Clinical Chemistry 45: 2073-2078, 1999;
(Clinical Chemistry. 1999;45:2073-2078.)
© 1999 American Association for Clinical Chemistry, Inc.
Biochemical and Molecular Genetic Characteristics of the Severe Form of Tyrosine Hydroxylase Deficiency
Christa Bräutigam1,
Gerry C.H. Steenbergen-Spanjers3,
Georg F. Hoffmann1,
Carlo Dionisi-Vici2,
Lambert P.W.J. van den Heuvel3,
Jan A.M. Smeitink3 and
Ron A. Wevers3,a
1
University Hospital, Department of Neuropediatrics and Metabolic Diseases, D-35037 Marburg, Germany.
2
Bambino Gesù Hospital, Department of Metabolism,
I-00165 Rome, Italy.
3
University Hospital Nijmegen, Laboratory of Paediatrics
and Neurology, NL-6500 HB Nijmegen, The Netherlands.
a Address correspondence to this author at: University Hospital Nijmegen, Laboratory of Pediatrics and Neurology, Institute of Neurology, Reinier Postlaan 4, 6525 GC Nijmegen, The Netherlands. Fax 31-24-3540297; e-mail r.wevers{at}ckslkn.azn.nl
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Abstract
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Background: Tyrosine hydroxylase (TH) catalyzes the rate-limiting
step in the biosynthesis of the catecholamines dopamine,
norepinephrine, and epinephrine. Recently, mutations were identified in
cases of autosomal recessive dopa-responsive dystonia and infantile
parkinsonism. We describe a patient with severe symptoms and a new
missense mutation in TH.
Methods: Relevant metabolites in urine and cerebrospinal fluid
were measured by HPLC with fluorometric and electrochemical detection.
All exons of the TH gene were amplified by PCR and
subjected to single-strand conformation polymorphism analysis.
Amplimers displaying aberrant migration patterns were analyzed by DNA
sequence analysis.
Results: The patient presented with severe axial hypotonia,
hypokinesia, reduced facial mimicry, ptosis, and oculogyric crises from
infancy. The major metabolite of dopamine, homovanillic acid, was
undetectable in the patient’s cerebrospinal fluid. A low dose of
L-dopa produced substantial biochemical but limited
clinical improvement. DNA sequencing revealed a homozygous 1076G
T
missense mutation in exon 10 of the TH gene. The
mutation was confirmed with restriction enzyme analysis. It was not
present in 100 control alleles. Secondary structure prediction based on
Chou-Fasman calculations showed an abnormal secondary structure of the
mutant protein.
Conclusions: We describe a new missense mutation (1076GT,
C359F) in the TH gene. The transversion is present in
all known splice variants of the enzyme. It produces more severe
clinical and biochemical manifestations than previously described in
TH-deficient cases. Our findings extend the clinical and the
biochemical phenotype of genetically demonstrated TH deficiency.
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Introduction
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Tyrosine hydroxylase
(TH,1
EC 1.14.16.2) catalyzes the hydroxylation of
L-tyrosine to L-dihydroxyphenylalanine
(L-dopa), the rate-limiting step in the biosynthesis of the
catecholamines dopamine, norepinephrine, and epinephrine (Fig. 1
). The iron-containing mixed function oxidase requires molecular
oxygen and the cofactor tetrahydrobiopterin (BH4)
for activity. TH is expressed mainly in specific brain areas and in the
adrenal medulla (1).
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Figure 1. Metabolic pathway of serotonin and the catecholamines.
AADC, aromatic L-amino acid decarboxylase;
alcd., alcohol dehydrogenase; ALD,
intermediate aldehyde (3-methoxy-4-hydroxyphenyl-hydroxyacetaldehyde);
aldd., aldehyde dehydrogenase; B6,
Vitamin B6; COMT,
catechol-o-methyltransferase, DOPAC,
3,4-dihydroxyphenylacetic acid; DßH,
dopamine-ß-hydroxylase; M, metanephrine;
MAO, monoamine oxidase; 3MT,
3-methoxytyramine; NM, normetanephrine;
PNM,
phenylethanolamine-N-methyltransferase;
TR, tryptophan hydroxylase. Solid
arrows indicate that several steps were involved; dashed
arrows indicate a single step.
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A central role of TH for prenatal development and postnatal survival
was indicated by the nonviability of TH-knockout mice (2).
In humans, secondary impairment of TH enzymatic activity occurs in
defects of BH4 synthesis and recycling, mostly
referred to as variant phenylketonurias. The first indication of
primary genetic TH deficiency (THD) in humans was provided in 1994 by
Clayton et al. (3). To date, four different mutations have
been described in six index cases from unrelated families. In two
siblings, a point mutation in exon 11c.1141CA (Q381K)
(4)(5) and in another girl a point mutation in
exon 5c.614TC (L205P) (3)(6) have been
identified. Recently, a missense mutation in exon 6c.698GA (R233H) a
"common" mutation in The Netherlands and a deletion delC291 in exon
3 could be identified in patients with autosomal recessive
L-dopa-responsive infantile parkinsonism
(7)(8). Patients were described as having
autosomal recessive L-dopa-responsive dystonia,
or Segawa syndrome, or as
L-dopa-responsive parkinsonism in infancy
(4)(5)(6)(7). Clinical symptoms of dystonia, hypokinesia,
rigidity, and truncal hypotonia were reported to develop in early
childhood. All patients showed marked clinical improvement on low doses
of L-dopa together with the decarboxylase
inhibitor carbidopa.
We identified a new mutation in a new case of THD with a very severe
clinical and biochemical picture. The case extends both the biochemical
and the clinical phenotype of the disease.
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Patient and Methods
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case report
The boy was born prematurely (33rd week of gestation) to healthy
consanguineous Italian parents. Severe respiratory distress complicated
the perinatal period. Moderate hypotonia and swallowing difficulties
were present since birth. Marked axial hypotonia, severe hypokinesia,
and reduced facial mimicry increased over the first months of life.
Prolonged diurnal periods of lethargy with increased sweating
alternated with irritability and rare sporadic dystonic movements and
prompted further investigation. The routine clinical chemistry
investigations for neurometabolic disorders and the
electroencephalogram were normal. Magnetic resonance imaging at
5 months of age revealed an unexpected degree of cerebral atrophy. A
diagnosis of THD was suggested on the basis of cerebrospinal fluid
(CSF) investigations of neurotransmitter metabolites, and therapy with
a low dose of L-dopa (6 mg/kg body weight per day) together
with the decarboxylase inhibitor carbidopa was initiated. After 10
months of treatment, there was only partial clinical improvement of
axial tone, appearance of spontaneous movements, and reduced sweating.
The child tolerated only a very gradual increase of medication,
complicated by dose-dependent side effects, mainly hyperkinesia and
irritability.
biochemical investigations
The neurotransmitter metabolites 5-hydroxyindoleacetic acid
(5-HIAA), homovanillic acid (HVA), and
3-methoxy-4-hydroxyphenylglycol (MHPG) in CSF and 5-HIAA, HVA,
and vanillylmandelic acid (VMA) in urine were measured with HPLC and
electrochemical detection, and the metabolites
3-o-methyldopa and L-dopa in
CSF and dopamine, epinephrine, and norepinephrine in urine were
measured with HPLC and fluorometric detection. The CSF samples were
collected according to a standardized protocol for lumbar puncture
(9). The catecholamines were measured in an acidified 24-h
urine. The analytical techniques used for the biochemical
investigations recently have been described in detail (9).
mutation detection studies
Genomic DNA was extracted from leukocytes by standard methods. All
exons of the TH gene were amplified by PCR. The
amplimers obtained were subjected to single-strand confirmation
polymorphism analysis by the Pharmacia Phast System. Running
conditions for exon 10 were as follows: 12.5% polyacrylamide gel,
20 °C, 400 V, 5 mA, and 1 W (prerun at 100 V-h and separation at 135
V-h). The primers used for PCR amplification and sequence analysis of
exon 10 were as follows: forward primer,
5'-GCACTCCCCTGAGCCGTGAG-3'; and reverse primer,
5'-GAGCAGGCAGCACACTTCACC-3'. Cycle sequencing of the coding and
the noncoding strands of exon 10 was carried out by the Taq Dye Deoxy
Terminator method in an ABI DNA sequencer (Applied Biosystems type
377). To confirm the mutation in genomic DNA, the 265-bp amplimers of
exon 10 of the index patient and the parents were digested with the
restriction enzyme ItaI, which spliced the wild-type
allele seven times (fragments of 79, 59, 41, 33, 31, 10, 9, and 3 bp)
and the mutant allele six times (fragments of 79, 59, 51, 33, 31, 9,
and 3 bp).
mutation nomenclature
The reports by of Lüdecke and co-workers
(4)(6)(10) and Knapskogg et
al. (5) have used a nomenclature strategy based on human
mRNA type 1. We have used a nomenclature strategy for indicating
TH mutations based on human mRNA type 4 as published by
Nagatsu et al. (11). In the human mRNA type 1, a part of
exon 1 and the full-length exon 2 are missing (11). This has
consequences for the numbering of the exons, nucleotides, and amino
acids. A table with the known mutations in the TH gene
comparing both nomenclature strategies has been published
(8).
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Results
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biochemical investigations
Routine clinical investigations and investigations for
neurometabolic disorders in our patient, including organic acids in
urine and amino acids in urine, blood, and CSF, were all normal. Pterin
concentrations in the urine were within the reference interval.
Biopterin in the CSF was borderline increased together with a low
normal dihydropteridine reductase activity in blood (Dr. N. Blau,
Zurich, Switzerland), excluding a defect in BH4
biosynthesis.
Analysis of the CSF revealed a severe impairment of dopamine
biosynthesis with undetectable HVA (lower limit of detection, 5 nmol/L)
and a very low MHPG concentration (6% of the lower reference range;
Table 1
). The concentrations of 5-HIAA, the end product of the
serotonin pathway, and 3-o-methyldopa in CSF as well as the
urinary excretion of vanillyllactic acid, a metabolite of
L-dopa, were within the appropriate reference
intervals, which excluded aromatic L-amino
acid decarboxylase deficiency and pointed to THD as the primary defect
(12).
Urine analysis (Table 1
) revealed a decreased concentration of HVA (6%
of the lower reference range). The concentration of 5-HIAA deriving
from serotonin and of VMA as the main metabolite of norepinephrine in
the periphery were within the reference ranges. The concentrations of
dopamine and epinephrine were in the lower reference range, and only
the concentration of norepinephrine was decreased (15% of the lower
reference range). The ratio of epinephrine to norepinephrine was
increased (4.8; reference, <1). The excretion of the free
metanephrines was very low; however, total normetanephrine and
metanephrine were within the reference range.
After treatment with increasing doses of L-dopa (up to 6
mg/kg body weight per day) with decarboxylase inhibitor, the HVA
concentration in the CSF increased but remained far below the reference
range (32% of the lower reference range). In urine, the treatment led
to an increase of HVA (48% of the lower reference range). The
catecholamines norepinephrine and epinephrine and the ratio
epinephrine/norepinephrine normalized, and dopamine increased (371%
above the upper reference range) after treatment with
L-dopa. Higher doses of L-dopa led to severe
adverse clinical symptoms of irritability and violent, abrupt
alternating flinging of the arms (ballism).
molecular analysis
Single-strand confirmation polymorphism analysis was carried out
on all exons of the TH gene under at least two different
conditions (temperature and gel type). Only exon 10 displayed an
aberrant migration pattern in the patient (Fig. 2
A) and in both parents (not shown). Direct sequencing revealed
that the patient has a novel, homozygous missense mutation, 1076GT
(Fig. 2B
). Both parents were heterozygous for this mutation. The
finding of a homozygous mutation is in line with parental
consanguinity. This transversion produces an amino acid exchange from
cysteine to phenylalanine at codon 359 (C359F). The mutation abolishes
an ItaI restriction site, producing a 51-bp fragment in the
patient DNA instead of the 41- and 10-bp fragments in wild-type DNA
(Fig. 3
). The mutation was not found in 100 control alleles. In
addition, the patient is homozygous for the common polymorphism V112M
(10).
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Figure 2. Single-strand confirmation polymorphism analysis of the
amplimers of exon 10 (A) and DNA sequence
(B) of the region of the coding strand of exon 10 in
which the mutation (arrow) was found.
(A), lane 1, patient; lanes 2 and
3, controls. (B), top,
wild type; bottom, patient.
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Figure 3. Electrophoresis on a 20% polyacrylamide gel of the
amplimers of exon 10.
Lane 1, control before digestion with
ItaI; lanes 2–5, control, father,
mother, and patient amplimers, respectively, after digestion with
ItaI. Numbers on right indicate base
pairs.
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Secondary structure prediction according to Chou and Fasman
(13) and Garnier et al. (14) predicted an extra
turn in the secondary structure of the mutant protein (not shown). The
mutation is present in all different splice variants known to be
present for human TH (15). Human TH has seven cysteine
residues. Six of the seven cysteine residues are conserved in other
species (rat, bovine, and quail) (11), including the
cysteine residue in our point mutation. The cysteine residues are
located in the carboxy-terminal half of the enzyme where the catalytic
domain is situated (11). The 20 amino acids around cysteine
359 are highly (90–100%) conserved among species (rat, bovine, and
quail; Fig. 4
). The mutant cysteine residue is one of five cysteine residues
also conserved in the other human aromatic amino acid hydroxylases,
tryptophan hydroxylase and phenylalanine hydroxylase (Fig. 4
). The
percentage of homology of the 20 amino acids surrounding this cysteine
residue is 71% for human phenylalanine hydroxylase and 67% for human
tryptophan hydroxylase. This part of the protein and the cysteine
residue therefore seem pivotal for enzymatic function of aromatic amino
acid hydroxylases.
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Figure 4. Comparison of the structures of the patient’s TH;
wild-type human TH; rat, bovine, and quail TH; human
phenylalanine hydroxylase (PAH); and human tryptophan
hydroxylase (TPH).
The mutation site C359F (C329F based on human mRNA type 1) is indicated
by the box. Amino acids of rat, bovine, and quail TH;
the patient’s TH; and human PAH identical with those of human
wild-type TH are indicated by dashes.
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Discussion
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THD has hitherto been described as a rare cause of autosomal
recessive dopa-responsive dystonia or L-dopa-responsive
infantile parkinsonism (4)(5)(7).
The diagnosis was suspected in our patient because the concentration of
HVA in the CSF was undetectable (<5 nmol/L) at the time of diagnosis.
In previously described patients, HVA in the CSF was between 8% and
30% of the lower reference range (9) or 5% of the lower
reference range (6). The biochemical results in the CSF
together with the clinical picture indicated a severe deficiency of TH.
The point mutation 1076GT in the TH gene probably has a
profound effect on the catalytic activity of the enzyme, and when
present in the homozygous form, it does not seem to allow substantial
residual enzymatic activity. The 1076GT transversion produced an
amino acid change from cysteine to phenylalanine at codon 359 mRNA type
4 (codon 329 in mRNA type 1). TH is composed of two functional
domains, i.e., a catalytic domain, which is located proximal to the
C-terminal region, and a regulatory domain, which is located at the
NH2 terminus. The catalytic domain of the enzyme
contains residues 188–456, and any truncation within these residues
produces a protein that expresses extremely poorly and has no
detectable activity (16). The six cysteine residues that are
conserved in their positions in other species are located in the
C-terminal half and form a catalytic domain. In addition, the amino
acids around them are highly conserved among species. This suggests
that these cysteine residues may play an important role in enzymatic
function by keeping the conformation of the protein by means of intra-
or intermolecular S–S bonds and/or by interacting with ferrous
ion, which is an essential component for the catalytic action of TH
(11). The C359F mutation seems to be the most severe
disease-causing mutation described to date in THD.
Our findings extend the biochemical phenotype of THD. Because HVA was
undetectable in the CSF of our patient and MHPG was very low, both
dopamine and norepinephrine biosynthesis are severely impaired in our
patient. There seems to be hardly any flux through the catecholamine
biosynthesis pathway in the brain. In all earlier studies on patients
in whom a defect in TH was genetically confirmed, there seemed to be
some degree of residual TH activity. As evidenced by the CSF HVA
concentrations in these patients, they all had the capability of
synthesizing catecholamines to some extent. This also may explain the
limited beneficial reaction to L-dopa in our patient. The
side effects of very low L-dopa doses may be explained by
receptor up-regulation. Iolopride-Spect scanning, however, did not show
evidence for dopamine D2 receptor up-regulation.
The biochemical findings in our patient are in line with the obviously
very severe clinical signs and symptoms that include structural
abnormalities in the brain as observed in the magnetic resonance
imaging. Therefore, our patient also extends the clinical phenotype of
THD. THD in most cases reacts favorably to low-dose L-dopa
therapy and is considered a treatable disease. Therefore, it is
important to know the various possible clinical presentations of the
disease. The present case illustrates that THD should be considered in
all children with severe encephalopathy, especially when dominated by
extrapyramidal signs and hypokinesia even when there are magnetic
resonance imaging abnormalities in the central nervous system.
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Acknowledgments
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We thank Dr. S. Mentzel (University Nijmegen, Department of
Pathology, Nijmegen, The Netherlands) for the Chou-Fasman calculation,
Dr. N. Blau (University Children’s Hospital Zürich, Clinical
Chemistry and Biochemistry, Zürich, Switzerland) for pterin
measurements in CSF and measurement of the dihydropteridine reductase
activity, Dr. R. Duran (University Children’s Hospital, Utrecht, The
Netherlands) for measurements of the urinary pterins, and Dr. N.
Abeling (Academic Medical Centre Amsterdam, Clinical Chemistry and
Paediatrics, Amsterdam, The Netherlands) for metanephrine measurements
in urine.
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Footnotes
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1 Nonstandard abbreviations: TH, tyrosine hydroxylase; L-dopa, L-dihydroxyphenylalanine; BH4, tetrahydrobiopterin; THD, tyrosine hydroxylase deficiency; CSF, cerebrospinal fluid; 5-HIAA, 5-hydroxyindoleacetic acid; HVA, homovanillic acid; MHPG, 3-methoxy-4-hydroxy-phenylglycol; and VMA, vanillylmandelic acid. 
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Abstract
Introduction
