-methylcrotonylglycine,
usually in conjunction with severe secondary carnitine
deficiency. Indeed, acyl-CoAs that accumulate in this
disorder are readily transesterified to acylcarnitine
species
with
3-hydroxyisobutyrylcarnitine
characteristically present in blood and urine. This
explains the acute carnitine depletion. At present
more than fifty patients with an isolated deficiency
in 3-methylcrotonyl-CoA carboxylase have been
reported. The phenotypic presentation is highly
variable. Some patients present in the neonatal period
with seizures and muscular hypotonia, whereas others
demonstrated severe psychomotor retardation, but
most children manifest acute episodes of metabolic
decompensation followed by complete recovery. These
episodes often arise after an infection or fasting, and
include feeding problems, vomiting, failure to thrive,
lethargy, apnea, hypotonia and infantile spasms. For
definitive diagnosis of isolated 3-methylcrotonylCoA carboxylase deficiency, one should demonstrate
deficient 3-methylcrotonyl-CoA carboxylase activity
with normal enzyme activities of at least one of the
other carboxylases.
3-methylglutaconic aciduria type I (MIM 250950) 3Methylglutaconic aciduria type I represents the isolated
deficiency of 3-methylglutaconyl-CoA hydratase.
Sixteen patients with 3-methylglutaconic aciduria
type I have been reported.28-39 Whereas the clinical
manifestations in some patients is confined to just a mildly
delayed speech development, several other patients
have been reported with severe (leuko)encephalopathy
in both the neonatal period and late adulthood. No
episodes of acidosis have been reported. In addition,
three patients with 3-methylglutaconyl-CoA hydratase
deficiency were initially misdiagnosed as suffering
from multiple sclerosis.28,30,31 In these patients
12
magnetic resonance imaging of the brain has shown
widespread involvement of the cerebral white matter
and bilateral involvement of the cerebellar peduncles,
closely resembling the brain abnormalities observed
in multiple sclerosis. Three additional forms of 3methylglutaconic aciduria have been recognized all
characterized by normal hydratase activities. Moreover,
patients with type I additionally excrete increased
amounts of 3-hydroxyisovaleric acid and therefore, 3hydroxyisovaleric acid is an important parameter for
differentiation between isolated 3-methylglutaconylCoA hydratase deficiency and the other types. More
importantly, the advent of a specific enzymatic assay to
determine 3-methylglutaconyl-CoA hydratase activity
has greatly facilitated the identification of patients
with 3-methylglutaconic aciduria type I (see chapter
4).40 In 1995 Nakagawa purified an AU-specific RNAbinding protein which was believed to be involved in
the turnover of mRNA.41-43 In addition, this protein
demonstrated enoyl-CoA hydratase activity. In fact,
we have shown that this protein is responsible for the
3-methylglutaconyl-CoA hydratase activity in leucine
degradation and that mutations in the AUH gene cause
3-methylglutaconic aciduria type I (see chapter 3).33
3-hydroxy-3-methylglutaryl-CoA lyase deficiency (MIM
246450) More than forty patients with 3-hydroxy3-methylglutaryl-CoA lyase deficiency have been
reported and most of them clinically present within
the first year of life.44-47 This disorder is often a lifethreatening condition in the neonatal period. The key
clinical features include severe infantile hypoglycemia,
metabolic acidosis, hepatomegaly, lethargy or coma and
a characteristic absence of ketosis. The neurological
complications in this disorder due to hypoketotic
hypoglycemia and acidosis may result in a severe and
permanent handicap. Patients may also manifest Reye
syndrome-like episodes characterized by increased
levels of ammonia and abnormal liver function tests,
fever, hepatomegaly, altered levels of consciousness
and seizures. 3-Hydroxy-3-methylglutaryl-CoA lyase
catalyses the ultimate step of both leucine degradation
and ketogenesis, and therefore plays an important role
in ketone body production. Nonetheless, the diagnosis
is often made from the typical abnormal organic acid
profile dominated by leucine metabolites. Indeed, the
pattern of urinary organic acids includes 3-hydroxy3-methylglutaric acid, 3-methylglutaconic acid, 3methylglutaric acid and 3-hydroxyisovaleric acid. In
common with other (branched chain) organic acidurias,
patients with 3-hydroxy-3-methylglutaryl-CoA lyase
deficiency usually show an elevated acyl-carnitine to
free carnitine ratio, with the most prominent carnitine
species in this disorder being 3-methylglutarylcarnitine.
Ultimately, the diagnosis is best confirmed by measuring
lyase activity48,49 in conjunction with molecular analysis.
Indeed, pathogenic mutations in the encoding HL gene
have been identified, but revealed considerable genetic
heterogeneity.
Inborn Errors of Isoleucine Degradation
2-methylbutyryl-CoA
dehydrogenase
deficiency
(MIM 610006) 2-Methylbutyryl-CoA dehydrogenase
deficiency, also referred to as short/branched chain
acyl-CoA dehydrogenase (SBCAD) deficiency, has
been described as an autosomal recessive disorder
of L-isoleucine catabolism. Although 18 individuals
have been diagnosed with SBCAD deficiency up
till now, the clinical presentation of this disorder
is still not well defined ranging from completely
asymptomatic to severe neurological handicaps.50-55
The lack of clinical symptoms in most patients should
not give the paediatrician a false sense of security, as
environmental factors are often crucial determinants
of the clinical course of inborn errors of metabolism.
Moreover, in other (branched chain) organic acidurias
acute decompensation and death have occurred with
the first severe catabolic episode. More importantly,
those patients who are clinically symptomatic often
demonstrated neurological involvement with epilepsy
being the key feature. This may indicate that when
SBCAD deficiency manifests itself clinically, the
brain and the spinal cord are primarily affected.
Although the underlying cause of the central nervous
system dysfunction in SBCAD deficiency remains
unclear, it is noteworthy that in most branched chain
organic acidurias neurological handicaps appear to
be common. 2-Methylbutyryl-CoA dehydrogenase
catalyses the conversion of 2-methylbutyryl-CoA to
tiglyl-CoA and a deficiency of this enzyme results in
the accumulation of its precursors. It appears that the
major fate of 2-methylbutyryl-CoA that accumulates
in this disorder is not deacylation to 2-methylbutyric
acid but rather transesterification with glycine to form
2-methylbutyrylglycine. Indeed, SBCAD deficiency
is characterized by persistently increased urinary
excretion of 2-methylbutyrylglycine. In addition,
eight additional patients with SBCAD deficiency were
detected through prospective newborn screening using
tandem mass spectrometry as their plasma acylcarnitine
analysis revealed elevated levels of C5-acylcarnitine,
i.e. 2-methylbutyrylcarnitine. The molecular basis
of this inborn error of metabolism has been resolved
by Andresen and colleagues. 2-Methylbutyryl-CoA
dehydrogenase is encoded by the ACADSB gene located
at chromosome 10q26.13 and contains 11 exons. The
gene encodes a mitochondrial precursor protein which
is cleaved upon mitochondrial import and predicted
to yield a mature peptide of approximately 44 kDa. At
present, a total of nine distinct mutations have been
reported in eighteen patients.
2-methyl-3-hydroxybutyryl-CoA
dehydrogenase
deficiency (MIM 300438) 2-Methyl-3-hydroxybutyrylCoA dehydrogenase deficiency has been described
as a X-linked neurodegenerative disorder with
psychomotor regression. Since its initial report in
2000 by Zschocke and co-workers56 nine additional
13
affected individuals, including two female patients,
have been described.57-63 These female patients
demonstrated a less severe phenotype without loss of
developmental milestones which has been attributed
to lyonization (X-chromosome inactivation) in most
but not all body tissues. Although considerable clinical
heterogeneity is observed among all patients, this
disorder is characterized by normal early development
followed by a progressive loss of acquired mental and
motor skills, hypotonia, loss of vision and seizures.
The onset of the regression appears to be variable
ranging from the neonatal period to as late as five years
of age. Furthermore, virtually all patients developed
neurological symptoms associated with changes in
MRI. The most common MRI finding was a pronounced
atrophy of the brain, usually manifesting itself as
frontotemporal atrophy. 2-Methyl-3-hydroxybutyrylCoA dehydrogenase deficiency is biochemically
characterized by moderately elevated urinary excretion
of tiglylglycine and 2-methyl-3-hydroxybutyric acid.
Furthermore, a number of additional metabolites have
been found, including elevated CSF or blood lactate,
increased urinary amounts of 3-hydroxyisobutyric
acid, 2-ethylhydracrylic acid and 3-methylglutaconic
acid using urinary organic acid analysis. In addition,
analysis of plasma acylcarnitine levels may show
a mild increase in C5:1-acylcarnitine and C5hydroxyacylcarnitine, representing tiglylcarnitine and
2-methyl-3-hydroxybutyrylcarnitine,
respectively.
Molecular analysis in eight patients with this disorder
revealed only three mutations in the encoding HADH2
gene, all being missense mutations.61 One missense
mutation (c.388C>T; R130C), was identified in five
of the eight patients, which is remarkable in a severe
X-linked disorder. Expression studies of the mutant
cDNAs demonstrated that two missense mutations
generated a fully inactive protein, whereas some residual
activity (2-3% of the wildtype enzyme) was found for
the third. There are speculations on an alternative role
of the HADH2 protein, possibly associated with other
mechanisms of neurological regression.
β-ketothiolase deficiency (MIM 203750) β-Ketothiolase
deficiency is an autosomal recessive disorder of ketone
body metabolism and isoleucine catabolism and is
caused by a deficiency in the mitochondrial acetoacetylCoA thiolase.64 This enzyme catalyzes the final step in
isoleucine degradation which is the conversion of 2methylacetoacetyl-CoA into acetyl-CoA and propionylCoA. In ketone body metabolism its substrate is
acetoacetyl-CoA rather than 2-methylacetoacetylCoA. The major clinical manifestations of this disorder
are intermittent episodes of acute ketoacidosis
which typically occur in a previously healthy child.
The age of onset is often between the age of six and
twenty four months and is usually triggered by a
febrile illness. However, there is considerable clinical
heterogeneity in β-ketothiolase deficiency.53,65-67
Vomiting and dehydration often occur and the patient
becomes progressively lethargic, sometimes leading
to coma. In contrast, some patients have died or had
severe neurological sequelae as a result of recurrent
or prolonged episodes of ketoacidosis. Occasionally
an affected but asymptomatic sibling or even parent
is identified within the same family. During an acute
episode patients excrete massive amounts of ketone
bodies (3-hydroxybutyric acid and acetoacetic acid),
2-methylacetoacetic acid, 2-methyl-3-hydroxybutyric
acid and tiglylglycine. Remarkably, rather than 2methylacetoacetic acid the most prominent metabolites
in ß-ketothiolase deficiency are tiglylglycine and
2-methyl-3-hydroxybutyric
acid.
ß-Ketothiolase
deficiency is caused by mutations in the ACAT1 (acetylCoA acetyltransferase 1) gene mapped to chromosome
11q22-q23.66,68 Over 40 mutations have been identified
which revealed a biochemical phenotype-genotype
correlation in this disorder. Those patients whose
mutation(s) resulted in residual activities of the thiolase
protein often demonstrated no increased excretion
of tiglylglycine even during ketoacidotic episodes and
(borderline) normal urine organic acid and plasma
acylcarnitine profiles when in remission. However,
no phenotype-genotype correlation has been found.
Prompt diagnosis and aggressive treatment will often
lead to a rapid and full recovery. In general, treatment
with a low protein diet can prevent the recurrence
of ketoacidotic episodes and improve the long-term
outcome of this disorder.
Inborn Errors of Valine Degradation
Isobutyryl-CoA dehydrogenase deficiency (MIM
604773) Isobutyryl-CoA dehydrogenase deficiency
was first reported in 199869 and to date only nine
patients have been reported. In 2000, Andresen et al
demonstrated that acyl-CoA dehydrogenase (ACAD) 8
is in fact isobutyryl-CoA dehydrogenase, after which
the molecular basis of isobutyryl-CoA dehydrogenase
deficiency was solved by the identification of diseasecausing mutations in the ACAD8 gene.51,70-72 Although
significant clinical symptoms, which included dilated
cardiomyopathy, failure to thrive and carnitine
deficiency, were observed in the first patient reported,
the isobutyryl-CoA dehydrogenase deficient newborns
identified by screening either remained asymptomatic
or presented with mild clinical abnormalities.
Examination of the urinary organic acid profile of
these patients showed marginally elevated excretion of
isobutyrylglycine. In addition, acylcarnitine profiling
has demonstrated elevated levels of C4-acylcarnitines
and an increased C4/C2-acylcarnitine ratio in this
disorder. As mentioned below (see ‘diagnosis of
branched chain organic acidurias’), tandem-MS does
not differentiate between isomers and therefore C4acylcarnitine could represent either butyryl-CoA or
isobutyryl-CoA. Accumulation of butyrylcarnitine
is suggestive of short chain acyl-CoA dehydrogenase
(SCAD) deficiency, an enzymatic defect in fatty acid
14
β-oxidation that may cause severe clinical symptoms.
Surprisingly, although disease-causing mutations
were found in the ACAD8 gene in the aforementioned
patients, SCAD gene analysis revealed heterozygosity
for the prevalent c.625G>A susceptibility variation all
individuals investigated. This variant of the SCAD gene
is frequent in the European population and is considered
to confer increased susceptibility to clinical SCAD
deficiency in combination with either environmental
factors or other genetic factors, such as perhaps
mutations in the ACAD8 gene. However, more patients
with isobutyryl-CoA dehydrogenase deficiency should
be identified and subsequently investigated to clarify
if isobutyryl-CoA dehydrogenase deficiency and the
SCAD c.625G>A variation are associated. In addition,
several reports have described patients with increased
excretion of C4-acylcarnitine and homozygosity for
the c.625G>A variant. However, isobutyryl-CoA
dehydrogenase deficiency has not been excluded and
therefore it could prove prudent to analyse the ACAD8
gene and isobutyryl-CoA dehydrogenase activity in
these patients.
3-hydroxyisobutyryl-CoA
hydrolase
deficiency
(MIM 250620) 3-Hydroxyisobutyryl-CoA hydrolase
deficiency is very rare with only two reported patients
sofar.4 Both male patients clinically presented with
poor feeding, hypotonia, developmental delay of motor
skills and neurological regression in infancy. Whereas
one patient demonstrated episodes of ketoacidosis
and Leigh-like changes in the basal ganglia, the
other patient presented with dysmorphic features
and died at an early age. The biochemical basis of 3hydroxyisobutyryl-CoA hydrolase deficiency was
solved in the early 1980s after the finding of elevated
urinary levels of S-2-carboxypropyl-cysteine and S2-carboxypropyl-cysteamine.73,74 These derivatives
are formed through the addition of cysteine and
cysteamine across the carbon-carbon double bond
of methacrylyl-CoA. Normal enoyl-CoA hydratase
activities in the patient’s fibroblasts suggested a
deficiency in 3-hydroxyisobutyryl-CoA hydrolase
which was subsequently confirmed enzymatically. In
contrast, no unusual sulphur containing amino acids
were identified in the urine of the second patient. It
was the detection of persistently elevated hydroxy-C4carnitine species in blood that suggested a deficiency
of 3-hydroxyisobutyryl-CoA hydrolase. Indeed,
enzymatic analysis demonstrated the complete absence
of 3-hydroxyisobutyryl-CoA hydrolase activity. The
molecular basis of this disorder was only recently
resolved by the identification of disease-causing
mutations in the encoding HIBCH gene (chapter 7).4
3-hydroxyisobutyric aciduria (MIM 236795) 3Hydroxyisobutyric aciduria is a rare metabolic anomaly
in valine degradation and today only thirteen patients
have been described in literature.75-83 These patients
usually present with dysmorphic features including
microcephaly, a small triangular face, low set ears and
long philtrum. Patients may show widely different
phenotypes ranging from mild vomiting attacks with
normal brain and cognitive development, to delayed
motor development, profound mental impairment
and early death. Patients with 3-hydroxyisobutyric
aciduria usually excrete elevated amounts of 3hydroxyisobutyric acid (3-HIBA) in urine, ranging
from 60 to 390 mmol/mol of creatinine (normal < 40
mmol/mol of creatinine) when in stable condition and
increasing up to 10,000 mmol/mol creatinine during
acute ketoacidotic episodes. In half of the reported
cases elevated lactate levels were observed. Other, less
frequently observed abnormal metabolites in urine are
3-hydroxypropionic acid, 2-ethyl-3-hydroxypropionic
acid, 2-hydroxybutyric acid, S-(2-carboxypropyl)cysteine and 3-hydroxyisovaleric acid. Although
the responsible defect has not been identified yet, a
deficiency of 3-hydroxyisobutyrate dehydrogenase has
been suggested as the most likely enzyme defect in
patients with 3-hydroxyisobutyric aciduria.
Methylmalonic
semialdehyde
dehydrogenase
deficiency (MIM 603178) So far, only a single patient
with methylmalonic semialdehyde dehydrogenase
deficiency has been described in literature.84-86
Routine newborn screening demonstrated a marked
hypermethioninemia. The male patient was hospitalized
at the age of three weeks with an unexplained episode
of diarrhoea and vomiting. Subsequent urinary organic
acid analysis revealed extremely elevated levels of
3-hydroxyisobutyric acid. Further investigations
also demonstrated increased urinary excretion of ßalanine, 3-hydroxypropionic acid, and both isomers
of 3-aminoisobutyric acids, suggesting methylmalonic
semialdehyde dehydrogenase deficiency. Molecular
analysis of the human MMSDH gene revealed
homozygosity for a missense mutation (c.1336G>A;
G446R) and the deletion of exon 10.84,87 No expression
studies were performed with the mutant cDNA.
Inborn Errors of Valine-Isoleucine Degradation
Propionic acidemia (MIM 606054) and methylmalonic
aciduria (MIM 251000) Propionic acidemia and
methylmalonic aciduria are the most frequent forms
of branched chain organic acidurias (reviewed in 8895
). These autosomal recessive disorders are caused
by deficiencies of propionyl-CoA carboxylase and
methylmalonyl-CoA mutase, respectively. PropionylCoA carboxylase is a heteromeric mitochondrial
enzyme comprising biotin-containing α subunits and
smaller β subunits, encoded by the PCCA and PCCB
genes, respectively.91,96 Propionic acidemia is caused
either by mutations in the structural genes PCCA
or PCCB, or by deficient activities of the enzymes
involved in the metabolism of its cofactor biotin. In
addition to propionyl-CoA carboxylase, three other
biotin-dependent carboxylases exist in human – 315
methylcrotonyl-CoA carboxylase (see above), pyruvate
carboxylase, and acetyl-CoA carboxylase – and a
deficit in biotin will result in multiple-carboxylase
deficiency. Methylmalonic aciduria includes disorders
of methylmalonate and cobalamin metabolism. Isolated
methylmalonic aciduria is caused by mutations in
the MUT gene, which encodes methylmalonyl-CoA
mutase.
Remarkably, patients with propionic acidemia
or methylmalonic aciduria manifest similar clinical
and biochemical abnormalities and therefore these
disorders are discussed here simultaneously. Patients
with propionic acidemia persistently demonstrate
increased urinary excretion of methylcitric acid
often in combination with 3-hydroxypropionic
acid and other propionyl-CoA metabolites which
include propionylglycine, tiglylglycine, 2-methyl3-hydroxybutyric acid and 2-methylacetoacetate.
Episodes of ketoacidosis occur frequently and are
accompanied by the excretion of abnormal, but highly
characteristic ketones such as 3-oxovaleric acid and 3hydroxyvaleric acid. Methylmalonic aciduria will show
large amounts of methylmalonic acid in conjunction
with the propionyl-CoA metabolites which are the
result of a secondary inhibition of propionyl-CoA
carboxylase. Plasma acylcarnitine analysis in both
propionic acidemia and methylmalonic aciduria shows
elevation of propionylcarnitine as the predominant
acylcarnitine species. Free carnitine is reduced and the
ratio of propionylcarnitine to free carnitine is greatly
increased. There are two vitamin B12 responsive forms
of methylmalonic aciduria, denoted CblA and B; both
involving defects in adenosylcobalamin metabolism.
Methylmalonic aciduria with homocystinuria is caused
by cobalamin C/D defects which impair the conversion
of vitamin B12 into its two metabolically active forms,
methylcobalamin and adenosylcobalamin. This disease
usually presents with a different – predominantly
neurological – clinical picture distinct from isolated
methylmalonic aciduria.
In most cases of propionic acidemia and
methylmalonic aciduria the onset of the disease is
in the neonatal period in which the patient rapidly
deteriorates for no apparent reason. After an initial
unremarkable interval the symptoms proceed from
vomiting, poor feeding and weight loss to progressive
neurologic sequelae which include mental retardation,
motor dysfunction, (epileptic) seizures, hypotonia and
lethargy. If left untreated, the patient’s condition could
progress to coma with brain oedema and respiratory
arrest, and ultimately permanent brain damage or
death. Normochloremic metabolic acidosis, ketonuria,
hyperuricemia, hyperammonemia, thrombocytopenia,
leukopenia and anemia are typically found which often
results in the false diagnosis of sepsis. In the late onset
forms, the clinical manifestations range from acute
encephalopathy to intermittent or chronic symptoms
which include intermittent ataxia, abnormal behaviour
and feeding with the selective refusal of protein-
rich foods, anorexia, recurrent vomiting, failure to
thrive and (neuro)developmental delay. The recurrent
episodes are often triggered by stress and between
these intervals the patient may appear normal.
Management of patients with propionic acidemia
and methylmalonic aciduria is difficult. Therapy
includes a low-protein high-energy diet and carnitine
supplementation. Some patients with methylmalonic
aciduria respond to pharmacological doses of
vitamin B12. Therefore a trial with vitamin B12
supplementation should always be made in newly
diagnosed methylmalonic aciduria patients. Propionic
acidemia is theoretically amenable to biotin treatment.
No biotin-responsive propionic acidemia patient
has been reported thus far, however. Given the poor
long-term prognosis, liver transplantation has been
attempted recently as an alternative therapy, but this
approach has not been proven efficient.
Biochemical Diagnosis of
Branched Chain Organic Acidurias
Among the organic acidurias genetic defects in the
degradation of branched chain amino acids are most
frequent. Two thirds of the cases manifest themselves
during the neonatal period, most of them with an
acute onset. Prompt diagnosis of organic acidurias
depends on their early identification of children with
suspect clinical symptoms. Once an inborn error of
metabolism is suspected from the clinical presentation,
the diagnosis should be corroborated further through
laboratory analysis. First, the biochemical evaluation of
patients with a potential metabolic disorder should be
aimed at the identification of either abnormal levels of
a physiological compound(s) or abnormal metabolite(s)
in body fluids. Indeed, accumulation of intermediates
prior to an obstruction in a metabolic pathway will
provide the paediatrician with clues for the underlying
enzymatic defect. The rapid differential diagnosis is
best accomplished by determination of an abnormal
pattern of urinary organic acids determined by gas
chromatography – mass spectrometry. The major fates
of the acyl-CoA thioesters that accumulate as a result of
a metabolic block in branched chain organic acidurias
are a) hydrolysis to the corresponding organic acid
and free coenzyme A, and b) transesterification with
glycine and/or carnitine to form acylglycines and/or
acylcarnitines, respectively. Although the acylglycines
can be detected by routine urinary organic acid analysis
some patients may have been overlooked as a result of
the insufficient sensitivity of acylglycine detection and
the inadequate interpretation of the acylglycine profile.
In contrast, organic acids are often easily detected.
During acute episodes, patients often excrete massive
amounts of accumulated metabolites. However, the
elevation, if any, may be very modest when stable. Thus,
a diagnosis could be missed when an interval sample is
analyzed.
In recent years acylcarnitine profiling by means of
16
tandem mass spectrometry has become an increasingly
powerful tool in the diagnosis of inborn errors of
metabolism.17,20,50,53,68,70,95,97-102 Indeed, this technique
has proven crucial in the identification of additional
patients with a branched chain organic aciduria,
including SBCAD deficiency and 3-hydroxyisobutyrylCoA hydrolase deficiency. Moreover, acylcarnitine
profiling has the additional advantage that it can be
applied on dried bloodspots obtained for routine
newborn screening. This can ultimately lead to a
very early diagnosis of many of the branched chain
organic acidurias. The acyl-CoA thioesters that
accumulate as a result of a metabolic block are in
equilibrium with their corresponding acylcarnitines.
These carnitine species are present in plasma and
readily excreted in urine. However, routine combined
high performance liquid chromatography - tandem
mass spectrometric analysis as used in many presentday laboratories does not differentiate between
isomers and, for example, a C5-acylcarnitine species
could represent either 2-methylbutyryl-, isovaleryl, valeryl- or pivaloylcarnitine or even a combination
of these isomers, whereas hydroxy-C5-acylcarnitine
could represent either 3-hydroxyisovaleryl- or 2methyl-3-hydroxybutyrylcarnitine. Consequently, the
contribution of this technique in the diagnosis of inborn
errors of metabolism, such as branched chain organic
acidurias, is rather limited. Nevertheless, in conjunction
with a carefully interpreted urinary organic acid profile
one should be able to determine the exact diagnosis.
Ultimately, enzymatic studies have to be performed
to identify the enzymatic defect, which then must be
followed by molecular analysis of the gene involved.
Implications for therapy
Accumulated intermediates proximal to the enzyme
deficiency are usually considered toxic and thus
the cause of the primary pathological effects. In
addition, much of the physiological abnormalities
may result from either endogenous compensatory
mechanisms, inhibition of general metabolism, or the
reduced availability of pivotal compounds such as free
coenzyme A and reducing equivalents. As branched
chain amino acids are essential amino acids, an
increased flux through the catabolic pathway can only
be accomplished via dietary intake or via endogenous
breakdown of protein. Thus, treatment of patients with
an inborn error of BCAAs degradation should be aimed
at removal of toxic compounds, i.e. minimizing the flux
through the catabolic pathway in both the acute and
chronic state.
In the critically ill, immediate treatment should be
initiated even if a definitive diagnosis may not yet be
established. Within 72 hours, the results of amino acid
and organic acid analysis should be available, allowing
diagnostic confirmation in most cases. Prompt and
aggressive treatment before the confirmation of a
diagnosis may be life-saving and could prevent or
reduce the neurological sequelae of some of these
disorders. First, the patient should be stabilized
by protein restriction, administration of fluids and
intravenous glucose. Insulin could be added to utilize
its anabolic effect. If warranted, (hemo)dialysis should
be instituted immediately without waiting a response
to the dietary manipulation or medication, providing
maximal supportive care simultaneously. Altogether,
this will often lead to a rapid and complete recovery
provided that treatment is initiated without delay.
Although this approach is very effective on short
terms, protein degradation will escape the restraint
of limited endogenous catabolism within 3 to 5 days
and the preceding improvement will arrest or, for
worse, the patient will deteriorate. Fortunately, the
introduction of formulas deficient in given amino
acid enables one to keep the level of the amino acid in
question within acceptable limits, while maintaining
amino acid homeostasis. This will result in improved
growth and fewer and less severe episodes of catabolic
decompensation associated with fasting or infection,
provided that a high carbohydrate intake is maintained.
Thus, selective amino acid restriction in conjunction
with a high carbohydrate or lipid intake is an important
strategy in the long-term management of many
branched chain organic acidurias.
Other eminent therapeutic approaches for long-term
removal of toxic metabolites include the administration
of detoxifying agents. Treatment with either glycine or
carnitine could increase the elimination of acyl-CoAs
as urinary acylglycines or acylcarnitines and, thus,
prevent the formation of toxic compounds. In addition,
organic acidurias often cause elevated amounts of acylCoAs, acylglycines and acylcarnitine species, which
affect the availability of free coenzyme A, glycine and
carnitine. As acylcarnitine species are not reabsorbed
in the renal tube, a further decrement in the amount
of carnitine is often observed, which could impair
substrate transport across the mitochondrial membrane
and subsequent energy production. Administration of
exogenous carnitine corrects this deficit and may also
shift the acyl-CoA:CoA ratio to free coenzyme A. In
addition, provision of pantothenate (vitamin B5) could
compensate for the fact that at least part of intracellular
CoA will be trapped in the form of acyl-CoA esters.
Once we have gained more experience with the
natural history of branched chain organic acidurias,
the role of therapy in these inborn error of metabolism
will become clearer.
References
1. Zello GA, Wykes LJ, Ball RO, Pencharz PB. 1995. J Nutr 125:
2907-15
2. Young VR, Pellett PL. 1985. Am J Clin Nutr 41: 1077-90
3. Zello GA, Menendez CE, Rafii M, Clarke R, Wykes LJ, et al.
2003. Pediatr Res 53: 338-44
4. Loupatty FJ, Clayton PT, Ruiter JP, Ofman R, Ijlst L, et al. 2007.
Am J Hum Genet 80: 195-9
17
5. Chuang DT, Chuang JL, Wynn RM. 2006. J Nutr 136: 243S-9S
6. Chuang DT. 1998. J Pediatr 132: S17-23
7. Chuang JL, Cox RP, Chuang DT. 1996. Am J Hum Genet 58:
1373-7
8. Chuang JL, Davie JR, Chinsky JM, Wynn RM, Cox RP, Chuang
DT. 1995. J Clin Invest 95: 954-63
9. Chuang DT, Fisher CW, Lau KS, Griffin TA, Wynn RM, Cox RP.
1991. Mol Biol Med 8: 49-63
10.Fisher CW, Chuang JL, Griffin TA, Lau KS, Cox RP, Chuang DT.
1989. J Biol Chem 264: 3448-53
11.Menkes JH. 1959. Pediatrics 23: 348-53
12.Kumta NB. 2005. Indian J Pediatr 72: 325-32
13.Scriver C. 2001. The Metabolic and Molecular Basis of Inherited
Diseases: McGraw-Hill
14.Buss P, Tornberg DN. 2006. Am J Transplant 6: 1982; author
reply 3
15.Nyhan WL, Rice-Kelts M, Klein J, Barshop BA. 1998. Arch
Pediatr Adolesc Med 152: 593-8
16.Tanaka K, Budd MA, Efron ML, Isselbacher KJ. 1966. Proc Natl
Acad Sci U S A 56: 236-42
17.Vockley J, Ensenauer R. 2006. Am J Med Genet C Semin Med
Genet 142: 95-103
18.Tokatli A, Coskun T, Ozalp I. 1998. Turk J Pediatr 40: 111-9
19.Tanaka K. 1990. Prog Clin Biol Res 321: 273-90
20.Ensenauer R, Vockley J, Willard JM, Huey JC, Sass JO, et al.
2004. Am J Hum Genet 75: 1136-42
21.Gregersen N, Kolvraa S, Mortensen PB. 1986. Biochem Med
Metab Biol 35: 210-8
22.Roe CR, Millington DS, Maltby DA, Kahler SG, Bohan TP. 1984.
J Clin Invest 74: 2290-5
23.Tanaka K, Isselbacher KJ. 1967. J Biol Chem 242: 2966-72
24.Cohn RM, Yudkoff M, Rothman R, Segal S. 1978. N Engl J Med
299: 996-9
25.Yudkoff M, Cohn RM, Puschak R, Rothman R, Segal S. 1978. J
Pediatr 92: 813-7
26.Baumgartner MR, Almashanu S, Suormala T, Obie C, Cole RN,
et al. 2001. J Clin Invest 107: 495-504
27.Gallardo ME, Desviat LR, Rodriguez JM, Esparza-Gordillo J,
Perez-Cerda C, et al. 2001. Am J Hum Genet 68: 334-46
28.Arbelaez A, Castillo M, Stone J. 1999. Neuroradiology 41: 9412
29.Duran M, Beemer FA, Tibosch AS, Bruinvis L, Ketting D,
Wadman SK. 1982. J Pediatr 101: 551-4
30.Engelke UF, Kremer B, Kluijtmans LA, van der Graaf M, Morava
E, et al. 2006. NMR Biomed 19: 271-8
31.Eriguchi M, Mizuta H, Kurohara K, Kosugi M, Yakushiji Y, et al.
2006. Neurology 67: 1895-6
32.Gibson KM, Elpeleg ON, Jakobs C, Costeff H, Kelley RI. 1993.
Pediatr Neurol 9: 120-3
33.IJlst, Loupatty FJ, Ruiter JP, Duran M, Lehnert W, Wanders RJ.
2002. Am J Hum Genet 71: 1463-6
34.Illsinger S, Lucke T, Zschocke J, Gibson KM, Das AM. 2004.
Pediatr Neurol 30: 213-5
35.Lehnert W, Scharf J, Wendel U. 1985. Eur J Pediatr 143: 301-3
36.Ly TB, Peters V, Gibson KM, Liesert M, Buckel W, et al. 2003.
Hum Mutat 21: 401-7
37.Narisawa K, Gibson KM, Sweetman L, Nyhan WL, Duran M,
Wadman SK. 1986. J Clin Invest 77: 1148-52
38.Pantaleoni C, D’Arrigo S, D’Incerti L, Rimoldi M, Riva D. 2000.
Pediatr Neurol 23: 442-4
39.Shoji Y, Takahashi T, Sawaishi Y, Ishida A, Matsumori M, et al.
1999. J Inherit Metab Dis 22: 1-8
40.Loupatty FJ, Ruiter JP, L IJ, Duran M, Wanders RJ. 2004. Clin
Chem 50: 1447-50
41.Kurimoto K, Fukai S, Nureki O, Muto Y, Yokoyama S. 2001.
Structure 9: 1253-63
42.Nakagawa J, Moroni C. 1997. Eur J Biochem 244: 890-9
43.Nakagawa J, Waldner H, Meyer-Monard S, Hofsteenge J, Jeno P,
Moroni C. 1995. Proc Natl Acad Sci U S A 92: 2051-5
44.Mitchell GA, Ozand PT, Robert MF, Ashmarina L, Roberts J, et
al. 1998. Am J Hum Genet 62: 295-300
45.Ozand PT, al Aqeel A, Gascon G, Brismar J, Thomas E, Gleispach
H. 1991. J Inherit Metab Dis 14: 174-88
46.Gibson KM, Breuer J, Nyhan WL. 1988. Eur J Pediatr 148: 1806
47.Wysocki SJ, Hahnel R. 1986. J Inherit Metab Dis 9: 225-33
48.Wanders RJ, Zoeters PH, Schutgens RB, de Klerk JB, Duran M,
et al. 1990. Clin Chim Acta 189: 327-34
49.Wanders RJ, Schutgens RB, Zoeters PH. 1988. Clin Chim Acta
171: 95-101
50.Matern D, He M, Berry SA, Rinaldo P, Whitley CB, et al. 2003.
Pediatrics 112: 74-8
51.Andresen BS, Christensen E, Corydon TJ, Bross P, Pilgaard B, et
al. 2000. Am J Hum Genet 67: 1095-103
52.Gibson KM, Burlingame TG, Hogema B, Jakobs C, Schutgens
RB, et al. 2000. Pediatr Res 47: 830-3
53.Korman SH. 2006. Mol Genet Metab 89: 289-99
54.Madsen PP, Kibaek M, Roca X, Sachidanandam R, Krainer AR,
et al. 2006. Hum Genet 118: 680-90
55.Korman SH, Andresen BS, Zeharia A, Gutman A, Boneh A, Pitt
JJ. 2005. Clin Chem 51: 610-7
56.Zschocke J, Ruiter JP, Brand J, Lindner M, Hoffmann GF, et al.
2000. Pediatr Res 48: 852-5
57.Perez-Cerda C, Garcia-Villoria J, Ofman R, Sala PR, Merinero B,
et al. 2005. Pediatr Res 58: 488-91
58.Poll-The BT, Wanders RJ, Ruiter JP, Ofman R, Majoie CB, et al.
2004. Mol Genet Metab 81: 295-9
59.Sass JO, Forstner R, Sperl W. 2004. Brain Dev 26: 12-4
60.Sutton VR, O’Brien WE, Clark GD, Kim J, Wanders RJ. 2003. J
Inherit Metab Dis 26: 69-71
61.Ofman R, Ruiter JP, Feenstra M, Duran M, Poll-The BT, et al.
2003. Am J Hum Genet 72: 1300-7
62.Ensenauer R, Niederhoff H, Ruiter JP, Wanders RJ, Schwab KO,
et al. 2002. Ann Neurol 51: 656-9
63.Olpin SE, Pollitt RJ, McMenamin J, Manning NJ, Besley G, et al.
2002. J Inherit Metab Dis 25: 477-82
64.Fukao T, Yamaguchi S, Kano M, Orii T, Osumi T, Hashimoto T.
1992. Prog Clin Biol Res 375: 573-81
65.Fukao T, Scriver CR, Kondo N. 2001. Mol Genet Metab 72: 10914
66.Fukao T, Yamaguchi S, Orii T, Hashimoto T. 1995. Hum Mutat
5: 113-20
67.Ozand PT, Rashed M, Gascon GG, al Odaib A, Shums A, et al.
1994. Brain Dev 16 Suppl: 38-45
68.Pasquali M, Monsen G, Richardson L, Alston M, Longo N. 2006.
Am J Med Genet C Semin Med Genet 142: 64-76
69.Roe CR, Cederbaum SD, Roe DS, Mardach R, Galindo A,
Sweetman L. 1998. Mol Genet Metab 65: 264-71
70.Pedersen CB, Bischoff C, Christensen E, Simonsen H, Lund AM,
et al. 2006. Pediatr Res 60: 315-20
71.Sass JO, Sander S, Zschocke J. 2004. J Inherit Metab Dis 27: 7415
72.Nguyen TV, Andresen BS, Corydon TJ, Ghisla S, Abd-El Razik
N, et al. 2002. Mol Genet Metab 77: 68-79
73.Brown GK, Hunt SM, Scholem R, Fowler K, Grimes A, et al.
1982. Pediatrics 70: 532-8
74.Truscott RJ, Malegan D, McCairns E, Halpern B, Hammond J, et
18
al. 1981. Biomed Mass Spectrom 8: 99-104
75.Loupatty FJ, van der Steen A, Ijlst L, Ruiter JP, Ofman R, et al.
2006. Mol Genet Metab 87: 243-8
76.Sasaki M, Yamada N, Fukumizu M, Sugai K. 2006. Brain Dev 28:
600-3
77.Shield JP, Gough R, Allen J, Newbury-Ecob R. 2001. Clin
Dysmorphol 10: 189-91
78.Sasaki M, Iwata H, Sugai K, Fukumizu M, Kimura M, Yamaguchi
S. 2001. Brain Dev 23: 243-5
79.Yoshida I. 1998. Ryoikibetsu Shokogun Shirizu: 317-9
80.Sasaki M, Kimura M, Sugai K, Hashimoto T, Yamaguchi S. 1998.
Pediatr Neurol 18: 253-5
81.Boulat O, Benador N, Girardin E, Bachmann C. 1995. J Inherit
Metab Dis 18: 204-6
82.Chitayat D, Meagher-Villemure K, Mamer OA, O’Gorman A,
Hoar DI, et al. 1992. J Pediatr 121: 86-9
83.Ko FJ, Nyhan WL, Wolff J, Barshop B, Sweetman L. 1991. Pediatr
Res 30: 322-6
84.Gibson KM, Lee CF, Bennett MJ, Holmes B, Nyhan WL. 1993. J
Inherit Metab Dis 16: 563-7
85.Roe CR, Struys E, Kok RM, Roe DS, Harris RA, Jakobs C. 1998.
Mol Genet Metab 65: 35-43
86.Gray RG, Pollitt RJ, Webley J. 1987. Biochem Med Metab Biol
38: 121-4
87.Zhang YX, Tang L, Hutchinson CR. 1996. J Bacteriol 178: 490-5
88.Deodato F, Boenzi S, Santorelli FM, Dionisi-Vici C. 2006. Am J
Med Genet C Semin Med Genet 142: 104-12
89.Feliz B, Witt DR, Harris BT. 2003. Arch Pathol Lab Med 127:
e325-8
90.Ohura T, Miyabayashi S, Narisawa K, Tada K. 1991. Hum Genet
87: 41-4
91.Ugarte M, Perez-Cerda C, Rodriguez-Pombo P, Desviat LR,
Perez B, et al. 1999. Hum Mutat 14: 275-82
92.Acquaviva C, Benoist JF, Pereira S, Callebaut I, Koskas T, et al.
2005. Hum Mutat 25: 167-76
93.Kasahara M, Horikawa R, Tagawa M, Uemoto S, Yokoyama S, et
al. 2006. Pediatr Transplant 10: 943-7
94.Kobayashi A, Kakinuma H, Takahashi H. 2006. Pediatr Int 48:
1-4
95.Dionisi-Vici C, Deodato F, Roschinger W, Rhead W, Wilcken B.
2006. J Inherit Metab Dis 29: 383-9
96.Lamhonwah AM, Barankiewicz TJ, Willard HF, Mahuran DJ,
Quan F, Gravel RA. 1986. Proc Natl Acad Sci U S A 83: 4864-8
97.Heldt K, Schwahn B, Marquardt I, Grotzke M, Wendel U. 2005.
Mol Genet Metab 84: 313-6
98.Wei CC, Lin WD, Tsai FJ, Wu JY, Peng CT, Tsai CH. 2004. Acta
Paediatr Taiwan 45: 236-8
99.Lin JF, Chiu PC, Hsu HY, Lin SM, Chen YY, Hsieh KS. 2004.
Acta Paediatr Taiwan 45: 287-9
100.
Koeberl DD, Millington DS, Smith WE, Weavil SD,
Muenzer J, et al. 2003. J Inherit Metab Dis 26: 25-35
101.
Ogier de Baulny H, Saudubray JM. 2002. Semin Neonatol
7: 65-74
102.
Wiley V, Carpenter K, Wilcken B. 1999. Acta Paediatr
Suppl 88: 48-51
Chapter 3
3-Methylglutaconic Aciduria Type I is Caused by Mutations in AUH
Ference J. Loupatty, Lodewijk IJlst, Jos P.N. Ruiter, Marinus Duran, Willy Lehnert
and Ronald J.A. Wanders
3-Methylglutaconic aciduria type I is an autosomal recessive disorder clinically characterized by
various symptoms ranging from delayed speech development to severe neurological handicap.
This disorder is caused by a deficiency of 3-methylglutaconyl-CoA hydratase, one of the keyenzymes of leucine degradation. This results in elevated urinary levels of 3-methylglutaconic acid,
3-methylglutaric acid and 3-hydroxyisovaleric acid. By heterologous expression in E. coli, we show
that 3-methylglutaconyl-CoA hydratase is encoded by the AUH gene, whose product previously had
been reported as an AU-specific RNA-binding protein. Mutation analysis of AUH in two patients
revealed a nonsense mutation (R197X) and a splice site mutation (IVS8-1G>A) demonstrating that
mutations in AUH cause 3-methylglutaconic aciduria type I.
3-Methylglutaconic aciduria is a group of metabolic
disorders characterized by increased urinary excretion
of 3-methylglutaconic acid and 3-methylglutaric acid.
At present, four distinct forms have been recognized.
Type I (MIM 250950) represents the isolated 3methylglutaconyl-CoA hydratase deficiency. This
mitochondrial enzyme catalyses the fifth step in
leucine catabolism, as shown in figure 1, which is the
conversion of 3-methylglutaconyl-CoA to 3-hydroxy3-methylglutaryl-CoA (HMG-CoA). Type II (MIM
302060), also referred to as Barth syndrome, is an
X-linked mitochondrial cardiomyopathy associated
with neutropenia and growth retardation and caused
by mutations in the gene encoding tafazzin (TAZ).
Type III (MIM 258501) or Costeff syndrome, is an
autosomal recessive disorder caused by mutations in
the OPA3 gene which leads to bilateral optic atrophy.
Type IV or “unspecified” 3-methylglutaconic aciduria
(MIM 250951) comprises a heterogeneous group of
patients with progressive neurological symptoms.
The hydratase activity in Barth syndrome, Costeff
syndrome and type IV 3-methylglutaconic aciduria is
normal and the excretion of 3-methylglutaconic acid
and 3-methylglutaric acid is secondary. Amongst the
four types, patients with the type I excrete the highest
levels of 3-methylglutaconic acid and 3-methylglutaric
acid. Furthermore, these patients additionally excrete
increased amounts of 3-hydroxyisovaleric acid and
therefore, 3-hydroxyisovaleric acid is an important
parameter for differentiation between the isolated
3-methylglutaconyl-CoA hydratase deficiency and
the other types. The phenotypic presentation of 3methylglutaconyl-CoA hydratase deficiency varies
from mild, including delayed speech development
and hyperchloremic acidosis associated with
gastroesophageal reflux to a much more severe
phenotype, including seizures and cerebellar
abnormalities. Although the hydratase was purified
partially in the late 1950s1, the gene encoding the
protein had not been identified.
Several mitochondrial enzymes with enoyl-CoA
hydratase activity have been characterized. The first
one, called crotonase, is most active towards short
chain enoyl-CoAs and participates in the β-oxidation
of short-chain fatty acids. Studies performed by Hilz H
et al. showed that an enzyme other than crotonase is
responsible for the hydratase step in the degradation of
leucine.1 Indeed, our preliminary studies showed that
crotonase is not active with HMG-CoA when measured
in the reverse direction. A second enzyme harbouring
hydratase activity is mitochondrial trifunctional protein
(MTP), which also catalyses dehydrogenation and
thiolytic cleavage of long-chain fatty acids. Together
with very long-chain acyl-CoA dehydrogenase, this
L-Leucine
2-Ketoisocaproic acid
Isovaleryl-CoA
3-Methylcrotonyl-CoA
HOOC
3-Hydroxyisovaleric acid
S-CoA
CH3 O
3-Methylglutaconyl-CoA
3-Methylglutaric acid
3-Methylglutaconic acid
3-Methylglutaconyl-CoA Hydratase
HOOC
OH
S-CoA
CH3 O
3-Hydroxy-3-methylglutaryl-CoA
Acetoacetic acid + Acetyl-CoA
Figure 1. Catabolism of L-leucine. The names of the intermediates in the pathway for catabolism of leucine are shown
on the left with solid arrows indicating enzymatic reactions.
The structures of 3-methylglutaconyl-CoA and HMG-CoA are
depicted on the left. The names of the metabolites that are
elevated due to 3-methylglutaconyl-CoA hydratase (italic) deficiency are shown by dashed arrows to the right.
From the Departments of Clinical Chemistry and Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.;J.P.N.R.;L.IJ.;M.D.;R.J.A.W.); University Children’s Hospital Freiburg, Germany (W.L)
19
protein is the main enzyme in the long-chain fatty acid
β-oxidation. It is unlikely, however, that this enzyme is
involved in the degradation of leucine, because patients
with MTP deficiency (MIM 143450, 600890) have no
abnormal urinary excretion of 3-methylglutaconic
acid.
Nakagawa and co-workers purified an AU-specific
RNA binding protein, designated AUH, using an affinity
column displaying A+U-rich elements.2 The protein
contained a hydratase motif and shared 32 % sequence
identity with crotonase. The enoyl-CoA hydratase
activity with butenoyl-CoA (C4:1-CoA) as substrate was
thousand-fold lower when compared to crotonase. The
three-dimensional structure of human AUH has been
determined at 2.2 Å resolution and predicted a high
affinity for short chain substrates.3
To investigate whether AUH has 3-methylglutaconylCoA hydratase activity we expressed AUH as a fusion
protein with maltose binding protein (MBP). To this
end, the ORF of AUH was amplified from human
cDNA by use of the primers –3AUHfEcoRI 5’-ATA
TGA ATT CAA CAT GGC GGC CGC GGT GG-3’
and 1097AUHrXbaI 5’-ATA TTC TAG ACA TAT AGT
GGA TCC GAA AGA C-3’, digested with EcoRI and
XbaI, and subsequently cloned into the EcoRI and XbaI
restriction sites of the pMAL-C2X vector (New England
Biolabs). The cloned PCR products were sequenced to
assess the integrity of the PCR process. Next, the MBPAUH was expressed in E. coli and affinity-purified to
homogeneity according to the manufacturer’s protocol
(fig. 2).
kDa
T
P
99.8
71.4
44.2
28.5
19.8
Figure 2. Expression and purification of MBP-AUH fusion protein in E. coli. Total E. coli extract (T) and affinity-purified
fusion protein (P) were analyzed by 10 % SDS-PAGE gel electrophoresis and stained with Coomassie Brilliant blue.
20
The 3-methylglutaconyl-CoA hydratase activity of the
expressed protein was measured in the reverse direction
using HMG-CoA as substrate. The reaction mixture
contained 50 mM Tris pH 7.4, 10 mM EDTA, 1 mg/ml
BSA, 0.1 mM HMG-CoA and was started by addition of
purified protein. The formation of 3-methylglutaconylCoA was followed spectrophotometrically at 260 nm
using a molar extinction coefficient of 6200 mol/L/
cm. The short-chain enoyl-CoA hydratase activity
was measured using butenoyl-CoA as a substrate as
described.4 For comparison, both activities were also
measured using bovine liver crotonase obtained from
Sigma.
As reported previously for AUH, we also found very
low hydratase activity with C4:1-CoA. In addition, we
found that AUH has high 3-methylglutaconyl-CoA
hydratase activity. Indeed, the HMG/C4:1 activityratio was more than 20. In contrast, crotonase has no
detectable 3-methylglutaconyl-CoA hydratase activity
(table 1). Therefore, AUH is a strong candidate for the
underlying enzymatic defect of 3-methylglutaconic
aciduria type I.
Table 1. Hydratase activity measurements of AUH and
crotonase with HMG-CoA and butenoyl-CoA (C4:1-CoA) as
substrate (nmol/min/mg)
Hydratase activity
HMG-CoA
C4:1-CoA
HMG-CoA/C4:1-CoA
MBP-AUH
110
4.8
23
Bovine liver crotonase
<1
8.3 * 105
< 10-6
To date, eleven patients have been described
with isolated 3-methylglutaconyl-CoA hydratase
deficiency.5-10 The hydratase deficiency in these patients
has been determined by use of an overall enzyme
assay measuring three steps of leucine degradation,
from 3-methylcrotonyl-CoA to acetoacetic acid.11 We
reinvestigated fibroblasts from two patients5,6 with
our novel enzyme assay using the same conditions as
described above, but the reaction was started with 0.3
mg/ml fibroblast homogenate. The reaction mixture
was incubated for 30 minutes at 37 °C, stopped with 0.2
M hydrochloric acid and subsequently neutralized with
sodium hydroxide. Formation of 3-methylglutaconylCoA was quantified on HPLC using a reverse-phase
C18-column (Supelcosil LC-18-DB, 250 mm X 10
mm, 5 µm, Supelco). Separation of the acyl-CoAs was
achieved by elution with a linear gradient of methanol
(10% to 37.6% in 22 min) in potassium phosphate buffer
(50 mM, pH 5.3). Both patients showed clear deficiency
of 3-methylglutaconyl-CoA hydratase (patients <0.1;
controls 2.8 ± 0.6 nmol/min/mg protein), whereas the
crotonase activity was normal for both patients.
To determine whether 3-methylglutaconic aciduria
type I is caused by mutations in AUH, the ORF of AUH
was amplified from human cDNA in two overlapping
fragments using the following M13-tagged primers:
fragment A: -28AUHf-21M13 5’- tgt aaa acg acg gcc
C1
C2
P
Figure 3. Mutation analysis of AUH in patient with 3methylglutaconic aciduria type I. PCR fragment B amplified
from cDNA of two control subjects (C1 and C2) and patient 1
(P) showing the 42 bp deletion (exon 9) as a consequence of a
IVS8-1G>A mutation.
431
383
References
agt TCG CAG GTC CAC GCC GTA AAC-3’ and
680AUHrM13 5’- cag gaa aca gct atg acc CGC GTG
GCA ATC GCT GTG TC-3’; fragment B: 505AUHf21M13 5’- tgt aaa acg acg gcc agt GCT ATT CTT CCA
GTG CCA AC-3’ and 1097AUHrM13 5’- cag gaa aca
gct atg acc CAT ATA GTG GAT CCG AAA GAC-3’.
PCR fragments obtained from patients and two control
subjects were sequenced using Big Dye Terminators and
analyzed on an Applied Biosystems 377A automated
DNA sequencer following the manufacturer’s protocols
(PE Applied Biosystems, Foster City).
The first patient was the third son of consanguineous
parents of Afghan origin with retardation in speech
development. A delayed motor development became
evident in retrospect.5 The amplification product of
fragment B was approximately 50 bp smaller than found
in controls (figure 3). Sequence analysis revealed that
this was due to skipping of exon 9. The splice acceptor
and donor site neighboring exon 9 conform to the AG/
GT rule. To identify the underlying mutation causing
missplicing we amplified splice sites flanking exon
9 from genomic DNA using the following primers:
In8AUHf-21M13 5’ tgt aaa acg acg gcc agt GAC ATG
CCT CTT TGA AGC AC 3’ and In9AUHrM13 5’ cag
gaa aca gct atg acc GCA AGG GTA ATC TTG CTC AG
3’. In patient 1 we identified a homozygous IVS8-1G>A
mutation, which disrupts the consensus sequence of the
splice acceptor site. Exon 9 encodes α-helix H93, which
crystal structure analysis suggests to be a constituent of
the active-site pocket, and is also necessary for subunit
interaction and trimer stability.
The second patient is the younger of two affected
brothers of healthy nonconsanguineous Moroccan
parents. He had no physical abnormalities, only his
speech development was retarded.6 Sequence analysis
at the cDNA level showed an apparently homozygous
nonsense mutation c.589C>T. Homozygosity for the
nonsense mutation was confirmed at the genomic level.
The R197X nonsense mutation causes the translation
to be terminated before glutamate 209, which together
with glutamate 189 forms the catalytic group of the
active-site pocket.
To conclude, our data show that mutations in AUH
cause 3-methylglutaconic aciduria type I.
21
1. Hilz H, Knappe J, Ringelmann E, Lynen F (1958)
Methylglutaconase, eine neue hydratase, die am stoffwechsel
verzweigter carbonsäuren beteiligt ist. Biochem Z 329:476-89
2. Nakagawa J, Waldner H, Meyer-Monard S, Hofsteenge J, Jeno P,
Moroni C (1995) AUH, a gene encoding an AU-specific RNA
binding protein with intrinsic enoyl-CoA hydratase activity.
Proc Natl Acad Sci 92:2051-5
3. Kurimoto K, Fukai S, Nureki O, Muto Y, Yokoyama S (2001)
Crystal structure of human AUH protein, a single-stranded RNA
binding homolog of enoyl-CoA hydratase. Structure 9:1253-63
4. Wanders RJA, IJlst L, Poggi F, Bonnefont JP, Munnich A, Brivet
M, Rabier D, Saudubray JM (1992) Human trifunctional protein
deficiency: a new disorder of mitochondrial fatty acid oxidation.
Biochem Biophys Res Commun 188:1139-45
5. Ensenauer R, Muller CB, Schwab KO, Gibson KM, Brandis
M, Lehnert W (2000) 3-Methylglutaconyl-CoA hydratase
deficiency: a new patient with speech retardation as the leading
sign. J Inherit Metab Dis 23:341-4
6. Duran M, Beemer FA, Tibosch AS, Bruinvis L, Ketting D,
Wadman SK (1982) Inherited 3-methylglutaconic aciduria in
two brothers- Another defect of leucine metabolism. J Pediatr
101:551-4
7. Gibson KM, Lee CF, Wappner RS (1992) 3-Methylglutaconylcoenzyme-A hydratase deficiency: a new case. J Inherit Metab
Dis 15:363-6
8. Gibson KM, Wappner RS, Jooste S, Erasmus E, Mienie LJ,
Gerlo E, Desprechins B, De Meirleir L (1998) Variable clinical
presentation in three patients with 3-methylglutaconylcoenzyme A hydratase deficiency. J Inherit Metab Dis 21:631-8
9. Narisawa K, Gibson KM, Sweetman L, Nyhan WL (1986)
Deficiency of 3-methylglutaconyl-coenzyme A hydratase in two
siblings with 3-methylglutaconic aciduria. J Clin Invest 77:114852
10. Shoji Y, Takahashi T, Sawaishi Y, Ishida A, Matsumori M, Shoji
Y, Enoki M, Watanabe H, Takada G (1999) 3-Methylglutaconic
aciduria type I: clinical heterogeneity as a neurmetabolic disease.
J Inherit Metab Dis 22:1-8
11. Narisawa K, Gibson KM, Sweetman L, Nyhan WL (1989)
3-Methylglutaconyl-CoA
hydratase,
3-methylcrotonylCoA carboxylase and 3-hydroxy3-methylglutaryl-CoA lyase
deficiencies: a coupled enzyme assay useful for their detection.
Clin Chim Acta 184:57-64
Chapter 4
Direct Non-isotopic Assay of 3-Methylglutaconyl-CoA Hydratase
Ference J. Loupatty, Jos P.N. Ruiter, Lodewijk IJlst, Marinus Duran and Ronald J.A. Wanders
Our objective was to develop a specific and sensitive assay for the determination of 3-methylglutaconylCoA hydratase activity. The 3-methylglutaconyl-CoA hydratase activity in fibroblast homogenates
was measured in the reverse direction using 3-hydroxy-3-methylglutaryl-CoA as a substrate. The
formation of 3-methylglutaconyl-CoA was quantified by use of reversed-phase HPLC with UV
detection at 260 nm. Baseline separation of substrate and product was achieved. Little variation of
3-methylglutaconyl-CoA hydratase activity was observed between pH 7.0 and 9.0. The assay was
linear in time up to 60 min using 50 µg protein/mL. The intraassay and interassay coefficients of
variation were 4.0% (n=10) and 4.8 % (n=11), respectively. The detection limit was 20 pmol/min/
mg protein. Mean (SD) control 3-methylglutaconyl-CoA hydratase activity was 2.1 (0.7) nmol/min/
mg protein (range 1.0-3.6; n=13). Patients with proven 3-methylglutaconic aciduria type I had 3methylglutaconyl-CoA hydratase activities below the detection limit. This rapid and nonradioactive
enzyme assay allows the specific and sensitive determination of 3-methylglutaconyl-CoA hydratase
activity in cultured human skin fibroblasts. The enzyme assay can be used for the reliable diagnosis of
3-methylglutaconic aciduria type I and their differentiation from other forms of 3-methylglutaconic
aciduria.
3-Methylglutaconic aciduria (3MGA) type I (MIM
250950) is biochemically characterized by increased
excretion of 3-methylglutaconic acid, 3-methylglutaric
acid and 3-hydroxyisovaleric acid in urine. Affected
individuals display a range of clinical manifestations
varying from mildly delayed speech development to
severe neurological involvement.1,2 3MGA type I is an
autosomal recessive disorder caused by a deficiency
of 3-methylglutaconyl-CoA hydratase (3MGH, E.C.
4.2.1.18). Three additional forms of 3-methylglutaconic
aciduria have been recognized (type II, Barth syndrome,
MIM 302060; type III, Costeff syndrome, MIM 258501;
type IV, “unspecified”, MIM 250951) all characterized
by normal hydratase activities.3 Recently, the gene
encoding 3MGH was identified by two independent
groups.4,5 As shown in figure 1, this mitochondrial
enzyme catalyzes the penultimate step in leucine
catabolism, which is the reversible conversion of 3methylglutaconyl-CoA to 3-hydroxy-3-methylglutarylCoA (HMG-CoA).
Eleven patients have been described with isolated 3methylglutaconyl-CoA hydratase deficiency.1,2,5-10 The
hydratase deficiency in these patients was determined
by use of a radioactive enzyme assay measuring three
consecutive steps of leucine degradation, from 3methylcrotonyl-CoA to acetoacetic acid.11 However,
this procedure lacks specificity and is laborious
because of the need to purify the coupling enzyme 3methylcrotonyl-CoA carboxylase (E.C. 6.3.4.14.) from
bovine liver. Furthermore, the assay is impractical
because it involves the use of radiochemicals.
The need to differentiate 3MGA type I patients
from others with 3-methylglutaconic aciduria
requires the availability of a sensitive and specific
enzyme assay. Based on the knowledge that in general
hydratase reactions are readily reversible and 3methylglutaconyl-CoA is not commercially available,
we studied the 3MGH activity in the reverse direction,
using HMG-CoA as a substrate. The formation of 3methylglutaconyl-CoA was quantified by reversedphase high performance liquid chromatography with
L-Leucine
2-Ketoisocaproic acid
Isovaleryl-CoA
3-Methylcrotonyl-CoA
HOOC
3-Hydroxyisovaleric acid
S-CoA
CH3 O
3-Methylglutaconyl-CoA
3-Methylglutaric acid
3-Methylglutaconic acid
3-Methylglutaconyl-CoA Hydratase
HOOC
OH
S-CoA
CH3 O
3-Hydroxy-3-methylglutaryl-CoA
Acetoacetic acid + Acetyl-CoA
Figure 1. Catabolism of L-leucine. The names of the intermediates in the pathway for catabolism of leucine are shown
on the left with solid arrows indicating enzymatic reactions.
The structures of 3-methylglutaconyl-CoA and HMG-CoA are
depicted on the left. The names of the metabolites that are
elevated due to 3-methylglutaconyl-CoA hydratase (italic) deficiency are shown by dashed arrows to the right.
From the Departments of Clinical Chemistry and Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.;J.P.N.R.;L.IJ.;M.D.;R.J.A.W.)
23
UV detection. The described assay allows the precise
measurement of residual 3MGH activity in cultured
human skin fibroblasts.
We obtained potassium dihydrogen phosphate
(KH2PO4), phosphoric acid (H3PO4), Tris, EDTA,
hydrochloric acid, potassium hydroxide, 2morpholinoethanesulfonic acid (MES) and acetonitrile
(chromatography grade) from Merck. Bovine serum
albumin essentially fatty acid free (BSA), 3-hydroxy3-methylglutaryl-coenzyme A (HMG-CoA) and
bicinchoninic acid were purchased at Sigma.
Skin fibroblasts were obtained from two subjects with
proven 3MGA type I 1,6, five subjects with proven Barth
syndrome12, two subjects with 3-methylglutaconic
aciduria type IV13 and thirteen control subjects with no
evidence of an inborn error of branched chain amino
acid oxidation or mitochondrial fatty acid oxidation.
All patient cells were initially obtained in the process of
diagnosing inborn errors of metabolism.
Fibroblasts were grown and harvested as described
elsewhere.14 For determination of the intraassay
(within-run) and interassay (between-day) variation,
six cell cultures of three different control subjects were
harvested, pooled, divided in 20 pellets and stored
frozen at -80 °C.
Cell pellets were suspended in 200 µL of phosphatebuffered saline by repeated pipetting and sonicated
three times on ice for 15 seconds at 8 W at 45 sec
intervals. Protein was determined by the bicinchoninic
acid assay (Sigma) using BSA as standard. The assay
mixture contained, in a final volume of 100 µL: 100
mmol/L TRIS, pH 8.0, 10 mmol/L EDTA, 1g/L BSA,
100 µmol/L HMG-CoA and 10-50 mg/L of fibroblast
protein. After incubation at 37 °C for 60 min the
reaction was terminated by addition of 10 µL of 2 mol/
L HCl. The samples were homogenized and the assay
tubes placed on ice. After 5 min, the homogenates were
brought to a pH 6 with (2 mol/L KOH)/ (1 mol/L MES
pH 6) and centrifuged for 10 min at 4 °C at 14.000 rpm.
Finally, 100 µL of the supernatant was transferred to a
HPLC vial.
HPLC separation was performed at ambient
temperature with a Perkin Elmer pump (PE series 200)
and a Gilson 234 auto-sampling injector. A frit C-402X
Signal output (mV)
30.0
(Upchurch scientific), a 4.6 mm x 20 mm SUPELCOSIL
LC-18-DB (5µM) guard column (Supelco) and a 4.6
mm x 250 mm column filled with the same packing
material were used. Compounds were detected with a
UV detector (SPD-10A VP UV-VIS, Shimadzu) at 260
nm.
For gradient elution, we used a binary solvent system.
Solvent A was 100 mmol/L KH2PO4 adjusted to pH 4
with 100 mmol/L H3PO4, filtered before use through
a 0.45 µm nitrocellulose membrane under reduced
pressure. Solvent B was a 4:1 mixture (by volume) of
solvent A, and acetonitrile. Both solvents were degassed
for 15 min in an ultrasonic bath (Branson 3510). 50 µl of
sample was injected and the acyl-CoAs were eluted at a
flow rate of 1 mL/min by a linear gradient of 20 % B to
100 % B in 15 min. Peak areas of interest were integrated
using Chromeleon software package (Dionex).
We first developed a HPLC-based method to
allow baseline separation between HMG-CoA and 3methylglutaconyl-CoA. Separation of acyl-CoA esters
using reversed-phase HPLC is dependent on the salt
concentration and the pH of the eluent.15 We found that
a linear gradient of acetonitrile in potassium phosphate
buffer (100 mmol/L, pH 4.0) allowed baseline separation
between substrate and product.
Typical chromatograms for incubations with
fibroblast extracts of a control subject and a 3MGA
type I patient are shown in figure 2. Using acyl-CoA
standards we were able to identify free CoA (9.0 min)
and HMG-CoA and acetyl-CoA which coeluted at 11.8
min. Comparison of the chromatograms of a 3MGA
type I patient and a control subject suggested the extra
peak at 16.5 min to be 3-methylglutaconyl-CoA. Indeed,
after hydrolysis and derivatization of its contents, the
presence of 3-methylglutaconate was identified on
the basis of its retention time and confirmed by mass
spectrometry (results not shown) as described by
Röschinger et al.16 Thus, the acyl-CoA which eluted at
16.5 min was the product 3-methylglutaconyl-CoA.
HMG-CoA is converted into 3-methylglutaconylCoA by the action of 3MGH and its activity can
be quantified by determining the amount of 3methylglutaconyl-CoA formed. The acyl-CoAs
present in the reaction mixture are susceptible to
A
B
25.0
20.0
15.0
C
10.0
5.0
0.0
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
22.0
24.0
Retention time (min)
Figure 2. Separation of HMG-CoA and 3-methylglutaconyl-CoA on HPLC. HPLC chromatograms of incubations with fibroblast extracts of a control subject (upper, 30.1 µg protein/assay), and a 3MGA type I patient with less than 1% residual 3MGH activity
(lower, 22.4 µg protein/assay). Compounds were detected with a UV detector at 260 nm. Peaks: A, free CoA (CoASH); B, coelution
of the substrate 3-hydroxy-3-methylglutaryl-CoA and acetyl-CoA; C, the product 3-methylglutaconyl-CoA.
24
acetyl-CoA according to the following equation, which
expresses the 3MGH activity as nmol/min/mg protein:
1.5
E 1 s 3MG CoA
Specific activity 3MGH
(nmol/min/mg protein)
;(E 1 s 3MG CoA) E 2 s ( HMG CoA acetyl CoA)=s T s P
1.0
0.5
0.0
7.0
7.5
8.0
8.5
9.0
pH
Figure 3. Effect of the pH on 3-methylglutaconyl-CoA hydratase (3MGH) activity.
enzymatic and chemical hydrolysis, but HMG-CoA
and 3-methylglutaconyl-CoA were equally hydrolyzed
(results not shown). Therefore, the 3MGH activity was
calculated as the ratio of product to the sum of product
and substrate. However, since HMG-CoA is a substrate
for other enzymes, calculating the 3MGH activity in
such manner would result in an overestimation of the
3MGH activity.
Preliminary studies revealed that, under the
incubation conditions described above, < 5 % of HMGCoA was converted into acetyl-CoA by the action of
HMG-CoA lyase (results not shown). Second, HMGCoA could be converted into mevalonic acid by the
action of HMG-CoA reductase (E.C.1.1.1.34) using
NADPH as a coenzyme. However, since NADPH was
not added, the action of the reductase on HMG-CoA
under the assay conditions was negligible. Therefore
the 3MGH activity was calculated as the ratio of the
amount of 3-methylglutaconyl-CoA to the sum of the
amounts of 3-methylglutaconyl-CoA, HMG-CoA and
where ε1 = 22.6 cm2/µmol 17, ε2 = 16.0 cm2/µmol 17, T =
incubation time (min), P = the amount of protein (mg),
and the acyl-CoAs are expressed as peak areas.
The activity of 3-methylglutaconyl-CoA hydratase
in human fibroblast extracts showed little variation as
a function of pH in the range from 7.0 to 9.0 (figure 3)
as reported by others using the coupled enzyme assay.18
For the standard assay we selected a pH of 8.0.
The assay was linear with an incubation time up to
at least 60 min with 50 mg fibroblast protein/L assay
(figure 4). For the standard assay, an incubation time
of 60 min and a protein content of 10-50 mg fibroblast
protein/L assay was chosen.
Using optimized assay conditions, we reinvestigated
fibroblasts from two patients with established
3MGA type I1,6 and detected no formation of 3methylglutaconyl-CoA in either case. Inclusion of 1 %
control homogenate in a homogenate of a 3MGA type I
patient, corresponding to a 3MGH activity of 20 pmol/
min/mg protein, could readily be detected. Hence, the
3MGH activity in both patients was < 20 pmol/min/mg
protein.
The intraassay variation, estimated by assaying 10 of
the pooled control pellets in a single experiment, was
4.0 % [mean (SD) = 2.26 (0.09) nmol/min/mg protein).
The interassay variation was 4.8 % [mean (SD) = 2.2
(0.1) nmol/min/mg protein], n = 10 days
Measurement of 3MGH activity in thirteen control
fibroblast homogenates revealed a mean (SD) 3MGH
activity of 2.1 (0.7) nmol/min/mg protein (range 1.03.6). The mean (SD) 3MGH activity in fibroblasts of five
Barth syndrome patients was 2.8 (0.8) nmol/min/mg
protein (range 2.2-4.2). The two patients with proven
3MGA type IV demonstrated 3MGH activities of
1.59 and 1.02 nmol/min/mg protein. These values for
1.0
0.6
3-Methylglutaconyl-CoA (nmol)
3-Methylglutaconyl-CoA (nmol)
0.7
0.5
0.4
0.3
0.2
0.1
0
15
30
45
60
75
90
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
A
0.0
0.9
B
0.0
105
0
Time (min)
25
50
75
100 125 150 175 200
Fibroblast protein (µg/mL assay)
Figure 4. A, Effect of time on 3-methylglutaconyl-CoA hydratase activity; Fibroblast extracts containing 50 µg protein/mL assay
were incubated at 37 °C. The values are the mean ± SD of three independent experiments. B, Effect of protein on 3-methylglutaconyl-CoA hydratase activity. Fibroblast extracts were incubated at 37 °C for 60 min.
25
3MGH activity were within the control range. Hence,
the 3MGH activity in cultured fibroblast homogenates
of patients with Barth syndrome or 3-methylglutaconic
aciduria type IV is normal. This corresponds with the
finding that normal 3MGH activity has been found in
Barth syndrome and 3MGA type IV using the coupled
enzyme assay.3
In conclusion, we present a sensitive and specific
enzymatic assay for 3-methylglutaconyl-CoA hydratase
that enables the rapid enzymatic diagnosis of 3methylglutaconic aciduria type I in cultured human skin
fibroblasts without the involvement of radiochemicals.
Baseline separation between the substrate HMGCoA and the product 3-methylglutaconyl-CoA was
achieved. Our novel procedure allows detection of 1%
residual 3MGH activity in 3MGA type I patients. So
far, all 3MGA type I patients demonstrated a 3MGH
activity below this limit. Our method could be useful
for studies of genotype / phenotype correlation as
well as studies for the enzymatic characterization of
mutated proteins as expressed in E.coli.
References
1. Duran M, Beemer FA, Tibosch AS, Bruinvis L, Ketting D,
Wadman SK. Inherited 3-methylglutaconic aciduria in two
brothers--another defect of leucine metabolism. J Pediatr
1982;101:551-4.
2. Shoji Y, Takahashi T, Sawaishi Y, Ishida A, Matsumori M, Shoji Y
et al. 3-Methylglutaconic aciduria type I: clinical heterogeneity
as a neurometabolic disease. J Inherit Metab Dis 1999;22:1-8.
3. Gibson KM, Sherwood WG, Hoffman GF, Stumpf DA, Dianzani
I, Schutgens RB et al. Phenotypic heterogeneity in the syndromes
of 3-methylglutaconic aciduria. J Pediatr 1991;118:885-90.
4. IJlst L, Loupatty FJ, Ruiter JP, Duran M, Lehnert W, Wanders
RJ. 3-Methylglutaconic aciduria type I is caused by mutations in
AUH. Am J Hum Genet 2002;71:1463-6.
5. Ly TB, Peters V, Gibson KM, Liesert M, Buckel W, Wilcken B
et al. Mutations in the AUH gene cause 3-methylglutaconic
aciduria type I. Hum Mutat 2003;21:401-7.
6. Ensenauer R, Muller CB, Schwab KO, Gibson KM, Brandis M,
Lehnert W. 3-Methylglutaconyl-CoA hydratase deficiency: a
new patient with speech retardation as the leading sign. J Inherit
Metab Dis 2000;23:341-4.
7. Hou JW, Wang TR. 3-Methylglutaconic aciduria presenting
as Reye syndrome in a Chinese boy. J Inherit Metab Dis
1995;18:645-6.
26
8. Jooste S, Erasmus E, Mienie LJ, de Wet WJ, Gibson KM. The
detection of 3-methylglutarylcarnitine and a new dicarboxylic
conjugate, 3-methylglutaconylcarnitine, in 3-methylglutaconic
aciduria. Clin Chim Acta 1994;230:1-8.
9. Gibson KM, Lee CF, Wappner RS. 3-Methylglutaconylcoenzyme-A hydratase deficiency: a new case. J Inherit Metab
Dis 1992;15:363-6.
10.Arbelaez A, Castillo M, Stone J. MRI in 3-methylglutaconic
aciduria type 1. Neuroradiology 1999;41:941-2.
11.Narisawa K, Gibson KM, Sweetman L, Nyhan WL. 3Methylglutaconyl-CoA
hydratase,
3-methylcrotonyl-CoA
carboxylase and 3-hydroxy-3-methylglutaryl-CoA lyase
deficiencies: a coupled enzyme assay useful for their detection.
Clin Chim Acta 1989;184:57-64.
12.Valianpour F, Wanders RJ, Overmars H, Vreken P, Van Gennip
AH, Baas F et al. Cardiolipin deficiency in X-linked cardioskeletal
myopathy and neutropenia (Barth syndrome, MIM 302060): a
study in cultured skin fibroblasts. J Pediatr 2002;141:729-33.
13. Gibson KM, Nyhan WL, Sweetman L, Narisawa K, Lehnert W,
Divry P et al. 3-Methylglutaconic aciduria: a phenotype in which
activity of 3-methylglutaconyl-coenzyme A hydratase is normal.
Eur J Pediatr 1988;148:76-82.
14.Wanders RJ, Denis S, Ruiter JP, Schutgens RB, van Roermund
CW, Jacobs BS. Measurement of peroxisomal fatty acid betaoxidation in cultured human skin fibroblasts. J Inherit Metab
Dis 1995;18 Suppl 1:113-24.
15.Corkey BE. Analysis of acyl-coenzyme A esters in biological
samples. Methods Enzymol 1988;166:55-70.
16. Roschinger W, Muntau AC, Duran M, Dorland L, IJlst L, Wanders
RJ, Roscher AA. Carnitine-acylcarnitine translocase deficiency:
metabolic consequences of an impaired mitochondrial carnitine
cycle. Clin Chim Acta 2000;298:55-68.
17.Lazarow PB. Rat liver peroxisomes catalyze the beta oxidation of
fatty acids. J Biol Chem 1978;253:1522-8.
18. Narisawa K, Gibson KM, Sweetman L, Nyhan WL, Duran M,
Wadman SK. Deficiency of 3-methylglutaconyl-coenzyme A
hydratase in two siblings with 3-methylglutaconic aciduria. J
Clin Invest 1986;77:1148-52.
Chapter 5
Identification of a Novel Enoyl-CoA Hydratase Encoded by ECHDC2
Ference J. Loupatty, Rob Ofman, Jos P.N. Ruiter, Lodewijk IJlst, Marinus Duran and Ronald J.A. Wanders
In this study we describe the identification of a novel enoyl-CoA hydratase encoded by ECHDC2
and present new data on human 3-methylglutaconyl-CoA hydratase (E.C. 4.2.1.18.), the enzyme
which is deficient in patients with 3-methylglutaconic aciduria type I. By heterologous expression
in Escherichia coli, we show that the ECHDC2 protein has 3-methylglutaconyl-CoA, crotonylCoA and octenoyl-CoA hydratase activities, but no tiglyl-CoA or 3-hydroxyisobutyryl-CoA
hydratase activities. Comparison of the enzymatic characteristics of the ECHDC2 protein with
3-methylglutaconyl-CoA hydratase showed that both enzymes have highly similar substrate
specificities. Subcellular fractionation studies revealed that both enzymes are localized exclusively
in the mitochondria. Using mono-specific antibodies we studied the expression of the ECHDC2
protein and 3-methylglutaconyl-CoA hydratase in different types of tissue in humans, rats and
mice. 3-Methylglutaconyl-CoA hydratase is predominantly expressed in brain, kidney, heart, and
moderately expressed in liver, muscle, testis and skin fibroblasts. The ECHDC2 protein is expressed
in kidney, liver and heart, but is not expressed in brain and cultured human skin fibroblasts. As
the ECHDC2 protein is not expressed in human skin fibroblasts, whereas 3-methylglutaconyl-CoA
hydratase is, a deficiency of the ECHDC2 protein should be considered in patients with normal
3-methylglutaconyl-CoA hydratase activity in human skin fibroblasts, but with elevated urinary
excretion of 3-methylglutaconic acid.
3-Methylglutaconic aciduria type I (3MGA I, MIM
250950) is an autosomal recessive disorder caused
by a deficiency of 3-methylglutaconyl-CoA hydratase
(3MGH, E.C. 4.2.1.18). Affected individuals display a
range of clinical manifestations varying from mildly
delayed speech development to severe neurological
involvement. As shown in figure 1, 3MGH catalyzes
the penultimate step in leucine catabolism, which is the
reversible conversion of 3-methylglutaconyl-CoA into
3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). The
absence of 3-methylglutaconyl-CoA hydratase activity
will result in elevated excretion of 3-methylglutaconic
acid, 3-methylglutaric acid and 3-hydroxyisovaleric
acid in the urine of affected individuals.
We have recently shown that crotonase (E.C.
4.2.1.17), also referred to as enoyl-CoA hydratase,
harbors 3-methylglutaconyl-CoA hydratase activity,
although the catalytic efficiency of the enzyme is
much higher with crotonyl-CoA as compared to 3methylglutaconyl-CoA.1 Crotonase plays a key role in
fatty acid metabolism by catalyzing the second step
in the classical β-oxidation cycle. The enzyme is most
active towards short-chain substrates and acts with
decreasing efficiency on 2-enoyl-CoAs from crotonylCoA to hexadecenoyl-CoA, which is only hydrated at
a rate of 1-2 % of the rate of crotonyl-CoA.2 In 1996,
Wieringa and colleagues described the quaternary
structure of human crotonase as a dimer of trimers with
each subunit containing 261 amino acids.3,4 The active
site is primarily contained within one subunit, but
some amino residues that line the binding pocket are
contributed by a neighboring subunit in the hexamer.
On the basis of the three-dimensional structure of
crotonase, a catalytic mechanism has been proposed
with the key players being Glu144 and Glu164.5
In an effort to resolve the molecular basis of
3MGA I we screened the database of the National
Center for Biotechnology Information (NCBI) for
L-Leucine
2-Ketoisocaproic acid
Isovaleryl-CoA
3-Methylcrotonyl-CoA
HOOC
3-Hydroxyisovaleric acid
S-CoA
CH3 O
3-Methylglutaconyl-CoA
3-Methylglutaric acid
3-Methylglutaconic acid
3-Methylglutaconyl-CoA Hydratase
HOOC
OH
S-CoA
CH3 O
3-Hydroxy-3-methylglutaryl-CoA
Acetoacetic acid + Acetyl-CoA
Figure 1. Catabolism of L-leucine. The names of the intermediates in the pathway for catabolism of leucine are shown
on the left with solid arrows indicating enzymatic reactions.
The structures of 3-methylglutaconyl-CoA and HMG-CoA are
depicted. The names of the metabolites that are elevated due
to 3-methylglutaconyl-CoA hydratase (italic) deficiency are
shown by dashed arrows to the right.
From the Departments of Clinical Chemistry and Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.;R.O.;J.P.N.R.;L.IJ.;M.D.;R.J.A.W.)
27
3-methylglutaconyl-CoA hydratase using crotonase
(ECHS1; E.C. 4.2.1.17) as query which resulted
in several potential candidates. Molecular and
biochemical investigations performed by our group
demonstrated that one of the candidate genes, AUH,
encodes 3-methylglutaconyl-CoA hydratase (3MGH).
More importantly, mutations in this AUH gene cause 3methylglutaconic aciduria type I.1 Additionally, we also
found a homologue, referred to as ECHDC2, belonging
to the crotonase superfamily.
In the present study, we describe the molecular
cloning and characterization of AUH and ECHDC2
which encodes a novel enoyl-CoA hydratase. We
demonstrate that the ECHDC2 enzyme harbors
considerable
3-methylglutaconyl-CoA
hydratase
activity and is located in the mitochondria, similar to
3MGH. As 3-methylglutaconyl-CoA hydratase activity
is essential in the catabolism of leucine, the tissue
distribution of 3MGH and the ECHDC2 protein is of
considerable interest. To this end, antibodies were raised
against both proteins. Immunoblot analysis revealed
tissue-specific expression of 3MGH and ECHDC2 in
humans, rats and mice. Therefore, we suggest that the
ECHDC2 protein is a therapeutic target for patients
with 3-methylglutaconic aciduria type I.
Experimental procedures
General. E.coli INV-αF and BL21 cells were purchased
from Invitrogen (Carlsbad, USA). The Amylose Resin
Column, the restriction enzymes EcoRI, BamHI and
SalI, the pMAL-C2X and pGEX-4T-1 vectors were
obtained from New England Biolabs (Herts, United
Kingdom). I-BLOCK was from Tropix (Bedford, MA),
goat anti-rabbit IgG-alkaline phosphatase conjugate
from Santa Cruz Biotechnology (CA, USA). All other
reagents were purchased at Sigma (St. Louis, MO). For
sequence analysis, the tools provided by the National
Center for Biotechnology Information (available
at http://www.ncbi.nlm.nih.gov) and the ExPASy
Molecular Biology Server (available at http://www.
expasy.ch) were used.
BLAST search with human crotonase – The human_
EST database of the National Center for Biotechnology
Information (NCBI) was screened for sequences
homologous to human crotonase (ECHS1; AAH08906)
using a translated BLAST search. The following criteria
were used for evaluation; (1) considerable similarity of
the translated sequence, (2) conservation of the regions
that define the active site pocket, (3) conservation of
the catalytic group of crotonase (Glu144 and Glu164),
and (4) localization of the candidate proteins to the
mitochondria. Potential candidates were also evaluated
using a CLUSTALW multiple sequence alignment
program (available at http://www.ebi.ac.uk/clustalw/#).
Potential mitochondrial targeting sequences were
evaluated with the signal prediction program MitoProt
(available at http://ihg.gsf.de/ihg/mitoprot.html).
28
Cloning and expression of human AUH and human
ECHDC2 in E.coli. The complete open reading frames
(ORFs) of the candidate genes were amplified from
human liver cDNA by use of an EcoRI-tagged forward
primer 5’-atatgaattcAACATGGCGGCCGCGGTGG3’ and a XbaI-tagged reverse primer 5’atattctagaCATATAGTGGATCCGAAAGAC-3’
for
the AUH gene, and an EcoRI-tagged forward primer
5’-atatgaattcATGCTGCGCGTTCTGTGCCTC3’ and a XbaI-tagged reverse primer 5’atattctagaCTGGCCACAAATCTTCCTTC-3’ for the
ECHDC2 gene. Next, the PCR products were digested
with EcoRI and XbaI, and subsequently cloned into
the EcoRI and XbaI restriction sites of the pMAL-C2X
vector, to express the 3MGH and ECHDC2 as fusion
proteins with maltose binding protein (MBP). The
ORFs were sequenced to exclude errors introduced
by PCR after which the constructs were transformed
to the E.coli strain INV-αF in accordance with the
manufacturer’s protocol. Finally, the MBP-3MGH and
MBP-ECHDC2 were expressed in E.coli and affinitypurified to homogeneity using an Amylose Resin
Column following the instructions of the manufacturer
and stored at –80 °C in 2 mM DTT, 10 % glycerol (w/
v), PBS. In addition, the assessed constructs were also
digested with EcoRI and SalI and cloned into the EcoRI
and SalI restriction sites of the pGEX-4T-1 vector.
Next, the constructs were transformed to the E.coli
strain BL21, expressed as GST-fusion proteins. The
pellets were stored at –80 °C.
Generation of antisera. Female New Zealand white
rabbits were primed with 100 µg of the highly purified
MBP-fusion protein in 750 µl PBS mixed with an
equal volume of Freund’s complete adjuvant. The
emulsion, divided in two equal parts, was injected both
subcutaneously and intramuscular. After 4 weeks the
immunization was followed by a boost injection of 100
µg of purified MBP-fusion protein mixed with an equal
volume of Freund’s incomplete adjuvant. Two more
boost injections were given at 4-week intervals. One
week after each boost injection a 15-ml blood sample
was taken, and serum was prepared.
Density gradient analysis. Kidneys were obtained
from male Swiss mice, 4 - 6 months of age, and
homogenized in 5 mM morpholinopropanesulphonic
acid buffer, pH 7.4, containing 250 mM sucrose, 2 mM
EDTA and 0.1 % ethanol. A post-nuclear supernatant
was produced by centrifugation of the homogenate
at 600 x g for 10 min at 4°C and subfractionated
by equilibrium density gradient centrifugation in a
linear Nycodenz gradient as described.6 Glutamate
dehydrogenase, catalase, phosphoglucoisomerase, βhexosaminidase and esterase were used as markers for
mitochondria, peroxisomes, cytoplasm, lysosomes and
microsomes, respectively. The activity of these enzymes
was determined as described elsewhere.7,8 Protein
concentration was measured according to Bradford9.
Immunoblot analysis. Samples were separated by
12% SDS-PAGE and transferred onto nitrocellulose
by semidry blotting. Nonspecific binding sites were
blocked for one hour in block buffer (PBS containing
1 g/L Tween-20, supplemented with 2 g/L I-BLOCK).
Primary (polyclonal anti-MBP-3MGH and polyclonal
anti-MBP-ECHDC2) and secondary (goat antirabbit IgG-alkaline phosphatase conjugate) antibody
incubations were performed in block buffer. Polyclonal
antibodies raised against purified MBP-3MGH and
MBP-ECHDC2 were used at a 1:10.000 dilution.
Antigen-antibody complexes were visualized with goat
anti-rabbit IgG-alkaline phosphatase conjugate using
alkaline phosphatase staining in a buffer containing 0.1
M Tris-HCl (pH 9.5), 0.1 M NaCl, 5 mM MgCl2, 0.33 g/
L 4-nitro blue tetrazolium chloride, 0.17 g/L 5-bromo4-chloro-3-indolyl-phosphate (disodium salt). As a
control for transfer of protein, each blot was reversibly
stained with Ponceau S immediately after blotting and
prior to the incubation with antibodies.
Enzyme assay. Enoyl-coenzyme A hydratase activities
were measured spectrophotometrically at 260 nm
and a molar extinction coefficient of 6200 L·mol/
cm. 3-Hydroxy-3-methylglutaryl-CoA, butenoylCoA, octenoyl-CoA, 2-methyl-butenoyl-CoA and 3hydroxyisobutyryl-CoA were used as substrates. The
reaction mixture contained 50 mM Tris pH 7.4, 10 mM
EDTA, 1 mg/ml BSA, 0.2 mM substrate and was started
by addition of purified fusion proteins. The Michaelis-
Menten constants (Km) and maximal velocity (Vmax)
were determined for the highly purified MBP-fusion
proteins from Line weaver-Burke plots.
Results
BLAST search. We searched the NCBI database
by performing a translated BLAST search using
human crotonase as the query which resulted in the
identification of the ECHDC2 protein (accession no.
AAH44574) and the AU-specific RNA-binding enoylCoA hydratase (3MGH; accession no. CAA56260) as
candidates. As mentioned in the introduction this latter
protein is in fact 3-methylglutaconyl-CoA hydratase
(E.C. 4.2.1.18).
Both candidate proteins satisfied the criteria
mentioned in the experimental procedures. First, as
determined by the CLUSTALW multiple alignment
program, the ECHDC2 protein and 3MGH were 31%
and 27% identical to the protein sequence of human
crotonase. Furthermore, both candidate proteins shared
46% sequence identity. Second, as depicted in figure 2,
the counterparts of the catalytic group of crotonase
(Glu144 and Glu164) were conserved as Glu189 and
Glu209 in 3MGH, and as Glu142 and Glu162 in the
ECHDC2 protein. Also the regions that line the active
site pocket, i.e. the oxyanion hole, were conserved.
Finally, as predicted by the MitoProt program both
proteins display a mitochondrial leader sequence and
are likely located to the mitochondria. Taken together,
ECHS1
3MGH
ECHDC2
1 -------------------MAALRVLLSCVRGPLRPPVRCPAWRP---------- FASGA
1 MAAAVAAAPGALGSLHAGGARLVAACSAWLCPGLRLPGSLAGRRAGPAIWAQGWV PAAGG
1 --------------------MLRVLCLLRPWRPLRARGCASDG------------ -AAGG
ECHS1
3MGH
ECHDC2
32 N-------------FEYIIAEKRGKNNTVGLIQLNRPKALNALCDGLIDELNQAL KTFEE
61 PAPKRGYSSEMKTEDELRVRHLEEENRGIVVLGINRAYGKNSLSKNLIKMLSKAV DALKS
28 --------------SEIQVRALAGPDQGITEILMNRPSARNALGNVFVSELLETL AQLRE
ECHS1
3MGH
ECHDC2
79 DPAVGAIVLTGGDK-AFAAGADIKEMQNLSFQDC--YSSKFLKHWDHLTQVKKPV IAAVN
121 DKKVRTIIIRSEVPGIFCAGADLKERAKMSSSEVGPFVSKIRAVINDIANLPVPT IAAID
74 DRQVRVLLFRSGVKGVFCAGADLKEREQMSEAEVGVFVQRLRGLMDDIAAFPAPT IAAMD
ECHS1
3MGH
ECHDC2
136 GYAFGGGCELAMMCDIIYAGEKAQFAQPEILIGTIPGAGGTQRLTRAVGKSLAME MVLTG
181 GLALGGGLELALACDIRVAASSAKMGLVETKLAIIPGGGGTQRLPRAIGMSLAKE LIFSA
134 GFALGGGLELALACDLRVAASSAVMGLIETTRGLLPGAGGTQRLPRCLGVALAKE LIFTG
ECHS1
3MGH
ECHDC2
196 DRISAQDAKQAGLVSKICP----VETLVEEAIQCAEKIASNSKIVVAMAKESVNA AFEMT
241 RVLDGKEAKAVGLISHVLEQNQEGDAAYRKALDLAREFLPQGPVAMRVAKLAINQ GMEVD
194 RRLSGTEAHVLGLVNHAVAQNEEGDAAYQRARALAQEILPQAPIAVRLGKVAIDR GTEVD
ECHS1
3MGH
ECHDC2
252 LTEGSKLEKKLFYSTFATDDRKEGMTAFVEKRKANFKDQ
301 LVTGLAIEEACYAQTIPTKDRLEGLLAFKEKRPPRYKGE
254 IASGMAIEGMCYAQNIPTRDRLEGMAAFREKRTPKFVGK
Figure 2. Comparison of human enoyl-CoA hydratases. Amino acid sequence of crotonase (ECHS1; GenBank™/EBI Data Bank accession number NP_004083), 3-methylglutaconyl-CoA hydratase (3MGH; GenBank™/EBI Data Bank accession number NP_001689) and
the ECHDC2 protein (GenBank™/EBI Data Bank accessionnumber AAH44574) are aligned with identities between the hydratases
indicated by black boxes,similarities indicated by grey boxes. Amino acids are numbered on the left. Arrows above the alignment
indicate the positions of the conserved glutamate residues of the catalytic group.
29
in addition to 3MGH, the ECHDC2 protein is a strong
candidate for a mitochondrial enoyl-CoA hydratase.
The ECHDC2 protein harbors 3-methylglutaconylCoA hydratase activity. To investigate if the ECHDC2
protein harbors enoyl-CoA hydratase activity, and to
further characterize the substrate specificity of 3MGH,
both proteins were expressed as fusion proteins with the
maltose binding protein (MBP) in E.coli and purified to
apparent homogeneity using affinity chromatography.
By SDS-PAGE analysis (figure 3), the purified MBPfusion proteins of ECHDC2 and 3MGH migrated
with a monomer molecular weight of ~ 80 kDa, which
corresponds to their predicted molecular weight
activity was detected using 2-methyl-butenoyl-CoA
or 3-hydroxyisobutyryl-CoA as a substrate. These
data indicate that the ECHDC2 gene encodes a novel
enzyme with enoyl-CoA hydratase activities similar to
3-methylglutaconyl-CoA hydratase.
Generation of specific antibodies raised against both
enoyl-CoA hydratases are specific. The similarity in the
observed substrate specificities between 3MGH and
ECHDC2 makes it impossible to differentiate between
the two enzymes using our standard hydratase assay in
any given tissue. To solve this problem, we generated
antibodies against the MBP-fusion proteins and
their specificity was determined using E.coli lysates
overexpressing GST-ECHDC2 or GST-3MGH. Western
blot analysis (figure 4) of GST-ECHDC2 using the
ECHDC2 antiserum revealed a single immunoreactive
protein with an approximate molecular mass of 55
kDa, which is in good agreement with the calculated
Figure 3. Expression and purification of MBP-enoyl-CoA
hydratase fusion proteins in E.coli. Total E.coli extract (T) and
affinity-purified fusion protein (P) were analyzed by 10% SDSPAGE and stained with Coomassie brilliant blue.
Figure 4. Specificity of anti-bodies. Each lane corresponds to
50 ng of GST-fusion protein. Lane a is GST-fusion protein 3methylglutaconyl-CoA hydratase. Lane b is GST-fusion protein
ECHDC2 protein.
of 74,086 and 78,567 Da, respectively. The kinetic
properties of these highly purified fusion proteins were
determined using different acyl-CoAs as substrates.
Table 1 shows the kinetic parameters (Km and Vmax)
of the proteins with 3-hydroxy-3-methylglutaryl-CoA
(HMG-CoA), butenoyl-CoA (C4:1-CoA) and octenoylCoA (C8:1-CoA) as substrates. Clearly, both enzymes
show considerable hydratase activity towards HMG-CoA
and butenoyl-CoA, whereas no enoyl-CoA hydratase
molecular weight of 57,583 Da. Moreover, the antiserum
demonstrated no detectable immunoreactive response
towards GST-3MGH.
In contrast, immunoblot analysis using the 3MGH
antiserum identified a cross-reacting species with an
approximate size of ~ 50 kDa in both lysates, which is
probably due to nonspecific binding of the antiserum.
More importantly, however, only western blot analysis
of GST-3MGH using this antiserum revealed an
Table 1. Substrate specificity of heterologously expressed MBP fusion proteins. Enzyme assays were performed as described in
“Experimental Procedures,” and the results are the means of three independent measurements. Substrates used were 3-hydroxy3-methylglutaryl-CoA (HMG-CoA), butenoyl-CoA (C4:1-CoA) and octenoyl-CoA (C8:1-CoA).
MBP-ECHDC2
Km
Vmax
MBP-3MGH
Vmax/Km
Km
Vmax
Vmax/Km
substrate
μM
nmol/min/mg
μM
nmol/min/mg
HMG-CoA
39
3.2
0.08
44
9.0
0.20
C4:1-CoA
144
2.6
0.02
156
0.7
0.00
568
19.0
0.03
n.d.
n.d.
C8:1-CoA
n.d. not detectable
30
immunoreactive protein with an approximate size
of 62 kDa, which is in agreement with the calculated
molecular mass of 62,064 Da for GST-3MGH. These
data show that both the 3MGH and ECHDC2 antisera
are specific for detecting either 3-methylglutaconylCoA hydratase or the ECHDC2 protein, respectively.
with an approximate size of 36 kDa and 31 kDa for the
human 3-methylglutaconyl-CoA hydratase and the
ECHDC2 protein, respectively. The distribution profile
of both proteins corresponded to the mitochondrial
marker glutamate dehydrogenase, confirming that both
enzymes are exclusively located in the mitochondria.
ECHDC2 and 3MGH are localized to the mitochondria.
We verified the subcellular localization of ECHDC2 and
3MGH by performing a density gradient centrifugation
of a mouse kidney homogenate (figure 5). The
distinct activity patterns for the marker enzymes,
catalase (peroxisomes), glutamate dehydrogenase
(mitochondria),
β-hexosaminidase
(lysosomes),
esterase (microsomes) and phosphoglucoisomerase
(cytosol) indicated a good resolution between the
organelles (figure 5). Western blot analysis using the
antisera identified a single immunoreactive protein
Tissue-specific expression of ECHDC2 and 3MGH
human, mouse and rat. As 3-methylglutaconyl-CoA
hydratase activity is essential in the catabolism of
leucine, and both hydratases are localized to the
mitochondria, the tissue distribution of 3MGH and the
ECHDC2 protein is of considerable interest. Western
blot analysis demonstrated tissue-specific distribution
of stably expressed 3MGH and ECHDC2 in humans,
rats and mice (figure 6).
The tissue distribution of 3MGH appears to be
similar among humans, rats and mice. 3MGH is
Figure 5. Subcellular distribution of enoyl-CoA hydratases in mouse kidney. A post-nuclear supernatant of kidneys from nine male
mice was prepared and subjected to equilibrium density-gradient centrifugation using a linear 200–350 g/L Nycodenz gradient as
described in experimental procedures. After centrifugation, the gradient was fractionated, and marker enzymes activities were
measured in all 21 fractions: (II) b-hexosaminidase (lysosomes); (III) catalase (peroxisomes); (IV) phosphoglucoisomerase (cytosol);
(V) glutamate dehydrogenase (mitochondria); (VI) esterase (microsomes); and (I) total protein concentration. Results are percentages of total activity observed (II-VI) or mg/ml protein (I). (VII) Immunoblot analysis of gradient fractions with antibodies raised
against crotonase (ECHS1), 3-methylglutaconyl-CoA hydratase (3MGH) and the ECHDC2 protein (ECHDC2).
31
predominantly expressed in kidney, heart and brain
of these species. Only in rat liver there is considerable
expression of 3MGH as compared to human and mouse.
Furthermore, there is moderate expression of 3MGH in
the liver, muscle and testis of humans and rats. Analysis
of the various tissues with antibodies raised against
the ECHDC2 protein demonstrated that this protein
is highly expressed in liver and kidney, whereas it is
moderately expressed in the heart of humans, mice and
rats. More importantly, no expression of the ECHDC2
protein could be detected in the brain of human,
mouse and rat, in contrast to 3MGH which was highly
expressed in this type of tissue.
In addition, we also demonstrated that in human
skin fibroblasts 3MGH is expressed, but the ECHDC2
protein not.
Figure 6. Tissue distribution of 3-methylglutaconyl-CoA hydratase (A) and the ECHDC2 protein (B) in various human, rat
and mouse tissues. Each lane corresponds to 50 µg of extract
protein from the indicated tissues brain (B), kidney (K), liver
(L), heart (H), muscle (M) and testis (T). Lane with human skin
fibroblasts (HF) corresponds to 100 µg of extract protein.
Discussion
In this study we describe the molecular cloning of the
human ECHDC2 gene encoding a novel mitochondrial
enoyl-CoA hydratase, the ECHDC2 protein. In addition,
we present new data on human 3-methylglutaconylCoA hydratase (3MGH). The amino acid sequences of
3MGH and the ECHDC2 protein displayed features
typical of other well-defined members of the crotonase
superfamily, including the regions that define the active
site pocket. Members of the crotonase superfamily
catalyze a broad range of metabolic reactions, but
share a common structural solution to a mechanistic
problem.5 In this family the common theme is the need
to stabilize an enolate anion intermediate derived from
an acyl-coenzyme A substrate. In fact, stabilization of
this intermediate is accomplished by two structurally
conserved peptide NH groups, in a so-called oxyanion
hole, that provide hydrogen bonds to the carbonyl
moieties of the acyl-coenzyme A substrates. Amongst
this superfamily are enzymes that display hydratase,
32
dehalogenase, isomerase and thioesterase activities.
In addition, both the ECHDC2 protein and 3MGH
described in this paper also displayed two glutamate
residues that are conserved in all currently reported
enoyl-CoA hydratases. Therefore, it appears that these
two glutamate residues determine enoyl-CoA hydratase
activity. Indeed, members of this superfamily with
only one or none of the conserved glutamate residues
demonstrated no enoyl-CoA hydratase activity but have
isomerase, racemase or dehalogenase activity.5,10 More
importantly, both the ECHDC2 protein and 3MGH
demonstrated hydratase activities towards 3-hydroxy3-methylglutaryl-CoA and butenoyl-CoA. However,
at present, the relationship between structure and
enzymatic activity in the crotonase superfamily is still
too ill-defined to allow predictions about the potential
physiological function of the protein based on the
amino acid sequence.
No hydratase activity was found using 2-methylbutenoyl-CoA or 3-hydroxyisobutyryl-CoA as a
substrate, which strongly indicates that the ECHDC2
protein is not involved in the catabolic pathways
of the branched-chain amino acids isoleucine and
valine, respectively. In fact, the ECHDC2 protein
demonstrated considerable 3-methylglutaconyl-CoA
hydratase activity, raising the interesting question
if the ECHDC2 protein could be involved in the
catabolic pathway of leucine, which is generally
believed to be mitochondrial. For this reason, the
subcellular localization of 3MGH and the ECHDC2
protein becomes a topic of considerable interest. Our
data clearly show that both enzymes are located to the
mitochondria, indicating that the ECHDC2 protein
can relay 3-methylglutaconyl-CoA metabolism.
Interestingly, all reported patients with 3methylglutaconic aciduria type I clinically present
with various neurological handicaps, whereas no
abnormalities in peripheral organs are observed.
Immunoblot analysis revealed that both the ECHDC2
protein and 3MGH have a tissue-specific distribution.
Indeed, 3MGH is predominantly expressed in kidney,
heart and brain, and moderately expressed in skin
fibroblasts, liver, muscle and testis. Whereas the
ECHDC2 protein is expressed in kidney, liver and heart,
but no expression of this enzyme could be detected in
brain and skin fibroblasts. Given that 3MGH and the
ECHDC2 protein are expressed in peripheral tissues,
a deficiency of 3MGH in these organs in principle can
be corrected by the enzymatic actions of the ECHDC2
protein. Yet, in human brain there is no expression of
the ECHDC2 protein. Hence, in the brain of patients
with 3-methylglutaconic aciduria type I there is a total
block of 3-methylglutaconyl-CoA hydratase activity,
which will lead to neurological problems as seen in
patients with this deficiency.
Three additional forms of 3-methylglutaconic
aciduria have been recognized (Barth syndrome, MIM
302060; Costeff syndrome, MIM 258501; and type IV,
MIM 250951). Yet, all of these additional types are
characterized by normal 3-methylglutaconyl-CoA
hydratase activity in human skin fibroblasts. As the
ECHDC2 protein is not expressed in this tissue type, a
deficiency of the ECHDC2 protein should be considered
in patients with normal 3-methylglutaconyl-CoA
hydratase activity in human skin fibroblasts, but with
elevated urinary excretion of 3-methylglutaconic acid.
Fortunately, as the ECHDC2 protein is expressed in
other human tissues, such as liver, kidney and muscle,
a biopsy of these tissues could provide a solution or
diagnosis.
In conclusion, although the physiological function
of the ECHDC2 protein remains unresolved, it is
important to note that the ECHDC2 protein could,
in theory, relay 3-methylglutaconyl-CoA metabolism.
Hence, upregulation of the ECHDC2 protein in
human brain could be beneficial in patients with 3methylglutaconic aciduria type I.
References
2. Wanders, R. J., Vreken, P., den Boer, M. E., Wijburg, F. A., van
Gennip, A. H., and Ijlst, L. (1999) J. Inherit. Metab Dis. 22, 442.
3. Engel, C. K., Mathieu, M., Zeelen, J. P., Hiltunen, J. K., and
Wierenga, R. K. (1996) EMBO J. 15, 5135-5145
4. Engel, C. K., Kiema, T. R., Hiltunen, J. K., and Wierenga, R. K.
(1998) J. Mol. Biol. 275, 847-859
5. Holden, H. M., Benning, M. M., Haller, T., and Gerlt, J. A. (2001)
Acc. Chem. Res. 34, 145-157
6. Wanders, R. J., van Roermund, C. W., Schor, D. S., ten Brink, H.
J., and Jakobs, C. (1994) Biochim. Biophys. Acta 1227, 177-182
7. Wanders, R. J., van Roermund, C. W., de Vries, C. T., van den, B.
H., Schrakamp, G., Tager, J. M., Schram, A. W., and Schutgens,
R. B. (1986) Clin. Chim. Acta 159, 1-10
8. Wanders, R. J., Kos, M., Roest, B., Meijer, A. J., Schrakamp, G.,
Heymans, H. S., Tegelaers, W. H., van den, B. H., Schutgens, R.
B., and Tager, J. M. (1984) Biochem. Biophys. Res. Commun.
123, 1054-1061
9. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254
10. Wong, B. J. and Gerlt, J. A. (2004) Biochemistry 43, 4646-4654
1. Ijlst, L., Loupatty, F. J., Ruiter, J. P., Duran, M., Lehnert, W., and
Wanders, R. J. (2002) Am. J. Hum. Genet. 71, 1463-1466
33
Chapter 6
Clinical, Biochemical and Molecular Findings in Three Patients with
3-Hydroxyisobutyric Aciduria
Ference J. Loupatty, Annemarie van der Steen, Lodewijk IJlst, Jos P.N. Ruiter, Rob Ofman, Matthias
R. Baumgartner, Diana Balhausen, Seji Yamaguchi, Marinus Duran and Ronald J.A. Wanders
3-Hydroxyisobutyric aciduria is a rare entity and affected individuals display a range of clinical
manifestations including dysmorphic features and neurodevelopmental problems in the majority of
patients. Here we present two novel patients with 3-hydroxyisobutyric aciduria. To our knowledge,
these are the eleventh and twelfth cases of 3-hydroxyisobutyic aciduria reported. It is believed
that a deficiency in 3-hydroxyisobutyrate dehydrogenase is the most likely cause of this disorder.
Measurement of 3-hydroxyisobutyrate dehydrogenase activity in fibroblasts homogenates of the
two newly identified patients and a previously reported patient, however, revealed similar activities
as in control fibroblasts. Since other enzymes with overlapping substrate specificity could conceal
abnormal 3-hydroxyisobutyrate dehydrogenase activity, we cloned a candidate human cDNA
for 3-hydroxyisobutyrate dehydrogenase (HIBADH). By heterologous expression in Escherichia
coli, we showed that the product of the HIBADH gene indeed displays 3-hydroxyisobutyrate
dehydrogenase activity. Mutation analysis of the corresponding gene in the patients suffering from
3-hydroxyisobutyric aciduria revealed no mutations. We conclude that HIBADH is not the causative
gene in 3-hydroxyisobutyric aciduria.
3-Hydroxyisobutyric aciduria (MIM 236795) is a rare
organic aciduria and to date only ten patients have been
described in literature.1-7 These patients usually present
with dysmorphic features including a small triangular
face, low set ears, long philtrum and microcephaly.
Patients may show widely different phenotypes ranging
from mild vomiting attacks with normal brain and
cognitive development, to delayed motor development,
profound mental impairment and early death. Patients
with 3-hydroxyisobutyric aciduria usually excrete
elevated amounts of 3-hydroxyisobutyric acid (3HIBA) in urine, ranging from 60 to 390 mmol/mol of
creatinine (normal < 40 mmol/mol of creatinine) when
in stable condition and increasing up to 10,000 mmol/
mol creatinine during acute ketoacidotic episodes.1 In
half of the reported cases elevated lactate levels were
observed.1-7 Other, less frequent, abnormal metabolites
in urine are 3-hydroxypropionic acid, 2-ethyl-3hydroxypropionic acid, 2-hydroxybutyric acid, S-(2carboxypropyl)-cysteine and 3-hydroxyisovaleric acid.
The biochemical and molecular basis of 3hydroxyisobutyric aciduria is poorly understood.
3-hydroxyisobutyric acid is an intermediate in the
generally accepted pathway of valine oxidation (figure
1). Oral administration of valine in patients with 3hydroxyisobutyric aciduria led to a major increase in the
excretion of 3-HIBA and reproduced the clinical signs
of ketoacidosis, suggesting that the 3-hydroxyisobutyric
aciduria as observed in these patients is indeed due to
a block in valine oxidation.1 Although the responsible
defect has not been identified, a deficiency of 3-
hydroxyisobutyrate dehydrogenase has been suggested
as the most likely enzyme defect in patients with 3hydroxyisobutyric aciduria.4 In 1957, Robinson and
Coon were the first to isolate and characterize 3hydroxyisobutyrate dehydrogenase from pig kidney and
this NAD+-dependent enzyme has further been purified
Figure 1. Catabolism of L-valine. The names of the intermediates in the pathway for catabolism of L-valine are shown on
with solid arrows indicating enzymatic reactions. The structures of 3-hydroxyisobutyrate and methylmalonic semialdehyde are depicted. 3-Hydroxyisobutyrate dehydrogenase is
HIBADH.
From the Departments of Clinical Chemistry and Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.;J.P.N.R.;L.IJ.;M.D.;R.J.A.W.); Division of Metabolism and Molecular Pediatrics, University Children’s Hospital, Zurich, Switzerland (M.R.B.;D.B.); Department of Pediatrics, Shimane Medical University, Japan (S.Y.)
35
complicated by pericarditis and cardiac decompensation
from which he slowly recovered. Echocardiography
showed excentric hypertrophy of the right ventricle and
persistent pulmonary hypertension (pulmonary artery
pressure about half systemic pressure). A therapy with
an endothelin receptor antagonist (Bosentan, Tracleer®)
was started. A muscle biopsy performed at the age of 14
months showed reduced activities of respiratory chain
complex I and IV (NADH-CoQ-oxidoreductase 0.1 U/
U citrate synthase [controls 0.17-0.58] and cytochrome
c oxidase 0.8 U/U citrate synthase [controls 1.1-5.0],
respectively). Furthermore, mitochondrial proliferation
and ragged red fibers were observed upon enzyme
histochemistry suggestive of a respiratory chain defect.
A search for mitochondrial DNA mutations, insertions
and deletions was performed with a normal result. His
diet low in protein was changed to a diet enriched with
fat (60% of total energy), and a therapy with riboflavin
and coenzyme Q10 was started. Under this therapy
plasma lactate (≤ 5 mmol/l) and urinary organic acids
were essentially unchanged with intermittent mild
elevations of 3-hydroxyisobutyric acid (10 and 30
mmol/mol creatinine). He had no further hypoglycemia
or metabolic decompensation and his cardiomyopathy
and pulmonary hypertension remained stable. However,
despite some developmental progress, he remains
severely retarded. At 21 months of age he shows severe
failure to thrive (weight and length 1.7 cm below the
3rd percentile) and microcephaly (head circumference
3 cm below the 3rd percentile).
and characterized from Pseudomonas aeruginosa,
Candida rugosa, rabbit liver and rat liver.8-12 Only, the
rat 3-hydroxyisobutyrate dehydrogenase gene has been
cloned until now.
In this report, we present two novel patients
manifesting dysmorphic features and developmental
delay, in who urinary organic acid analysis revealed
abnormal excretion of 3-hydroxyisobutyric acid. To
our knowledge, these are the eleventh and twelfth
cases of 3-hydroxyisobutyic aciduria reported. In
an effort to clarify the abnormal metabolite profiles
detected in patients suffering from 3-hydroxyisobutyric
aciduria, we undertook the molecular and biochemical
characterization of the human 3-hydroxyisobutyrate
dehydrogenase in cultured fibroblasts derived from
patients with 3-hydroxyisobutyric aciduria and control
subjects.
Case reports
Patient 1. This boy is the first child of unrelated healthy
Swiss parents. The pregnancy was uneventful until
ultrasound displayed intrauterine growth retardation
at 33 weeks of gestation. He was born by C-section
at 39 weeks of gestation because of pathological
cardiotocogram. APGAR was 9/10/10. His weight was
2360 g (150 g below the 10th percentile), length was 44.5 cm
(2 cm below the 10th percentile) and head circumference
32 cm (0.8 cm below the 10th percentile). He was mildly
dysmorphic with unusually prominent eye brows and
lashes and displayed mild muscular hypotonia. No
hepatomegaly was noted. Echocardiography showed
persistent pulmonary hypertension. In the first days of
life he repeatedly had hypoglycemia (1.5 – 2.0 mmol/
L) and developed lactic acidosis with peak lactate
concentrations of up to 20 mmol/L. Plasma lactate/
pyruvate and 3-hydroxybutyrate/acetoacetate ratios
were elevated (43 and 4, respectively). While the
tendency to hypoglycemia resolved over time, lactate
remained elevated (5-10 mmol/L). There was no
hyperammonemia and no ketonuria. A plasma amino
acid profile displayed elevated alanine (940 μmol/
L) and mildly elevated branched chain amino acids.
Acylcarnitine analysis showed low free carnitine and
analysis of urinary organic acids repeatedly displayed
elevated concentrations of 3-hydroxyisobutyric acid (340
mmol/mol creatinine) and 2-ethyl-3-hydroxypropionic
acid (260 mmol/mol creatinine) combined with
lactic aciduria suggesting a defect at the level of 3hydroxyisobutyrate dehydrogenase. On carnitine
and a low protein diet supplemented with valine free
amino acid mixture he stabilized, but remained with
feeding difficulties and made only little developmental
progress. Organic acid excretion normalized. Lactate
remained mildly elevated (≤ 5 mmol/L) but no further
hypoglycemia was noted. At the age of 7 months he
was hospitalized because of persistent diarrhea and
vomiting. While hospitalized he suffered an acute
deterioration due to a gram positive sepsis which was
36
Patient 2. This male patient is the second child of nonconsanguineous parents of Swiss origin. His 5 years
older brother is healthy. In the 20th week of gestation
ultrasound showed significant growth retardation. The
child was born at 36 5/7 weeks of gestation with APGAR
9/9/10. He was small for date with weight 1450 g (550
g below the 10th percentile), length 40 cm (4 cm below
the 10th percentile) and head circumference 31 cm (10th50th percentile). Due to icterus neonatorum he received
phototherapy for 3 days. The child was very weak and
therefore was fed with bottle milk. A herniotomy
and orchidopexy on the left side was performed at 6
weeks. During operation it was noted that the left
vas deferens was missing. There was also agenesis of
the left kidney. Mild dysmorphic features including
ptosis of the left eye lid, clinodactyly of the fifth finger
and toe, and a syndactyly of the 2nd and 3rd toes were
present. Smith-Lemli-Opitz syndrome was excluded.
At the age of 10 weeks the patient was hospitalized
because of a severe failure to thrive (weight 2140 g).
He showed hyperaminoaciduria with prominent
glycine and proline. Urinary organic acids analysis
displayed repeated elevation of 3-hydroxyisobutyric
acid (50 mmol/mol creatinine) and lactate (450
mmol/mol creatinine). Plasma amino acid analysis
was unremarkable. Plasma lactate was only mildly
elevated (≤ 5 mmol/L). Acylcarnitine analysis showed
low free carnitine. A defect of 3-hydroxyisobutyrate
dehydrogenase was suspected and a therapy with a low
protein diet supplemented with a valine-free amino
acid mixture and carnitine was initiated at 4 months
of age. Under this regimen the metabolic parameters
normalized. Motor development was mildly delayed (
independent walking at 20 months). Mild spasticity of
the lower extremities was observed. Development of
speech was delayed with a remarkable deep voice. The
diet was slowly released and then stopped at the age of 2
years. Off diet mild elevations of 3-hydroxyisobutyrate
(≤ 20 mmol/mol creatinine) were again present. At
33 months of age all measures are still below the 3rd
percentile (weight 10.4 kg, length 86.3 cm, and head
circumference 46.6 cm).
Patient 3. The clinical details of this patient were
described previously by Sasaki and co-workers.4
Materials and methods
Cell culture. Primary skin fibroblasts were obtained from
the three subjects with 3-hydroxyisobutyric aciduria
mentioned above and four control subjects. Fibroblasts
were grown in HAM F10 medium, supplemented with
100 U/L penicillin G sodium, 100 U/L streptomycin
sulfate, 250 mg/L amphotericin B and 100 mL/L fetal
calf serum (Life technologies). Cultures were grown to
confluence in 162 cm2 disposable culture flasks (Costar)
and maintained at 37 °C in an atmosphere of 5% CO295% air with a relative humidity of 98%. Next, the cells
were harvested with trypsin-EDTA (Life technologies)
and the trypsin was inactivated by dilution in culture
medium. Finally, the cells were washed twice with
phosphate-buffered saline and stored as pellets frozen
at -80 °C.
Enzymatic
assay
for
3-hydroxyisobutyrate
dehydrogenase. S-3-Hydroxyisobutyric acid was
prepared from its methyl ester (Sigma-Aldrich) as
described by Rougraff and co-workers.11 Quantitative
analysis of S-3-hydroxyisobutyrate was done using GC/
MS as described by Röschinger and co-workers.13
3-Hydroxyisobutyrate dehydrogenase activity was
measured spectrophotometrically. The reaction mixture
contained 100 mmol/L Tris-CHES-CAPS buffer pH
9.5, 1 g/L Triton X-100, 100 mmol/L hydrazine pH 9.5,
1 mmol/L NAD+ and 0.9 mg/mL fibroblast homogenate
or recombinant protein. After a preincubation of five
minutes the reaction was started by the addition of 3hydroxyisobutyric acid at a final concentration of 2.5
mmol/L. The production of NADH was followed in
time on a Cobas-Fara centrifugal spectrophotometer
at 340 nm using a molar extinction coefficient of 6220
L.mol/cm. Protein was determined by the bicinchoninic
acid assay (Sigma) using BSA as standard.
Cloning and expression of HIBADH. First strand cDNA
was synthesized from RNA isolated from normal
cultured human skin fibroblasts as described by IJlst
37
and co-workers.14
The open reading frame (ORF) of HIBADH was
amplified from human cDNA by PCR using the primers:
+1HIBADHfBamHI: 5’-a tat gga tcc ATG GCA GCC
TCC TTA CGG CTC and +1124HIBADHrSalI: 5’a tat gtc gac GGT TCC CAA CAG TGT CCG TG.
The primer set introduced a 5’ BamHI and 3’ SalI
restriction site (underlined). The PCR product was
ligated into the pGEM-T vector (Promega, Madison,
USA) and sequenced to exclude errors introduced by
Taq polymerase.
The ORF of HIBADH was subsequently released
from the pGEM-T vector as a BamHI-SalI fragment
and ligated in-frame with the maltose binding protein
(MBP) ORF into the BamHI and SalI restriction sites
of pMAL-C2X (Promega, Madison, USA). pMALC2X plasmids containing the HIBADH ORF were
transformed into E.coli INV-α chemo-competent cells
(Invitrogen, Carlsbad, California, USA). Cells were
grown from a 100-fold diluted fresh overnight culture
for 4 hours in Luria-Bertani medium containing 100 µg/
mL ampicillin and 2 g/L glucose and grown to an OD600
0.4-0.6. Isopropyl-1-thio-β-D-galactopyranoside was
added to a final concentration of 1 mmol/L to induce
the production of the MBP-HIBADH fusion protein.
Cells were lysed by sonication (nine times on ice for 15
seconds at 8 W at 45 second intervals) and the fusion
proteins were affinity purified using an Amylose Resin
Column following the instructions of the manufacturer
(New England Biolabs, Ipswich, Massachusetts, USA).
Mutation analysis of human HIBADH on genomic
DNA. Genomic DNA was extracted from cultured
human skin fibroblasts using the Wizard Genomic
DNA purification Kit according to the instructions of
the manufacturer (Promega, Madison, USA). Eight
sets of HIBADH specific primers with –21M13 or
M13rev extensions were used for amplification of the
eight exons and their flanking intronic sequences. The
sequences of these primers on request.
The PCR reaction mixture contained 0.5 μmol/L of
each primer (Eurogentec, Liege, Belgium), 10 mmol/L
Tris/HCl pH 8.4, 50 mmol/L KCl, 1.5 mmol/L MgCl2,
0.1 g/L BSA, 0.2 mmol/L dNTP (Pharmacia, New
York, USA) and 2.5 U Taq DNA polymerase (Promega,
Madison, USA). With the following exceptions; for
exon 8 the reaction mixture contained 2.0 mmol/L
MgCl2, and the above mixture was supplemented with
5 % DMSO for exons 1 and 5.
Two different PCR programs were used to amplify
HIBADH from genomic DNA. For amplification of
exon 1 and 5 a touchdown approach was used for
increased specific primer annealing and started with
2 min of denaturation at 96 °C, followed by 10 cycles
during which the annealing temperature was lowered
with 1 °C per cycle from 65 °C to 55 °C. Every cycle
was initiated with 30 s of denaturation at 96 °C, 30 s
of annealing and 30 s of extension at 72 °C. These ten
‘touchdown’ cycles were followed by 23 cycles including
Results and Discussion
A
HIBADH activity (µmol/min/mg protein)
Here we present two patients biochemically
characterized by increased urinary excretion of 3hydroxyisobutyric acid and 2-ethyl-3-hydroxypropionic
acid. As our patients showed no elevated urinary
levels of 3-aminoisobutyrate the differential diagnosis
of methylmalonate-semialdehyde dehydrogenase
deficiency was excluded. In addition, our patients
showed lactic acidosis. The phenotypic presentation
of our patients includes minor dysmorphic features,
microcephaly, episodes of vomiting and developmental
delay. As the biochemical and clinical manifestations
are common in patients with 3-hydroxyisobutyric
aciduria, our patients were diagnosed with this
condition. As mentioned above, a deficiency of 3hydroxyisobutyrate dehydrogenase has been suggested
as the most likely enzyme defect in patients with 3hydroxyisobutyric aciduria.4 However, measurement
of 3-hydroxyisobutyrate dehydrogenase activity in
the patients showed normal activities (patient 1: 4.6;
patient 2: 4.2; index patient: 3.7 nmol/min/mg protein)
as compared to controls (4.7 ± 1.2 nmol/min/mg
B
1.6
1.4
1.2
1
12
10
8
1/V 6
4
2
0
0.8
0.6
0.4
0.2
0
0.0
protein (mean ± SD); range 2.9-6.6). These data suggest
that the 3-hydroxyisobutyrate dehydrogenase activity
in cultured human skin fibroblasts of three patients
with 3-hydroxyisobutyric aciduria is not affected.
However, the specificity of the enzymatic assay has
not been established, e.g. by immuno-precipitation
with antibodies raised against 3-hydroxyisobutyrate
dehydrogenase, and thus the presence of other enzymes
with overlapping substrate specificity could potentially
conceal a deficient activity of 3-hydroxyisobutyrate
dehydrogenase.
Another possibility for the normal activity found
in the patients’ fibroblasts is that the defect is not
expressed in cultured human skin fibroblasts due to the
presence of different isoforms of 3-hydroxyisobutyrate
dehydrogenase with a tissue-specific expression.
The elevated metabolites found in the plasma and
urine of patients with 3-hydroxyisobutyric aciduria
could originate from organs in which the amino
acid metabolism is high, e.g. liver and muscle. As a
consequence, these types of tissue are potentially the
best sources for diagnosis at the enzymatic level. As
these tissues were not available we decided to study the
defect on the genomic level.
HIBADH has been characterized in different
organisms and was cloned from a single mammalian
species, i.e. the rat, but the human 3-hydroxyisobutyrate
dehydrogenase gene has not been cloned. In the
Genbank database we identified a human candidate
gene (HIBADH; Genbank accession no. NM152740)
located on chromosome 7p15.2 and encoding a protein
with 70% identity to the rat 3-hydroxyisobutyrate
dehydrogenase protein. To investigate whether this
protein displays 3-hydroxyisobutyrate dehydrogenase
activity we expressed it as a fusion protein with
maltose binding protein in E. coli and affinity-purified
the fusion protein according to the manufacturer’s
protocol. The purified fusion protein indeed displayed
3-hydroxyisobutyrate dehydrogenase activity. The Km
values of 3-hydroxyisobutyric acid and NAD+ were
0.5
y = 0.0937x + 0.7641
R2 = 0.997
0
20
40
1/S
60
1.0
1.5
2.0
3-hydroxyisobutyric acid (mM)
80
100
HIBADH activity (µmol/min/mg protein)
30 s at 94 °C, 30 s at 55°C and 30 s at 72 °C, with a final
extension step of 5 min at 72 °C.
For the remaining exons the amplification program
started with 2 min of denaturation at 96 °C, followed by
5 cycles including 30 s of denaturation at 96 °C, 30 s of
annealing at 55 °C and 1 min extension at 72 °C. These
five cycles were followed by 25 cycles with 30 s at 94 °C,
30 s at 55 °C and 1 min at 72 °C, with a final extension
step of 7 minutes at 72 °C. With the exception that for
exon 8 the extension step was 30 s instead of 1 min.
Sequence analysis of these PCR fragments using Big
Dye fluorescent-labeled M13 primers was performed
on an Applied Biosystems 377A automated DNA
sequencer following the manufacturer’s protocols
(Perkin-Elmer Applied Biosystems, Massachusetts,
USA).
3.0
2.5
2.0
y = 0.1057x + 0.2142
8
1.5
1/V
1.0
2
R = 0.9998
6
4
2
0
0.5
0.0
0.0
2.5
10
0
0.2
0.4
20
0.6
40
1/S
0.8
60
80
1.0
NAD+ (mM)
Figure 2. Km-curves for the purified MBP-fusion HIBADH protein. A). The effect of the 3-hydroxyisobutyric acid concentration on
3-hydroxyisobutyrate dehydrogenase activity in the presence of 1 mmol/L NAD+. Km for 3-HIBA was 0.12 mM as determined from
the Lineweaver–Burke plot (inset). B) The effect of the NAD+ concentration on 3-hydroxyisobutyrate dehydrogenase activity in
the presence of 2.5 mmol/L 3-hydroxyisobutyric acid. Km for NAD+ was 0.5 mM as determined from the Lineweaver–Burke plot
(inset).
38
determined for the purified enzyme from LineweaverBurke double-reciprocal plots and were 0.12 mM and
0.5 mM respectively (figure 2).
Finally, all the eight exons of HIBADH and their
flanking intronic sequences were analyzed, but no
mutations were found in the three patients with 3hydroxyisobutyric aciduria. Hence, we conclude that
mutations in HIBADH are not common in patients
with 3-hydroxyisobutyric aciduria.
The finding of normal 3-hydroxyisobutyrate
dehydrogenase activity in the patients’ cells raises the
question whether 3-hydroxyisobutyric aciduria is due
to a secondary deficiency. It has been suggested that
accumulation of 3-hydroxyisobutyrate is the result of
an increased NADH/NAD+ ratio inhibiting the NAD+dependent 3-hydroxisobutyrate dehydrogenase.11,15
This occurs when the respiratory chain is blocked
due to a genetic deficiency of one or more of the
individual complexes thus leading to an impaired
oxidation of NADH and consequently, an elevated
NADH/NAD+-ratio. This is supported by the fact that
fifty percent of the patients with 3-hydroxyisobutyric
aciduria excreted elevated amounts of lactic acid in
their urine. In line with these observations, our first
patient is a further example of a respiratory chain
deficiency (in particular a complex I defect) leading to
impaired oxidation of NADH and secondary inhibition
of 3-hydroxyisobutyrate.15 Thus the presence of large
quantities of urinary 3-hydroxyisobutyrate together
with lactic acidemia should raise suspicion of a
respiratory chain deficiency.
In summary, although we did identify the human
gene encoding 3-hydroxyisobutyrate dehydrogenase,
this gene appeared not to be defective in patient with
3-hydroxyisobutyric aciduria, leaving the underlying
cause of this defect unknown.
References
1 F.J.Ko, W.L.Nyhan, J.Wolff, B.Barshop, and L.Sweetman,
3-Hydroxyisobutyric aciduria: an inborn error of valine
metabolism, Pediatr.Res. 30 (1991) 322-326.
2 D.Chitayat, K.Meagher-Villemure, O.A.Mamer, A.O’Gorman,
D.I.Hoar, K.Silver, and C.R.Scriver, Brain dysgenesis and
congenital intracerebral calcification associated with 3hydroxyisobutyric aciduria, J.Pediatr. 121 (1992) 86-89.
3 O.Boulat, N.Benador, E.Girardin, and C.Bachmann, 3hydroxyisobutyric aciduria with a mild clinical course, J.Inherit.
Metab Dis. 18 (1995) 204-206.
39
4 M.Sasaki, M.Kimura, K.Sugai, T.Hashimoto, and S.Yamaguchi,
3-Hydroxyisobutyric aciduria in two brothers, Pediatr.Neurol.
18 (1998) 253-255.
5 M.Sasaki, H.Iwata, K.Sugai, M.Fukumizu, M.Kimura,
and S.Yamaguchi, A severely brain-damaged case of 3hydroxyisobutyric aciduria, Brain Dev. 23 (2001) 243-245.
6 J.P.Shield, R.Gough, J.Allen, and R.Newbury-Ecob, 3Hydroxyisobutyric aciduria: phenotypic heterogeneity within a
single family, Clin.Dysmorphol. 10 (2001) 189-191.
7 Mienie L.J. and Erasmus E. Biochemical studies on a patient with
a possible 3-hydroxyisobutyrate dehydrogenase deficiency. 5th
international congress on inborn errors of metabolism. 1990.
8 W.G.ROBINSON and M.J.Coon, The purification and properties
of beta-hydroxyisobutyric dehydrogenase, J.Biol.Chem. 225
(1957) 511-521.
9 J.W.Hawes, E.T.Harper, D.W.Crabb, and R.A.Harris, Structural
and mechanistic similarities of 6-phosphogluconate and 3hydroxyisobutyrate dehydrogenases reveal a new enzyme family,
the 3-hydroxyacid dehydrogenases, FEBS Lett. 389 (1996) 263267.
10 J.W.Hawes, D.W.Crabb, R.J.Chan, P.M.Rougraff, R.Paxton, and
R.A.Harris, Mammalian 3-hydroxyisobutyrate dehydrogenase,
Methods Enzymol. 324 (2000) 218-228.
11 P.M.Rougraff, R.Paxton, M.J.Kuntz, D.W.Crabb, and R.A.Harris,
Purification and characterization of 3-hydroxyisobutyrate
dehydrogenase from rabbit liver, J.Biol.Chem. 263 (1988) 327331.
12 P.M.Rougraff, B.Zhang, M.J.Kuntz, R.A.Harris, and D.W.Crabb,
Cloning and sequence analysis of a cDNA for 3-hydroxyisobutyrate
dehydrogenase. Evidence for its evolutionary relationship to
other pyridine nucleotide-dependent dehydrogenases, J.Biol.
Chem. 264 (1989) 5899-5903.
13 W.Roschinger, A.C.Muntau, M.Duran, L.Dorland, L.IJlst,
R.J.Wanders, and A.A.Roscher, Carnitine-acylcarnitine
translocase deficiency: metabolic consequences of an impaired
mitochondrial carnitine cycle, Clin.Chim.Acta 298 (2000) 5568.
14 L.IJlst, R.J.Wanders, S.Ushikubo, T.Kamijo, and T.Hashimoto,
Molecular basis of long-chain 3-hydroxyacyl-CoA dehydrogenase
deficiency: identification of the major disease-causing mutation
in the alpha-subunit of the mitochondrial trifunctional protein,
Biochim.Biophys.Acta 1215 (1994) 347-350.
15 M.J.Bennett, W.G.Sherwood, K.M.Gibson, and A.B.Burlina,
Secondary inhibition of multiple NAD-requiring dehydrogenases
in respiratory chain complex I deficiency: possible metabolic
markers for the primary defect, J.Inherit.Metab Dis. 16 (1993)
560-562.
Chapter 7
Mutations in the Gene Encoding 3-Hydroxyisobutyryl-CoA
Hydrolase Results in Progressive Infantile Neurodegeneration
Ference J. Loupatty, Peter T. Clayton, Jos P.N. Ruiter, Rob Ofman, Lodewijk IJlst, Garry K. Brown,
David R. Thorburn, Robert A. Harris, Marinus Duran, Carlos DeSousa, Steve Krywawych, Simon
J.R. Heales and Ronald J.A. Wanders
Only a single patient with 3-hydroxyisobutyryl-CoA hydrolase deficiency has been described in
literature and the molecular basis of this inborn error of valine catabolism has remained unsolved
thus far. Here, we present a second patient with 3-hydroxyisobutyryl-CoA hydrolase deficiency
who was identified through blood spot acylcarnitine analysis which showed persistently increased
levels of hydroxy-C4-carnitine. Both patients manifested hypotonia, poor feeding, motor delay and
subsequent neurological regression in infancy. Additional features in the newly identified patient
included episodes of ketoacidosis and Leigh-like changes in the basal ganglia on the MRI scan.
In cultured skin fibroblasts of both patients, the 3-hydroxyisobutyryl-CoA hydrolase activity was
deficient, and virtually no 3-hydroxyisobutyryl-CoA hydrolase protein could be detected by western
blotting. Molecular analysis in both patients uncovered mutations in the HIBCH gene, including one
missense mutation which is in a conserved part of the protein and two mutations affecting splicing.
A carefully interpreted acylcarnitine profile will allow more patients with 3-hydroxyisobutyryl-CoA
hydrolase deficiency to be diagnosed.
The catabolic pathway for the branched-chain amino
acid valine is exceptional, because it is believed that part
of the pathway between 3-hydroxyisobutyryl-CoA and
propionyl-CoA proceeds via free acids, thus requiring
a specific hydrolase. This is in marked contrast to the
degradation pathways of the other branched-chain
amino acids, i.e. leucine and isoleucine, in which the
intermediates distal to the 2-oxo-acids are all CoA
thioesters. Indeed, the hydrolysis of an activated
acyl group in the heart of a catabolic pathway is not
only uncommon, but also energetically unfavorable
especially when subsequent steps of the pathway again
involve CoA thioester intermediates. Nevertheless,
although the evidence for the need of a specific hydrolase
in the catabolic pathway of valine was poor, a strong
support for this hypothesis came from one case report
documenting 3-hydroxyisobutyryl-CoA hydrolase
(HIBCH) deficiency (MIM 250620) published in 1982.1
However, the molecular basis of 3-hydroxyisobutyrylCoA hydrolase deficiency has remained unsolved.
Brown and collegues1 described a male infant
(patient 1) born to consanguineous parents, who
clinically manifested various physical malformations
(dysmorphic facial features, multiple vertebral
anomalies, tetralogy of Fallot and - at post-mortem
examination - agenesis of the cingulate gyrus and
corpus callosum), as well as failure to thrive and
marked hypotonia. More importantly, however,
the urine of this patient persistently demonstrated
increased levels of S-2-carboxypropyl-cysteamine and
S-2-carboxypropyl-cysteine.1,2 As the latter compound
can be formed by the condensation between cysteine
and methacrylyl-CoA, an intermediate in valine
oxidation, the abnormal levels suggested a defect in the
valine catabolic pathway. In valine catabolism (fig. 1),
methacrylyl-CoA is converted to 3-hydroxyisobutyrylCoA by crotonase, but fibroblast and liver homogenates
of patient 1 showed normal hydratase activities
towards methacrylyl-CoA (as well as crotonyl-CoA and
tiglyl-CoA) when compared to controls. This finding
suggested that if a defect in the catabolic pathway of
valine were to exist, it would be distal to this hydration
reaction. Indeed, through a coupled enzyme assay
using methacrylyl-CoA and an excess of the enzyme
crotonase, Brown and co-workers demonstrated a 20 %
residual activity of 3-hydroxyisobutyryl-CoA hydrolase
in cultured skin fibroblasts.1 No additional patients
with 3-hydroxyisobutyryl-CoA hydrolase deficiency
have been reported so far.
In this report we describe the second patient with
From the Departments of Clinical Chemistry and Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.;J.P.N.R.;R.O.;L.IJ.;M.D.;R.J.A.W.); University College London Institute of Child Health with Great Ormond Street Hospital for Children, London WC1, UK (P.T.C.;C.D.;S.K.); University College London Institute of Neurology with the National Hospital for Neurology and Surgery, Queen Square, London WC1, UK
(S.J.R.H.); Genetics Unit, Department of Biochemistry, University of Oxford, Oxford, UK (G.K.B.); Murdoch Children’s Research Institute, Royal Children’s Hospital (D.R.T.) and Department of Pediatrics, University of Melbourne, Melbourne, Australia (D.R.T.); Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, USA (R.A.H.)
41
thiamine or a combination of vitamin C, vitamin E and
ubiquinone. As he recovered from his episode of acute
encephalopathy, he developed dystonia predominantly
affecting the right arm and leg. A�������������������
��������������������
CT scan performed
during the episode of acute encephalopathy revealed
generalised oedema of the brain with loss of grey /
white differentiation in the basal ganglia. In addition,
an MRI scan (fig. 2) detected signal
������������������������
abnormalities in
the globus pallidus and the midbrain with asymmetrical
involvement of the cerebral peduncles (right greater
than left). �����������������������������������������
No���������������������������������������
structural abnormalities of the brain
were observed.
A����������������������������������������������
s the neuroimaging was reminiscent of Leigh’s
disease, we investigated the activities of pyruvate
dehydrogenase and pyruvate carboxylase in cultured
Figure 1. Catabolism of L-valine. The names of the intermediates in the pathway for catabolism of L-valine are shown on
the left with solid arrows indicating enzymatic reactions. The
structures of methacrylyl-CoA, 3-hydroxyisubutyryl-CoA and
3-hydroxyisobutyrate are depicted. The names of the metabolites that are increased as result of 3-hydroxyisobutyryl-CoA
hydrolase deficiency are shown by dashed arrows to the right.
HIBCH deficiency (patient 2), who was�����������������
the first child
of healthy non-consanguineous Caucasian parents and
was born at term following an uneventful pregnancy.
Initially he fed poorly but otherwise appeared well until
4 months of age when his parents noted head bobbing.
This was followed by a delay in motor milestones, then
ataxia and a loss of skills. He was able to roll over at
5 months but lost this ability at 10 months. He was
reaching out and grasping objects at 4 months but
gradually lost this skill at 13 months. From the age of
9 months he started to have transient absences and
episodes of eye rolling. At the age of 10 months he had a
febrile illness during which he became irritable and more
wobbly. He lost the ability to finger feed. Examination
at 11 months revealed an alert interactive child who
had no nystagmus but constant titubation of the head.
He had marked truncal ataxia and was unable to sit
unsupported. Tone, power and reflexes were normal
in the upper limbs; tone was reduced, particularly
distally, in the lower limbs. He had marked dysmetria
and tremor on reaching out. At the age of 14 months
following two days of coryza and lethargy, he became
acutely unwell with a reduced level of consciousness
and metabolic acidosis, and required intubation and
ventilatory support. Blood pH was 7.29 with a base
deficit of 15.8 mmol/L. Blood lactate and ammonia
were normal. Echocardiography demonstrated no
anomalies. Examination following recovery from this
illness showed drowsiness, variable nystagmus, sluggish
papillary responses, pooling of secretions in the mouth,
fasciculation of the tongue, titubation of the head, and
upper and lower limb hypotonia with preservation of
reflexes. He showed no improvement on treatment with
42
Figure 2. MRI scans of patient 2 MRI scan undertaken when patient 2 was 14 months old. T2-weighted images. There is signal
abnormality in the brain regions indicated by the white arrows:
in the globi pallidi (upper image) and in the midbrain with asymmetrical involvement of the cerebral peduncles (R > L) (lower
image). The appearances were considered likely to represent a
neurometabolic disorder. No structural abnormality was noted.
human skin fibroblasts and measured the activities
of respiratory chain complexes in a muscle biopsy of
patient 2, as described elsewhere.3-7 Normal activities
were found for pyruvate carboxylase and pyruvate
dehydrogenase. The activities of the respiratory
chain complexes, expressed as a ratio to citrate
synthase, revealed a marked
������������������������������
reduction of complex I
(patient 0.089, controls 0.104-0.268) and a borderline
reduction in complex IV (patient 0.013, controls
0.014-0.034). These results were obtained on a muscle
biopsy obtained 3 months after the major episode of
metabolic decompensation. A second muscle biopsy
obtained 3 months later showed normal activities of
all four respiratory chain complexes. Urine organic
acid analysis during ketotic episodes showed ����������
excessive
excretion of 3-hydroxybutyrate and acetoacetate, and
moderate excretion of lactate, 2-hydroxyisovalerate,
2-oxoisocaproic acid, dicarboxylic acids (C6, C8, C10)
and methylmalonate with normal methylcitrate. In
contrast, he excreted normal levels of urinary organic
acids when well. Normal values were found for ����
nonfasting blood 3-hydroxybutyrate (<0.05mM), and blood
and CSF lactate. Urine amino acid analysis was normal,
except on one occasion when it showed increased ratios
to creatinine of several amino acids (including glycine,
alanine and taurine) plus one �����������������������
unidentified ninhydrin
positive compound.
Remarkably, elevated levels of hydroxy-C4carnitine were consistently found in blood spots of
patient 2. Its concentration ranged from 0.45-1.73
µM, controls <0.4 µM. Elevation
����������������������
of hydroxy-C4carnitine may occur in ketosis but the elevated blood
concentration persisted in this patient even when he
was not ketotic. Tandem mass spectrometric analysis
does not distinguish between 3-hydroxy-n-butyrylcarnitine or 3-hydroxyisobutyryl-carnitine. Either of
these hydroxy-C4-carnitine species could be increased
as a consequence of a specific enzyme defect, i.e. short
chain 3-hydroxyacyl-CoA dehydrogenase deficiency
(SCHAD; MIM 300256) or 3-hydroxyisobutyryl-CoA
hydrolase deficiency, respectively. Patients with SCHAD
deficiency uniformly suffer from hyperinsulinism8, a
phenotype very different from that observed in patient
2. As expected, a normal activity of short chain 3hydroxyacyl-CoA dehydrogenase was found in the
fibroblasts of this patient as well as of patient 1 (data
not shown), suggesting 3-hydroxyisobutyryl-CoA
hydrolase deficiency.
Therefore, we investigated fibroblasts of patients
1 and 2 for 3-hydroxyisobutyryl-CoA hydrolase
activity using a direct enzyme assay based on the use
of the physiological substrate S-3-hydroxyisobutyrylCoA. The thioester was synthesized from methyl
S-3-hydroxyisobutyrate as described elsewhere.
3-Hydroxyisobutyryl-CoA hydrolase activity was
subsequently
measured
spectrophotometrically.
The reaction mixture contained in a total volume
of 250 µL, 100 mM Tris.HCl pH 8.0, 1 mM EDTA, 1
g/L Triton X-100, 0.1 mM DTNB and 0.2-0.4 mg/ml
43
fibroblast homogenate. After a preincubation of ten
minutes the reaction was started by the addition of 3hydroxyisobutyryl-CoA at a final concentration of 0.2
mM. The reduction of DTNB was followed in time on
a Cobas-Fara centrifugal analyzer (Roche) at 412 nm
using a molar extinction coefficient of 13700 L/mol/
cm. Using this newly developed assay, an activity of 6.4
± 1.6 nmol/min/mg protein (mean ± SD) was found in
control fibroblasts (n=8) whereas in fibroblasts from
both patients no 3-hydroxyisobutyryl-CoA hydrolase
activity could be detected.
A human cDNA (HIBCH) encoding a protein with
3-hydroxyisobutyryl-CoA hydrolase activity has been
described previously.9 This HIBCH gene maps to
chromosome 2q32.2 and has an open reading frame
of 1161 base pairs encoding a protein of 386 amino
acid residues with a calculated molecular weight of 43
kDa. Immunoblot analysis using an antibody against 3hydroxyisobutyryl-CoA hydrolase revealed the absence
of the HIBCH protein in patient 1 (fig. 3). Fibroblast
lysates of patient 2 demonstrated an apparently lower
expression of the HIBCH protein as compared to
controls.
To establish that 3-hydroxyisobutyryl-CoA
hydrolase deficiency is caused by mutations in
HIBCH, we analyzed the gene at the genomic level
and the mRNA level in both patients. We identified
a homozygous IVS3-9T>G mutation in patient 1,
consistent with parental consanguity. The IVS3-9T>G
mutation was absent from 210 control chromosomes.
However, this T>G transversion only slightly weakens
the consensus sequence for a splice acceptor site,
since a pyrimidine is preferred over a purine at this
position in the consensus sequence. Hence, the
consequence of this missense mutation on the splicing
efficiency was investigated by analyzing HIBCH
HL
C1
C2
P1
P2
HIBCH>
Figure 3. Immunoblot analysis of 3-hydroxyisobutyryl-CoA hydrolase in fibroblast lysates and human liver homogenates. 25
µg of human liver (HL) protein and equal amounts of fibroblast
protein (100µg) of two control subjects (C1 and C2), the index patient (P1) and the newly identified patient (P2) were
subjected to SDS-PAGE and transferred onto nitrocellulose by
semidry blotting. Polyclonal antibodies raised against purified
rat liver 3-hydroxyisobutyryl-CoA hydrolase were used at a
1:5000 dilution. Antigen-antibody complexes were visualized
with goat anti-rabbit IgG-alkaline phosphatase conjugate.
As a control for transfer of protein, each blot was reversibly
stained with Ponceau S prior to the incubation with antibodies.
A
[Exon]..TATCCACAGCTAAAG
gtttgtaattttctt ..[ 2081 bp]..atgattgaatatatgtgcatatg
catataactttaatgagtttgtgtttataatatgcttataccatcttctgttacatttgaatag
AAGTGGGAA..[Exon]
B
[Exon]..TATCCACAGCTAAAG gtttg taattttctt ..[ 2081 bp]..atgattgaatatatgtgcatatg
catataactttaatgagtttgtgtttataatatgcttataccatcttctgttacag* ttgaatagAAGTGGGAA..[Exon]
Figure 4. Cryptic splice acceptor site in patient 1. (A) Sequence analysis of the genomic DNA region from which the 8 bp insertion originates and amplified by PCR from control subjects identified an intron of 2183 bp (small lettering) with consensus
splice donor and splice acceptor site sequences (italics and underlined) and branch point (underlined). The complete nucleotide
sequence of the intron can be obtained from GenBank under accession number NT_005403. (B) In patient 1, a T>G mutation
(*) at the -9 position of the authentic splice acceptor site (underlined) results in alternative splicing at a cryptic splice acceptor site located 9 bp 5’ of the authentic splice acceptor site and preceded by a consensus branch point sequence. The 8 bp
intron sequence, which is retained as a result of the aberrant splicing, is indicated in bold. Original exon sequences are indicated in capitals. Consensus sequences are defined as follows: splice donor site, [exon]..(C/A)AG gt(a/g)agt..[intron]; branch
point, (t/c)n(t/c)t(g/a)ac (18-40 bp 5’ of splice acceptor site); splice acceptor site, [intron]..(t/c)6n(t/c)ag G(G/T)..[exon].
cDNA obtained by reverse-transcriptase-PCR (RTPCR) from skin fibroblast RNA. Sequence analysis of
the amplicon revealed a homozygous 8 bp insertion
(c.219_220insTTGAATAG), after the last base of exon
3, causing a frame shift (K73fsX86). The 8 bp insertion
resulted from retention of the 3’ end of intron 3. Close
examination of the intronic DNA sequence adjacent to
the IVS3-9T>G mutation revealed a stronger homology
to the consensus sequence of a splice acceptor site than
present in the wild type intronic sequence, with identity
at 9 of 10 intronic bases instead of 7 of 10 (fig. 4). Both
the wild type and mutant sequence are preceded by a
DNA sequence with strong homology to the branch
point consensus sequence, thus constituting a rather
strong alternative cryptic splice acceptor site (fig. 4).
Missplicing was evident since only the aberrant spliced
transcript could be detected.
Sequence analysis revealed that patient 2 was
compound heterozygous for a missense mutation,
c.365A>G (Y122C), and a splice acceptor site mutation,
IVS2-3C>G. The latter mutation was paternal,
whereas the mother was a heterozygous carrier of the
c.365A>G mutation. The missense mutation predicts
the substitution of the bulky amino acid residue
tyrosine which is conserved among different species,
including Mus musculus (mouse), Rattus norvegicus
(rat), Arabidopsis thaliana (plant), Caenorhabditis
elegans (nematode), Gallus gallus (chicken), Xenopus
tropicalis (frog), Bos taurus (cow), Canis familiaris
(dog), Pongo pygmeus (orangutan), Danio rerio (zebra
fish), Saccharomyces cerevisiae (budding yeast) and
Schizosaccharomyces pombe (fission yeast). The IVS23C>G transversion disrupts the consensus sequence for
a splice acceptor site, given that a pyrimidine (cytosine
or thymine) is preferred over guanine at this position
in the consensus sequence. Thus, the significance of
this particular mutation on splicing was investigated
by analyzing HIBCH cDNA. Remarkably, sequence
analysis revealed a heterozygous 2 bp insertion resulting
from retention of the 3’ end of intron 2, causing a frame
44
shift (R27fsX50). Indeed, the intronic DNA sequence
adjacent to the IVS2-3C>G mutation presented a
strong homology to the consensus sequence of a splice
acceptor site with identity at 9 of 10 intronic bases. As
this sequence is preceded by a branch point consensus
sequence, it constitutes an alternative splice acceptor
site.C���������������������������������������������
ompound heterozygosity for the Y�������������
��������������
122����������
C���������
and the
IVS�����������������������������������������������
2-3C>G mutation resulted in a complete absence
of 3-hydroxyisobutyryl-������������������������������
CoA���������������������������
hydrolase activity in the
patient, indicative of a pathogenetic effect of both
mutations. �������������������������������������������
H������������������������������������������
owever, to exclude that the ��������������
Y�������������
122����������
C���������
and the
IVS2-3C>G mutations present polymorphic variants we
analysed 210 control chromosomes for both mutations,
assuming a general polymorphic variant frequency of
0.01 but did not detect any of these mutations.�������
Based
on these data, we conclude that 3-hydroxyisobutyrylCoA hydrolase deficiency is caused by mutations in the
HIBCH gene.
Until now, only one patient with 3-hydroxyisobutyrylCoA hydrolase deficiency had been described. The
urine of this patient showed abnormal levels of S2-carboxypropyl-cysteine and S-2-carboxypropylcysteamine which were found using a combination of
high voltage electrophoresis, paper chromatography
and ninhydrin-staining.2 However, in many presentday laboratories routine analysis of plasma and urinary
amino acids is performed by liquid chromatography
using lithium citrate buffers for elution.10 Unfortunately,
S-2-carboxypropyl-cysteine was not available as
a reference substance which precluded a correct
identification in patient 2. The concentration of S2-carboxypropyl-cysteine and S-2-carboxypropylcysteamine in patient 1’s urine2 was estimated to be
less than 10 µM. Furthermore, S-2-carboxypropylcysteine has a retention time close to glycine, alanine
and citrulline.10 As these amino acids are commonly
present in the urine of children, they can easily obscure
the presence of the cysteine adducts. As a consequence,
additional patients with 3-hydroxyisobutyryl-CoA
hydrolase deficiency may easily have remained
undetected using this amino acid analyzer approach.
This is exemplified by patient 2, in whom only one out
of four urine
�����������������������������������������������
samples showed an �����������������������
unidentified ninhydrin
positive compound. However, we were unable to
determine if this was S-2-carboxypropyl-cysteine or
S-2-carboxypropyl-cysteamine. Fortunately,������������
������������������������
it was the
detection of persistently elevated hydroxy-C4-carnitine
in the blood of this patient that led us to determine the
activity of 3-hydroxyisobutyryl-CoA hydrolase.
Both patients with HIBCH deficiency demonstrated
delayed development of motor skills, hypotonia, initial
poor feeding and a deterioration in neurological
function during the first stages of life. However, the
neuropathology of patient 1 included agenesis of the
cingulate gyrus and the corpus callosum, whereas no
structural brain abnormalities were observed in patient
no. 2. Moreover, the brain anomalies of patient 2 as
indicated on the CT and MRI scans was predominantly
in the basal ganglia – a picture commonly seen in
respiratory chain disorders (Leigh’s disease), glutaric
aciduria type I, methylmalonic acidaemia and priopionic
acidaemia. It is also important to note that��������������
, in contrast
to patient 1, patient 2 manifested no dysmorphic
(facial) features, nor congenital heart disease (tetralogy
of Fallot) and is still alive today.
Treatment of patients
��������������������������������������
with HIBCH deficiency should
be aimed at prevention of an increased flux through the
catabolic pathway of L-valine. Hence, it is advisable to
reduce the protein intake and maintain a high intake of
carbohydrates, especially during episodes of ketosis (as
is done for other disorders of branched chain amino acid
catabolism). Other possible therapeutic approaches
include, administration of carnitine to release coenzyme
A and increase the elimination of 3-hydroxyisobutyrylCoA as urinary 3-hydroxyisobutyrylcarnitine, together
with administration of pantothenate
�����������������������
(vitamin B5) to
compensate for the fact that at least part of intracellular
CoA will be trapped in the form 3-hydroxyisobutyryl��������������������
CoA�.
In conclusion, we have resolved the molecular basis
of 3-hydroxyisobutyryl-CoA hydrolase deficiency. By
doing so, we also provide conclusive evidence that
in humans, part of the catabolic pathway of valine
does indeed proceed via free acids, in contrast to
the degradation of the other branched-chain amino
acids leucine and isoleucine. Furthermore, we have
identified the second patient with HIBCH deficiency.
Both patients with HIBCH deficiency demonstrated
progressive neurodegeneration. O����������������������
ur data suggests that
analysis of blood spot acylcarnitines may circumvent
the problematic analysis of the amino acids S-2carboxypropyl-cysteine
and
S-2-carboxypropylcysteamine which previously led to the diagnosis
of 3-hydroxyisobutyryl-CoA hydrolase deficiency.
Moreover, as hydroxy-C4-carnitine could appear in
routine acyl-carnitine analysis we predict that more
45
patients with 3-hydroxyisobutyryl-CoA hydrolase
deficiency will be diagnosed as this technique becomes
more widespread available. Until we have more
experience with the natural history of this disorder and
the effect of therapeutic interventions, it is probably
wise to regard 3-hydroxyisobutyryl-CoA hydrolase
deficiency as a disorder associated with progressive
neurological damage ultimately leading to early death.
Fortunately, the full elucidation of the molecular and
metabolic basis of the disorder will allow prenatal
diagnosis.
References
1. Brown GK, Hunt SM, Scholem R, Fowler K, Grimes A, Mercer
JF, Truscott RM, Cotton RG, Rogers JG, Danks DM (1982) betahydroxyisobutyryl coenzyme A deacylase deficiency: a defect
in valine metabolism associated with physical malformations.
Pediatrics 70:532-538
2. Truscott RJ, Malegan D, McCairns E, Halpern B, Hammond J,
Cotton RG, Mercer JF, Hunt S, Rogers JG, Danks DM (1981)
Two new sulphur-containing amino acids in man. Biomed Mass
Spectrom 8:99-104
3. Ragan CI, Wilson MY, Darley-Usman VM (1988)
Subfractionation of mitochondria and isolation of proteins of
oxidative phosphorylation. In Darley VM, Rickwood D, Wilson
MT, eds. Mitochondria: A Practical Approach. Oxford: IRL
Press. 79-113
4. King TE (1967) Preparation of succinate cytochrome c reductase
and cytochrome b-c1 particle and reconstruction of Succinate
cytochrome c reductase. Methods Enzymol 10: 446-451
5. Wharton DC, Tzagoloff A (1967) Cytochrome oxidase from
beef heart mitochondria. Methods Enzymol 10: 245-250
6. Shepherd JA, Garland PB (1969) Citrate synthase activity from
rat liver. Methods Enzymol 13: 11-19
7. Heales SJR, Hargreaves IP, Olpin SE (1996) Diagnosis of
mitochondrial electron transport chain defects in small muscle
biopsies. J Inher Metab Dis 19:P151
8. Clayton PT, Eaton S, Aynsley-Green A, Edginton M, Hussain
K, Krywawych S, DattaV, Malingre HE, Berger R, van de Berg
I (2001) Hyperinsulinism in short-chain L-3-hydroxyacyl-CoA
dehydrogenase deficiency reveals the importance of betaoxidation in insulin secretion. J Clin Invest 108:457-465
9. Hawes JW, Jaskiewicz J, Shimomura Y, Huang B, Bunting J,
Harper ET, Harris RA (1996) Primary structure and tissuespecific expression of human beta-hydroxyisobutyryl-coenzyme
A hydrolase. J Biol Chem 271:26430-26434
10. Moore S, Spackman DH, Stein WH (1958) Automatic recording
apparatus for use in the chromatography of amino acids. Fed
Proc 17:1107-1115
Summary
Ference J. Loupatty
In recent years tremendous progress has been made with respect to the enzymology of the catabolic
pathway of the branched chain amino acids and defects therein. Nevertheless, at the start of this
project the metabolic and molecular basis of several inborn errors of branched chain amino acid
metabolism, including 3-methylglutaconic aciduria type I and 3-hydroxyisobutyryl-CoA hydrolase
deficiency, were still elusive. Therefore, it was the purpose of this study to examine and elucidate the
basis of these disorders and to devise new and improved means to identify branched chain organic
acidurias. Indeed, the need to distinguish isolated 3-methylglutaconyl-CoA hydratase deficiency
from secondary 3-methylglutaconic acidurias required a sensitive and specific enzyme assay. In
the past enzymatic confirmation depended on a complex assay which utilized radioactive markers
and commercially unavailable coupling enzymes. We developed a HPLC-method measuring 3methylglutaconyl-CoA hydratase activity in the reverse direction using 3-hydroxy-3-methylglutarylCoA (HMG-CoA) as substrate (chapter 4). The assay parameters were well within the accepted limits
for clinical diagnostic tests and the assay can readily be adopted by other laboratories. Moreover,
since its introduction several patients with 3-methylglutaconic aciduria type I have been identified
using this method.
HMG-CoA is a substrate for the enoyl-CoA hydratase referred to as crotonase, albeit a very poor
one. Patients with 3-methylglutaconic aciduria type I have normal crotonase activities. Accordingly,
we hypothesized that 3-methylglutaconyl-CoA hydratase should closely resemble crotonase. Thus,
we looked for sequences homologous to human crotonase which resulted in ECHDC2 and AUH
as candidates for 3-methylglutaconyl-CoA hydratase. Comparison of the corresponding products
revealed that both proteins are mitochondrial enoyl-CoA hydratases with highly similar substrate
specificities, but tissue-specific distribution (chapter 5). More importantly, molecular analysis
revealed that mutations in AUH cause 3-methylglutaconyl-CoA hydratase deficiency (chapter 3).
Although the physiological function of the ECHDC2 protein remains unclear, it could prove a
potential therapeutic agent for patients with isolated 3-methylglutaconyl-CoA hydratase deficiency
and is a candidate enzyme wich hydratase activities should be studied in patients suffering from one
of the unresolved 3-methylglutaconic acidurias.
After its initial and only case report in 1982, we resolved the molecular basis of 3-hydroxyisobutyrylCoA hydrolase deficiency which provide conclusive evidence that in humans, part of the catabolic
pathway of valine does indeed proceed via free acids (chapter 7). Moreover, we identified a second
patient with 3-hydroxyisobutyryl-CoA hydrolase deficiency through blood spot acylcarnitine
analysis which showed persistently increased levels of hydroxy-C4-carnitine. This is an important
finding as carnitine profiling may circumvent the problematic analysis of the amino acids S-2carboxypropyl-cysteine and S-2-carboxypropyl-cysteamine which previously led to the diagnosis
of 3-hydroxyisobutyryl-CoA hydrolase deficiency. Furthermore, as hydroxy-C4-carnitine could
appear in routine acylcarnitine analysis we predict that more patients with 3-hydroxyisobutyrylCoA hydrolase deficiency will be diagnosed.
Finally, we identified the human gene encoding 3-hydroxyisobutyrate dehydrogenase (chapter 6).
It is believed that a defect in this enzyme activity is the most likely cause of 3-hydroxyisobutyric
aciduria, a disorder of valine metabolism. This gene, however, appeared not to be defective in patient
with 3-hydroxyisobutyric aciduria. Moreover, measurement of 3-hydroxyisobutyrate dehydrogenase
activity in fibroblasts homogenates of these patients, revealed similar activities as in control
fibroblasts, leaving the underlying cause of this defect unknown.
From the Departments of Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam,
The Netherlands (F.J.L.)
46
Vertakte keten aminozuren: Feiten en fouten
Ference J. Loupatty
Het menselijk lichaam is opgebouwd uit cellen waarin zich organellen bevinden zoals peroxisomen,
mitochondria en lysosomen. Elk van deze organellen vervult één of meerdere specifieke taken in het
goed en volledig functioneren van de cel en derhalve van het menselijk lichaam. In de mitochondria
vindt afbraak plaats van vertakte keten aminozuren en dit proces is het centrale thema van het
proefschrift.
De drie vertakte keten aminozuren – leucine, isoleucine en valine – zijn essentiële aminozuren, d.w.z.
dat deze stoffen uit het dieet gehaald dienen te worden, omdat het menselijk lichaam niet in staat
is deze verbindingen zelf te maken. Eenmaal door het lichaam opgenomen worden ze gebruikt als
bouwstenen voor eiwitten of wordt een overmaat direct in de mitchondria via een reeks enzymreacties
omgezet tot energie. Wanneer de afbraak van vertakte keten aminozuren niet goed functioneert
door een blokkade in de degradatieroute kunnen tussenproducten stapelen en/of omgezet worden
via secundaire afbraakroutes. Deze intermediairen kunnen toxisch zijn of anderzins ernstige schade
toebrengen aan organen zoals het hart, de hersenen of de lever met bijbehorende complicaties.
Tevens kunnen deze metabolieten uitgescheiden worden in lichaamsvochten als bloed of urine. De
vertakte keten organische acidurieën zoals hier beschreven hebben een erfelijke achtergrond, d.w.z.
dat ze het gevolg zijn van mutaties in het ouderlijk DNA. Samengevat is het van (levens)belang
om de klinische, biochemische en moleculaire aspecten van dit soort stofwisselingsziekten goed te
begrijpen waardoor deze opgespoord en behandeld kunnen worden. Bovendien kan hierdoor een
reproductieve keuzemogelijkheid geboden worden.
Gelukkig is in de afgelopen decennia een enorme vooruitgang geboekt op het gebied van kennis,
diagnostiek en behandeling van erfelijke stofwisselingsziekten in de afbraak van vertakte keten
aminozuren. Met name de komst van tandem-massa spectrometrie en de inspanningen van een aantal
onderzoekgroepen, waaronder laboratorium Genetische Metabole Ziekten, hebben hieraan een
immense bijdrage geleverd. Echter, van de 15 vertakte keten organische acidurieën waren 12 volledig
opgehelderd. De uitdaging was om de laatste drie enzymdeficiënties – 3-methylglutaconyl-CoA
hydratase deficiëntie, 3-hydroxyisobutyryl-CoA hydrolase deficiëntie en 3-hydroxyisoboteracidurie
– zowel metabool als moleculair in kaart te brengen en methoden te onwikkelen waarmee deze
erfelijke aandoeningen opgespoord kunnen worden. Dit heeft geleid tot de volledige opheldering
van zowel 3-methylglutaconyl-CoA hydratase deficiëntie (hoofdstuk 3) als 3-hydroxyisobutyrylCoA hydrolase deficiëntie (hoofdstuk 7). Tevens zijn voor beide enzymdeficiënties diagnostische
methoden ontwikkeld (hoofdstukken 4 en 7) waarmee nieuwe patiënten zijn gevonden. Bovendien
is een humaan eiwit, ECHDC2, gekarakteriseerd welke sterke overeenkomsten vertoont met 3methylglutaconyl-CoA hydratase. Hoewel, de fysiologische functie hiervan onduidelijk is gebleven
kan het een potentiele therapie zijn voor patiënten met 3-methylglutaconyl-CoA hydratase
deficiëntie (hoofdstuk 5). Tot slot wordt een humaan gen beschreven welke codeert voor het enzym
3-hydroxyisobutyraat dehydrogenase. Algemeen wordt aangenomen dat een defect in dit enzym
leidt tot 3-hydroxyisoboteracidurie. Echter, dit gen lijkt niet defect te zijn in patiënten met 3hydroxyisoboteracidurie (hoofdstuk 6), waarmee de moleculaire basis van deze erfelijke aandoening
voorlopig onopgehelderd blijft.
From the Departments of Pediatrics, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam,
The Netherlands (F.J.L.)
47
Dankwoord
Allereerst wil ik zonder meer Ronald bedanken voor de vrijheid en het vertrouwen die ik al tijdens
mijn studententijd van je kreeg. Ik waardeer het zeer dat je mijn eigenzinnige karakter al die tijd
de ruimte hebt gegeven. Ik hoop dat ik jouw enthousiasme, gedrevenheid en literatuurkennis ooit
zal kunnen evenaren. Het is me een waar genoegen geweest om bij jou te mogen promoveren en ik
hoop van harte dat onze wegen elkaar snel zullen kruizen.
Ries, bedankt voor de fijne samenwerking zowel hier op lab GMZ als op het laboratorium
erfelijke stofwisselingsziekten van het Wilhemina KinderZiekenhuis in Utrecht. Jouw expertise
en ����������������������������������������������������������������������������������������������
patiëntengevoel�������������������������������������������������������������������������������
hebben een grote bijdrage geleverd aan mijn keus om de opleiding tot klinisch
chemicus te volgen.
Uiteraard wil ik graag mijn oprechte dank uitspreken aan mijn drie paranimfen zonder wie dit
boekje zeker niet tot stand was gekomen:
Jos, bedankt voor al jouw hulp, steun, kennis, begeleiding en geduld tijdens mijn worstelpartijen
met die verdraaide spectrofotometers, vooral de COBAS-Fara. Daarbij vergeleken is HPLC is een
eitje, behalve de eerste run. Wat fantastisch dat jij en Mirjam elkaar gevonden hebben. Ik wens jullie
veel liefde en goeds toe.
Lodewijk jouw kennis, kritische suggesties en veeleisendheid hebben mij niet alleen veel geleerd,
maar bovenal gemotiveerd om een wetenschappelijk vervolg te geven aan mijn stage op lab GMZ
met dit proefschrift als mooi resultaat. Gelukkig hebben we dezelfde filosofie wat betreft werken.
Gedoseerd ontspannen! Elke dag was een enorm feest (ik zeg altijd zo: als je een baan hebt waar je
van houdt hoef je nooit een dag te werken) Bedankt voor je hulp en je vriendschap.
Rob, held in eiwit- en antilichamenland. Bedankt voor je kritische ���������������������������
ideeën, praktische hulp en
schuine grappen. Ik ben blij en vereerd dat je mijn paranimf wil zijn.
Mijn studenten Jorrit, Annemarie en Sherien, bedankt voor jullie energieke en enthousiaste
bijdrage aan mijn onderzoek. Ik kijk met plezier terug op een leerzame en leuke periode. Ik hoop dat
jullie, ondanks de soms chaotische en lange dagen, dat ook zo ervaren hebben. Ontzettend bedankt
en ik wens jullie veel succes en goeds.
Een groot dankjewel aan iedereen van laboratorium GMZ. Jullie stonden altijd klaar voor het
beantwoorden van vragen, het geven van ideeën���������������������������������������������������
���������������������������������������������������������
en het verlenen van praktisch hulp. In bijzonder,
dank aan Petra en Patricia zonder wie veel proeven niet starten, Simone voor haar ongezoute en
kritische kanttekeningen, Janet K. voor heel veel (onmogelijk om te rekenen naar geld), Hans
voor de kritische suggesties en hulp bij het schrijven van artikelen, en Maddy voor de �������������
secretariële�
ondersteuning. Alle (ex-)AIO’s - Saskia (ik mis onze treinreisjes), Sietske (de eerste scheikundestapjes zijn KC-stappen geworden), Wouter (druivenplasma bestaat!!), (keukenprinses en snuzzlepuzzle wizzard) Naomi, Merel, Roos, Malika, Jolein, Robert-Jan (op naar nr. 3), Jeannette, Marit,
Linda, Jasper, (mi fratello) Daan, Riekelt, (fietspartner) Mark, Annemieke B., Annemieke de R.,
Pedro, Hidde (wanneer is de volgende fietsproef?), Nadia, Claire, Fred, Sacha, Stephan en Sander
- bedankt voor de gezelligheid, interessante discussies en AIO-etentjes.
Dank aan al mijn vrienden die ik de afgelopen jaren zo verwaarloosd heb. Het wordt tijd voor een
grote inhaalslag.
Tenslotte mijn familie...
Lieve pap, mam en Jermaine bedankt dat jullie er altijd voor me zijn geweest en dat jullie me altijd
hebben gesteund in de keuzes die ik gemaakt heb. Jullie geven me altijd het gevoel dat jullie trots op
me zijn. Bedankt voor alles.
Lieve Marian, jij mag natuurlijk niet ontbreken in mijn dankwoord. Ik ben er zeker van dat het me
zonder jouw onvoorwaardelijke steun en geduld nooit gelukt was. Ik ben blij dat we elkaar gevonden
hebben en dat o.a. Tristan het mooie resultaat daarvan is. Ik hou van jullie beiden.
Ference J. Loupatty
48