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Molecular Diagnostics and Genetics |
1
Department of Neuropaediatrics and Metabolic Diseases, University Hospital, D-35037 Marburg, Germany.
2
Department of Neurology, University Hospital Nijmegen,
6525 GC Nijmegen, The Netherlands.
3
Department of Neurology, Ignatius Hospital, 4800RK
Breda, The Netherlands.
a Address correspondence to this author at: University Hospital Nijmegen, Laboratory of Pediatrics and 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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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: L-dopa, L-dihydroxyphenylalanine • TH, tyrosine hydroxylase • HVA, homovanillic acid • CNS, central nervous system • VMA, vanillylmandelic acid • CSF, cerebrospinal fluid • MHPG, 3-methoxy-4-hydroxyphenylethyleneglycol • 5-HIAA, 5-hydroxyindolacetic acid • DRD, dopa-responsive dystonia • 3-OMD, 3-o-methyldopa • carbidopa, (S)-2-(3,4-dihydroxybenzyl)-2-hydrazinpropionic acid.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The major catabolic product in the central nervous system
(CNS) from norepinephrine is 3-methoxy-4-hydroxy-phenylethyleneglycol
(MHPG) (2) , whereas vanillylmandelic acid (VMA) is the major
norepinephrine catabolite outside the CNS.
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TH deficiency can cause the autosomal recessive form of dopa responsive dystonia (DRD); in its clinical form, it is also known as Segawa's disease. The autosomal dominant form of DRD results from a mutation in the GTP I cyclohydrolase gene (3) . Clayton et al. (4) were the first to suggest TH deficiency for the recessive form of DRD. The TH gene on chromosome 11p15.5 was sequenced by Lüdecke et al. (5) . A point mutation (Q381K) in exon 11 (5)(6) and a point mutation (L205P) in exon 5 (7) were found as disease-causing mutations in the two patients with TH deficiency described thus far.
In this study, we report the diagnostic methodology and the biochemical hallmarks of TH deficiency in the CNS. Four unrelated families with the characteristic clinical signs and symptoms of recessive Segawa's disease are described. The patients share a point mutation (R233H) in exon 6 in the TH gene recently identified by van den Heuvel et al. (8) . Three of our patients are homozygous for the R233H mutation, and one is compound heterozygous for this mutation and for another mutation in exon 3 of this gene. Specific biochemical analyses are compatible with a markedly reduced biosynthesis of dopamine in the CNS because of a deficient function of the TH enzyme system and may enable the diagnosis of additional patients worldwide.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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csf and urine
By using a standardized protocol, lumbar CSF was collected between
0830 and 1200 and stored at -70 °C until analyzed. At the age of
0–1 year, the first 2 mL were used for HVA, 5-HIAA, and MHPG
determination. At the age of 2–12 years, the first 5 mL of CSF were
used for routine investigations; subsequently, the 3 mL were used for
HVA, 5-HIAA, and MHPG determination. At the age >12 years, the first 8
mL were for routine CSF investigations, and the subsequent 3 mL were
used for measurement of neurotransmitter metabolites. By using the same
standardized protocol, reference values were established on CSF left
over from routine investigations. Retrospectively, these patients were
classified as suitable reference subjects because of lack of evidence
for endocrine or metabolic abnormalities. Patients with epilepsy or
extrapyramidal signs and symptoms were excluded. Appropriate studies
ruled out immunological or chronic infectious diseases, deficiencies,
and disorders caused by toxic agents. Samples containing red blood
cells were eliminated. The study was approved by the ethical committee
of the University of Marburg. CSF samples for the investigations of the
gradient in the CSF column were obtained with informed consent of the
patients involved.
Twenty-four-hour urine was collected into 5 mL of 3 mol/L hydrochloric acid and stored at -20 °C. Reference intervals for urine were obtained from children without metabolic disease, neuroblastoma, or phaochromocytoma.
procedures
(a) Measurement of HVA and 5-HIAA in CSF and urine.
We adapted
the method of Westerink and Mulder (9) for determination of
HVA and 5-HIAA in CSF and urine with the following modifications: to 1
mL of CSF samples, 20 µL of 12.6 mol/L formic acid was added, and the
pH ± 2.5 was controlled. The urine samples were centrifuged (10
min, 3000 rpm), and to 50 µL of supernatant, 5 mL Millipore water and
20 µL of 12.6 mol/L formic acid were added, and the pH ± 2.5
was controlled. Five hundred microliters of the CSF sample or urine
sample were applied to the Sephadex G-10 column. After washing the
Sephadex G-10 column successively with 3.5 mL of 25 mmol/L formic acid
and 1.5 mL of 0.2 mol/L phosphate solution, the metabolites were eluted
with 2 mL of 34 mmol/L ammonia in a tube containing 50 µL of 12.6
mol/L formic acid and 50 µL of 2.3 mmol/L ascorbic acid. HPLC was
performed using a mobile phase of 0.2 mol/L phosphate solution and 0.1
mol/L citric acid (30:70, by volume), pH 3.5, and 350 mL of methanol; a
Spectra-Physics SP 8800 gradient pump; a Spectra-Physics SP 8880
autosampler and a SP 8760 autosampler cooler (15 °C); and a
15-cm x 4.6-mm (i.d.) Nucleosil 5-µm RP-18 column. The flow
rate was 1.0 mL/min. One hundred microliters of the eluate were
injected into the system, and detection was by a Spark Holland
amperometric detector with the analytical electrode set at +0.64 V,
range x 1, offset x 0.1, and 50 nA full scale. Detector
output was integrated by using the PC 1000 software system, Ver. 3.01
(Thermo Separations).
(b) Measurement of MHPG in CSF.
We used the same method as
described above for HVA and 5-HIAA with the following modifications.
After the washing step of the Sephadex G-10 column, MHPG was eluted
with 2.0 mL of 25 mmol/L formic acid and 0.5 mL of 0.2 mol/L phosphate
solution. Chromatography was performed with a mobile phase of 6.25
mmol/L phosphate buffer, pH 4.0, containing, per liter, 6 mmol of
citric acid, 6 mmol of sodium chloride, 7 mmol of sodium perchlorate, 1
mmol of octyl sodium sulfate, 0.2 mmol of sodium EDTA, and 5 mL of
methanol; a Spectra-Physics SP8810 isocratic pump; a Rheodyne 7125
injector; and a 25-cm x 4.6-mm (i.d.) Nucleosil 5-µm C18
column. The flow rate was 0.6 mL/min. Fifty microliters of the eluate
were injected into the system, and detection was by an electrochemical
detector Decade (Antec) with the analytical cell set at +0.70 V,
range x 2, and damping 5. Detector output was integrated using
the PC 1000 software system, Ver. 3.01 (Thermo Separations).
(c) Measurement of L-dopa and 3-OMD in CSF.
L-dopa and 3-OMD were measured by HPLC on a 25-cm
x 4.6-mm (i.d.) Progidy 5-m 5 ODS-2 column in combination with
fluorescence detection (excitation, 278 nm; emission, 325 nm) as
described by Hyland (10) .
(d) Measurement of VMA in urine.
VMA in urine was determined
by HPLC on a Bio-Rad RP-ODS 5 column in combination with
electrochemical detection after sample preparation using a commercially
available kit (Bio-Rad, no. 1955001).
(e) Measurement of dopamine, norepinephrine, and epinephrine in
urine.
After extraction of the catecholamines with cation exchange
columns (Bio-Rad, no. 189-2202), we included an additional cleaning
step on a Sephadex G-10 column. Forty microliters of 86.1 mol/L
perchloric acid were added to 2 mL of the boric acid eluate, and after
mixing 1 mL of this solution, it was applied to a Sephadex G-10 column.
The column was washed with 2.5 mL of 25 mmol/L formic acid, and the
catecholamines were eluted with 2.5 mL of 25 mmol/L formic acid. The
catecholamine concentrations were determined with HPLC on a 15-cm
x 4.6-mm (i.d.) Supelcosil 5-µm C18 column with electrochemical
detection. The system was calibrated by injecting 50 µL of eluate
after cation exchange and by cleaning on Sephadex G-10 with a solution
containing, per liter, 200 nmol of dopamine, norepinephrine, and
epinephrine and 200 nmol of dihydroxybenzylamine (internal standard) in
4 mmol/L formic acid.
cases
Our patients came from four unrelated families from different
parts of the Netherlands. The parents were all healthy. All patients
presented with signs and symptoms characteristic of the recessive form
of DRD. A detailed clinical description will be published elsewhere. In
short, after normal pregnancy and delivery, progressive severe motor
retardation with predominant extrapyramidal symptoms first became
obvious at ages between 3 and 7 months, whereas psychosocial
development appeared relatively healthy. The children appeared
hypokinetic with mask face, rigidity of arms and legs, and axial
hypotonia. No diurnal fluctuation in the symptoms was observed. Routine
clinical chemistry, electroencephalogram, and magnetic resonance
imaging and computed tomography neuroimaging were within normal
reference intervals in all cases. After establishing the diagnosis of
TH deficiency and starting therapy with L-dopa together
with (S)-2-(3,4-dihydroxybenzyl)-2-hydrazinpropionic acid
(carbidopa) there was a clear improvement of symptoms. In all cases, a
G698A transition was found by direct sequencing of exon 6 of the TH
gene. This transition produces an amino acid change from arginine to
histidine (R233H) (8) . Patient I was heterozygous for the
G698A mutation and also for a one-base deletion in exon 3 of the TH
gene. The other three were homozygous for the G698A mutation. CSF and
urine of the children were investigated before any medication was
initiated and also during therapy with L-dopa.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows our age-related reference values of the CSF metabolites
HVA, 5-HIAA, HVA/5-HIAA ratio, and MHPG. All CSF samples included in
this reference range study were obtained with the standardized protocol
described in Materials and Methods. For the first 2 years of
life, there were only relatively few data points available that could
be included in the statistical calculation. There was a marked effect
of age on CSF metabolite concentrations. The median concentration of
free MHPG decreases rapidly in the first year of life. In later life,
the reference range for MHPG does not change substantially any more.
The metabolites HVA and 5-HIAA decrease rapidly in early neonatal life
and decrease steadily until they reach adult concentrations at the age
of 15 years. The HVA/5-HIAA ratio increases from birth up to the age of
12 years. Lower values for this ratio are found in adults.
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the rostro caudal gradient
To investigate a concentration gradient in CSF for the metabolites
HVA, 5-HIAA, and MHPG in CSF from different levels of the
spinocisternal system, consecutive CSF fractions from adults without
deficiency in the neurotransmitter metabolism were taken and analyzed.
There was a steep gradient for HVA and 5-HIAA in spinal CSF of two
adult patients, with an increase of 60% in HVA concentrations and of
95% in 5-HIAA concentrations in the first two CSF fractions. Fig. 2
gives a representative example. From the first fraction to the
last CSF fraction (35 mL), there was an increase of 124% for HVA and
173% for 5-HIAA. The HVA/5-HIAA ratio varied between 1.03 and 1.48 in
the various fractions. The metabolite MHPG was unaffected by the volume
fraction (Fig. 2
).
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the stability of the metabolites in CSF
To investigate the stability of the monoamine metabolites in CSF,
we took fresh CSF samples from two adult patients without
neurometabolic disease. One portion of the CSF was immediately stored
at -70 °C. Separate volumes of the sample of the first patient were
left at room temperature (25 °C) for 1.5, 4, 20, 27, and 51 h
and of the second patient for 1.5, 3, 4, 6, 23, and 27 h and
subsequently stored at -70 °C.
The CSF HVA, 5-HIAA, and MHPG concentrations of the first patient were stable at room temperature (25 °C) for at least 27 h (HVA: mean 303 nmol/L, SD 10; 5-HIAA: mean 129 nmol/L, SD 6; MHPG: mean 47 nmol/L, SD 2; HVA/5-HIAA ratio: mean 2.4, SD 0.1) after CSF collection without any addition of antioxidants or buffers in the analyzed samples. After 51 h, the HVA concentration increased >2 SDs. In the second CSF sample, the metabolites were also found to be stable during 27 h at room temperature.
pretreatment metabolite concentrations in body fluids
Clinical chemical routine investigations were within normal
reference intervals in all patients, as were metabolic investigations
including organic acids in urine and amino acids in urine, blood, and
CSF. Phenylalanine was within normal reference intervals in all body
fluids investigated. Tyrosine as substrate of tyrosine hydroxylase was
in the normal reference interval in urine, plasma, and CSF [for CSF,
patient I: 14 µmol/L (reference, 6–19 µmol/L); patient II, 13
µmol/L (reference, 6–19 µmol/L); patient III, 10 µmol/L
(reference, 5–13 µmol/L); and patient IV, 12 µmol/L (reference,
5–13 µmol/L] per method according to Gerrits et al.
(11) . The pterin concentrations in CSF and urine (biopterin,
neopterin, and their ratio) were all completely within the normal
reference interval. Determinations of the dihydropteridine reductase
activity in blood showed results within the normal reference intervals
in all four patients (data not given). These findings exclude a defect
of tetrahydrobiopterin biosynthesis or recycling.
Fig. 3
shows a characteristic chromatogram of the CSF metabolites HVA
and 5-HIAA of patient III and of a control subject. In four patients
with TH deficiency, analyses of the CSF revealed low concentrations of
the dopamine metabolite HVA and the norepinephrine metabolite MHPG,
whereas the serotonin metabolite 5-HIAA was always in the normal
reference interval (Table 2
). The HVA/5-HIAA ratio in CSF was abnormally low in all
patients. These results were confirmed in repeat CSF samples from all
four patients (data not shown).
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The CSF concentration of HVA ranged from 8% of the age-related lower
reference range limit (percentile, 2.5) in patient IV to 30% in
patient I. The CSF concentration of MHPG ranged from 6% to 37% of the
lower limit of the age-related reference range (percentile, 2.5).
Concentrations of CSF L-dopa were not detectable in
all patients (reference, 0–10 nmol/L). Similarly, 3-OMD, a metabolite
deriving from L-dopa (Fig. 1
), was not detectable in all
patients (reference, 0–50 nmol/L), and the concentration of
vanillactic acid in urine was not increased, thus excluding aromatic
L-amino acid decarboxylase deficiency.
Pretreatment urinary HVA (Table 3
) was found decreased in three patients and within normal
reference intervals in patient IV. The VMA concentration was decreased
in all four cases. 5-HIAA concentrations in all samples were within the
normal reference interval. Remarkable were the values within the normal
reference intervals of the catecholamines norepinephrine, dopamine, and
epinephrine in most of the urine samples, except the urine of patient
III, which had decreased values of norepinephrine and dopamine. In all
four cases, the epinephrine/norepinephrine ratio was increased (1.0 to
5.4; reference, <1). Unexpectedly, epinephrine was repeatedly found
increased in the urine of patient I (about 255% above the upper limit
of the age-related reference range). The concentrations of
normetanephrine and metanephrine in two different urine samples of
patient I were also measured: the normetanephrine concentrations were
within the normal reference intervals, and the metanephrine
concentrations were increased (about 38% to 257% above the upper
limit of the reference range in the two samples).
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metabolite concentrations during treatment in body fluids
In our patients, we initially used low dose
L-dopa of about 3 mg/kg/day, together with the
decarboxylase inhibitor carbidopa, of 0.75 mg/kg/day to block
peripheral conversion of L-dopa into dopamine. The
medication was given in three divided doses per day. This resulted in
an immediate clinical improvement.
In some patients, additional clinical improvements could be achieved by
further increasing the daily L-dopa dose after some
time. Table 4
gives the neurotransmitter metabolites in CSF under treatment.
Both HVA, MHPG, and the HVA/5HIAA ratio in CSF increased on treatment
with 2.9–4.4 mg/kg/day of L-dopa but remained below the
age-related reference range (compared with the lower reference range
limit of percentile 2.5: HVA, 35–51%; MHPG, 42–51%; and HVA/5-HIAA,
28–61%; Table 4
). When higher doses of L-dopa (5–6.8
mg/kg/day) together with carbidopa were given, a further increase of
CSF neurotransmitter metabolites could be observed (Table 4
). However,
their concentrations did not normalize (compared with the lower
reference range limit of percentile 2.5: HVA, 48–58%; MHPG, 51–94%;
and HVA/5-HIAA, 44–84%). The CSF concentrations of L-dopa
and 3-OMD were 1 to 55 times higher than the upper limit of the
reference range in different samples (Table 4
).
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Using the treatment with the low dose of L-dopa, we also analyzed the catecholamines and metabolites in the urine of the patients and found a similar picture in all samples. As expected, we found high concentrations of dopamine (6 to 25 times higher than the upper limit) and HVA (within normal reference intervals to 4 times higher than the upper limit) and values within normal reference intervals for 5-HIAA, VMA, norepinephrine, and epinephrine. Without exception, the ratio of epinephrine/norepinephrine reached values within the normal reference intervals. In patient I, the increased concentration of epinephrine that was observed in two samples before treatment also fully reached values within normal reference intervals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In CSF samples of two adult patients, we found the neurotransmitter metabolites HVA, 5-HIAA, and MHPG stable for at least 27 h at room temperature, in agreement with an earlier publication (18) . In contrast, pterin concentrations in CSF are unstable at room temperature and are also light sensitive (13) . Because pterin analysis is important in the differential diagnosis of genetic defects of biogenic monoamine metabolism, CSF samples for all of these investigations have to be kept in the dark and stored immediately at -70 °C.
When interpreting concentrations of conjugated catecholamines in urine, it is important to consider dietary influences. Particular caution is necessary with food known to contain biogenic amines, i.e., bananas (19)(20) . An influence of diet on CSF catecholamine metabolites has not yet been fully investigated.
TH is mainly expressed in brain and adrenal medulla. Therefore, direct measurement of TH enzymatic activity in tissue samples is not a diagnostic option, and final proof can only be obtained by molecular genetic analysis. Until now, only two patients have been described in the literature with DNA-confirmed defects in TH (6)(7) . Our experience suggests that TH deficiency is a rare but widely underdiagnosed inborn error of metabolism.
diagnostic hallmarks of th deficiency
TH deficiency leads to depressed concentrations of
L-dopa and consecutively to low concentrations of the
catecholamines dopamine, norepinephrine, and epinephrine. The
measurement of phenylalanine and tyrosine in body fluids does not
provide any clues to the diagnosis of TH deficiency. Furthermore,
urinary measurements of VMA, HVA, and catecholamines can give values
within normal reference intervals and lead to erroneous
interpretations. The ratio of urinary epinephrine/norepinephrine may
prove to be a useful indicator. As yet we conclude that the biochemical
diagnosis of TH deficiency can only be made reliably by measuring
neurotransmitter metabolites in CSF. Reduced dopamine synthesis leads
to decreased CSF concentrations of HVA and MHPG. Together with
unaffected pterin and CSF tyrosine and 5-HIAA concentrations, these
findings are the diagnostic hallmarks of isolated TH deficiency.
relation between mutation and csf neurotransmitter metabolites
For all four patients, the CSF HVA concentration ranged between
8% and 30% of the lower reference range limit, whereas MHPG ranged
from 6% to 37%. The patient (I) with the highest CSF HVA
concentration is heterozygous for the G698A mutation in exon 6 of the
TH gene and for another mutation in exon 3 of the TH gene. The other
three patients are homozygous for the same mutation. Different
mutations in the gene may produce a variable residual enzyme activity
and therefore various CSF HVA and MHPG concentrations between patients.
The different mutations may give rise to a wider clinical and
biochemical spectrum.
therapy
There was a marked improvement of all clinical signs and symptoms
under treatment with low doses of L-dopa/carbidopa.
HVA concentrations in CSF increased substantially but still were below
the lower reference range limit. After increasing the dose (Table 4
),
an additional clinical improvement could be achieved corresponding to
an additional increase in HVA concentrations in CSF but still below the
lower limit of the reference range. In earlier publications
(4)(7) , higher doses of L-dopa (up
to 10 mg L-dopa/kg/day) were given, and a healthy HVA
concentration in CSF could be achieved. However, some of our patients
responded with hyperkinesia to increasing doses. Also, it should be
considered that adverse effects may occur in the long run because the
L-dopa therapy has to be continued life-long. Therefore,
the therapy strategy in each patient has to find a balance between the
short-term beneficial aspects and the longer term side effects of the
therapy.
urine analysis
The biochemical picture in urine warrants additional comments. Low
to healthy HVA and VMA concentrations, together with healthy 5-HIAA
concentrations, occur in all samples. The catecholamines are in the
normal reference interval in many urine samples of our patients.
Because TH in the adrenals and in the CNS derives from the same gene,
TH in the adrenals is expected to be equally deficient in the patients.
Therefore, the often healthy urinary catecholamine concentrations and
the lack of clinical evidence for adrenal malfunction are
ill-understood. Dietary influences may play a role in the healthy
urinary concentrations of the catecholamines
(19)(20) . We have no explanation for the
increased concentration of epinephrine in patient I in different urine
samples and the disturbed epinephrine/norepinephrine ratio in all four
cases. The high epinephrine concentration in the urine of patient I was
also found in another laboratory using an independent technique.
Because we also found high concentrations of metanephrine in the urine
of the patient, these findings are unlikely to be artifacts. There has
been speculation in the literature about alternative pathways in
catecholamine metabolism (1)(21) . Attempts have
been made to find alternative pathways in vivo and to determine whether
they are functionally important. Biosynthetic pathways from
L-tyrosine to norepinephrine and epinephrine are possible
via p-tyramine, octopamine, and synephrine
(1)(21) . Such alternative pathways of
epinephrine and norepinephrine synthesis may become important in
TH-deficient patients.
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Acknowledgments |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  K. Hyland Clinical Utility of Monoamine Neurotransmitter Metabolite Analysis in Cerebrospinal Fluid Clin. Chem., April 1, 2008; 54(4): 633 - 641. [Abstract] [Full Text] [PDF]
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 W. F. Abdo, B.P.C. van de Warrenburg, M. Munneke, W. J.A. van Geel, B. R. Bloem, H. P.H. Kremer, and M. M. Verbeek CSF analysis differentiates multiple-system atrophy from idiopathic late-onset cerebellar ataxia Neurology, August 8, 2006; 67(3): 474 - 479. [Abstract] [Full Text] [PDF]
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 J L K Van Hove, J Steyaert, G Matthijs, E Legius, P Theys, R Wevers, A Romstad, L B Moller, K Hedrich, D Goriounov, et al. Expanded motor and psychiatric phenotype in autosomal dominant Segawa syndrome due to GTP cyclohydrolase deficiency J. Neurol. Neurosurg. Psychiatry, January 1, 2006; 77(1): 18 - 23. [Abstract] [Full Text] [PDF]
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 R. P. Carson and D. Robertson Genetic Manipulation of Noradrenergic Neurons J. Pharmacol. Exp. Ther., May 1, 2002; 301(2): 410 - 417. [Abstract] [Full Text] [PDF]
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 H. Hahn, M. R. Trant, M. J. Brownstein, R. A. Harper, S. Milstien, and I. J. Butler Neurologic and Psychiatric Manifestations in a Family With a Mutation in Exon 2 of the Guanosine Triphosphate-Cyclohydrolase Gene Arch Neurol, May 1, 2001; 58(5): 749 - 755. [Abstract] [Full Text] [PDF]
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J. F. de Rijk-van Andel, F. J.M. Gabreels, B. Geurtz;, G. C.H. Steenbergen-Spanjers;, L. P.W.J. van den Heuvel, J. A.M. Smeitink, and R. A. Wevers L-dopa-responsive infantile hypokinetic rigid parkinsonism due to tyrosine hydroxylase deficiency Neurology, December 26, 2000; 55(12): 1926 - 1928. [Abstract] [Full Text] [PDF]
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C. Brautigam, G. C.H. Steenbergen-Spanjers, G. F. Hoffmann, C. Dionisi-Vici, L. P.W.J. van den Heuvel, J. A.M. Smeitink, and R. A. Wevers Biochemical and Molecular Genetic Characteristics of the Severe Form of Tyrosine Hydroxylase Deficiency Clin. Chem., December 1, 1999; 45(12): 2073 - 2078. [Abstract] [Full Text] [PDF]
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, HVA concentrations; 
, 5-HIAA concentrations; 
, MHPG
concentrations; x, HVA/5-HIAA ratio x 10.