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Cerebrospinal fluid biogenic amines and biopterin in rett syndrome.

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Cerebrospinal Fluid Biogenic Armnes
and Biopterin in Rett Syndrome
Huda Y . Zoghbi, MD," Sheldon Milstien, PhD,S Ian J. Butler, MD,I E. OBrian Smith, PhD,"
Seymour Kaufman, PhD,S Daniel G. Glaze, MD," and Alan K. Percy, MD"
We evaluated cerebrospinal fluid biogenic amine metabolites in 32 patients with Rett syndrome. Significant reductions
were noted in the metabolites of norepinephrine, dopamine, and serotonin, as compared with controls of similar age ( p
< 0.001, p < 0.001, and p < 0.05, respectively). Tetrahydrobiopterin is an essential cofactor and may be rate-limiting
for the synthesis of catecholamines and serotonin. Accordingly, total cerebrospinal fluid biopterin was measured and
found to be elevated in patients compared with controls ( p < 0.002). These findings constitute the only biochemical
changes detected in Rett syndrome so far.
Zoghbi HY, Milstien S, Butler IJ, Smith EO, Kaufman S, Glaze DG, Percy AK. Cerebrospinal fluid
biogenic amines and biopterin in Rett syndrome. Ann Neurol 1989;25:56-60
Rett syndrome, which is seen in female patients only,
is characterized by cessation of normal development in
early infancy followed by regression, loss of communication, and acquisition of abnormal stereotypic hand
movements. Other features include ataxia, acquired
microcephaly, intermittent hyperventilation, and seizures [ 1-51. The pathophysiological process remains
unknown. Studies of cerebrospinal fluid (CSF)
biogenic amine neurotransmitter metabolites in 6
patients with Rett syndrome revealed a significant reduction in homovanillic acid (HVA) and 3-methoxy-4hydroxyphenylethylene glycol (MHPG), the metabolites of dopamine and norepinephrine, respectively
C61. These results led to the present study of CSF
biogenic amine metabolites and biopterin levels in a
larger group of patients with Rett syndrome. CSF biopterin levels were measured because tetrahydrobiopterin is the essential cofactor for hydroxylation of
tyrosine to L-dopa and tryptophan co 5-hydroxyuyptophan, the rate-limiting steps of catecholamine and
serotonin synthesis, respectively C71. Furthermore,
CSF biopterin levels have been reported to be decreased in Parkinson's disease and dystonia [S].
Methods
Thirty-two female patients with suspected Rett syndrome
(mean age 6.7 years; range 1.8-16 years) were evaluated
clinically to confirm the diagnosis of Rett syndrome. Thirty
of the 32 patients fulfilled the criteria for the classical form of
this disease [9}. The remaining 2 patients were considered
From the "Departments of Pediatrics and Neurology, Baylor College of Medicine, and the ?Department of Neurolom,
_. the UniversivOf Texas Health Science Center at Houston, Houston, m, and
the $Laboratory of Neurochemistry, National Institute of Mental
Health, Bethesda, MD.
atypical, based on later onset and slower progression of
neurological signs. Thirty-one patients were in a good state
of nutrition and were maintained on a regular diet. One
patient was severely malnourished. Sixteen patients were
taking anticonvulsants: 6, carbamazepine; 4 , valproate; 3,
phenobarbital; 2, phenytoin; 2, acetazolamide; 1, clorazepate; and 1, ethosuximide. None of the patients were taking
any other medication. Serum and urine amino acid analyses
were performed for all patients. Detailed electroencephalographic and polygraphic studies during sleep and wakefulness that included monitoring of respiratory effort were performed on 19 patients.
CSF was obtained from 32 patients by spinal puncture in
the lateral decubitus position. The initial 2 ml of CSF was
collected in a glass tube containing 2 mg/ml ascorbic acid as a
preservative and immediately frozen and stored at - 70°C.
HVA, the metabolite of dopamine; 5-hydroxyindole acetic
acid (5-HIAA),the metabolite of serotonin; and MHPG, the
metabolite of norepinephrine, were assayed by quantitative
gas chromatography-mass spectrometry [lo]. Total CSF
biopterin was measured in 19 patients by reverse-phasehighperformance liquid chromatography [ 111. Two CSF samples
were carefully collected for measurement of tetrahydrobiopterin. One-milliliter aliquots of these two samples
were placed into vials containing dithiothreitol and ethylenediaminetetraacetic acid (1 mg each), immediately frozen
on dry ice, and stored in the dark at - 70°C. The concentration of tetrahydrobiopterin was determined by high-performance liquid chromatography after the sample was subjected
to a differential oxidation procedure that preferentially destroyed only the fully reduced form of biopterin [l2]. Normal values for biogenic amine metabolites were determined
in a previous study in an age-matched control population
Received Jan 12, 1988, and in revised form May 25. Accepted for
Dublication T u l 2. 1988.
Address co~respondenceto Dr Zoghbi, Departmentof pediatrics,
Baylor
of Medicine, One Baylor plaza, Houston, Tx
77030.
56 Copyright 0 1989 by the American Neurological Association
tl3l. Since there are no established normal values for CSF
biopterin levels in children, CSF was collected from a control
population comparable in age to our 19 patients undergoing
CSF biopterin level determination. This control group included 62 patients less than a month to 16 years old who
were undergoing a lumbar puncture for the investigation of
headaches, fever, or disorders of peripheral nerve.
Neopterin, biopterin, and dihydropteridine reductase
(DHPR) levels were assayed in brain acquired from a 17year-old patient with Rett syndrome at autopsy. A control
brain was obtained from an adult patient at autopsy. DHPR
levels were also assayed in 5 frontal cortex biopsy specimens
obtained for diagnosis of nondegenerative neurological disease in children 5 to 15 years of age.
Brain samples were homogenized in three to five volumes
of phosphate-buffered saline in a glass homogenizer, then
centrifuged for 60 minutes at 15,000 g. Aliquots of the supernatants were adjusted to a 0.3 molar concentration in
perchloric acid and a 0.2 molar concentration in phosphoric
acid, denatured proteins were removed by centrifugation,
and total neopterin and biopterin content was determined by
the procedure used for analysis of CSF pterins. DHPR activity was determined as described previously [14].
u.
0
< 0.002).
The only known pathological condition that leads to
increases in levels of biopterin in physiological fluids is
DHPR deficiency [14}. In the absence of DHPR, large
amounts of dihydrobiopterin are formed and excreted.
Normally, almost all of the biopterin in CSF is in the
tetrahydric form. To determine if the increased CSF
biopterin levels in Rett patients were due to a central
nervous system deficit of DHPR activity, we measured
tetrahydrobiopterin concentrations in two CSF samples and also determined pterins and DHPR activity in
some brain samples obtained at autopsy. The teuahydrobiopterin content in the two CSF samples was
64.3% and 100% of the total biopterin present, com-
*
120-
A Atypical Pallent
a
2 3 4
.
.
a TypIcaI Patlent
1[
1
0
5 6
Control
7 8 910111213141516
Age in Years
A
16
14-
A
I
12.
0
0
a
5
.
' a
6-
I
a
0 .
a
0 . .
8-
A
P
0
10-
a
a
a
0
a
'a
4.-
a ~yp1c.1 Patient
A Atyplcal Patlent
2
o Control
-1
ReSUltS
Individual values for HVA, MHPG, and 5-HIAA
concentrations are shown in Figure 1, along with mean
values iz standard error of the mean for controls of
comparable age. Analysis of covariance of CSF monoamine metabolites revealed a significant decrease of
CSF HVA ( p < 0.001), MHPG ( p < 0.001), and 5HIAA ( p < 0.05) in the patient population as compared with control subjects. Of interest, in the 2 patients with atypical presentation of Rett syndrome the
absolute values for HVA, MHPG, and 5-HIAA were
within the normal ranges for the patient's age. The
biopterin value was normal in 1 of these 2 patients and
not determined in the other.
Total CSF biopterin levels for Rett syndrome patients are displayed in Figure 2, along with mean values
for controls of comparable age. Total CSF biopterin
levels were significantly increased in Rett syndrome
patients (14.1 rt 1.8 pmoYml) as compared with controls (6.2 & 0.5 pmoYml) by analysis of covariance ( p
140-
v)
o l l l l l l l l l l l l l l l l
1
2
4
3
5
6
7 8 910111213141516
Age In Years
B
a"
u.
*I
v)
0
40
B
P
A
0
.
30
0
0
:%
a
A*
I
a
0
20
a
a
0
a
0.
0
a Typical Patlent
A AtyplCal Patlent
o Control
10
0
h
1
1
2
3
4
5
6
7
8
I
Age In Years
L
1
1
1
I
I
1
9 10 11 12 13 14 15 16
Fig I. (A-C) Cerebrospinalfluid (CSF) biogenic amine metabolites in Rett syndrome. Control values are expressed as mean
SEM. There were 16 controls 2 to 4 years old, 1 1 controls 5 to
10years old, and 10 controls 11 to 16 years old. Different symbols are given for typical and atypical patients. HVA =
homouanillic acid; MHPG = 3-metboxy4-hydroxyphenylethylene gbcol; 5-HIAA = 5-hydroxyinhleaceticacid.
*
Zoghbi et al: Monoamines in Rett Syndrome 57
!$
30
Typlcal Patient
AAtyplcal Patient
o Control
I>
0
t '
0
1
2
3
4
s
6
7
a
9 i o i 1 1 2 i 3 1 4 i s i ~
Age In Years
Fig 2. Cerebrospinalfluid (CSF) biopterin leuels in Rett syndrome. Control values are expressed as mean t SEM. There were
20 controls 2 to 5 years old, 14 controls 5 to 10 years old, and 8
controls 1 1 t o 15 years old.
pared with a normal value of 75 to 80%. The total
neopterin and biopterin content of various brain regions and the DHPR activity are given in the Table. In
various brain regions from a single Rett patient there
were no significant decreases in DHPR activity, in
agreement with the CSF tetrahydrobiopterin measurements.
Serum and urine amino acids were normal in all patients. Detailed polygraphic studies revealed slowing
of the electroencephalogram in all 19 patients studied,
and epileptiform activity was found in 15 of 19 patients. During wakefulness, episodes of disorganized
breathing consisting of apnea followed by increased
respiratory effort and rate were noted in 17 of 19
patients; respiratory effort during sleep was continuous
and regular. Rapid eye movement sleep was reduced in
the same 17 patients 1151. The 2 atypical patients did
not have episodes of disorganized breathing and had
normal percentages of rapid eye movement sleep and
less marked electroencephalographic abnormalities.
Discussion
Cerebrospinal fluid HVA, MHPG, and 5-HIAA were
significantly reduced in patients with the typical form
of Rett syndrome. This finding, together with the
abnormal neurophysiological and respiratory studies,
suggests dysfunction in subcortical or brainstem areas
or both. The stereotypic hand movements may represent dysfunction in the basal ganglia. Abnormal CSF
biogenic amines have been reported in disorders that
share some clinical features with Rett syndrome, including Parkinson's disease, DHPR deficiency, Huntington's disease, and Lesch-Nyhan syndrome [16-221.
Abnormal CSF biogenic amine metabolism does not
necessarily indicate specific neuronal dysfunction C231.
Accordingly, factors that may affect monoamine metabolites, such as diet, drug intake, and nutritional
status, were considered [241. None of the patients had
unusual diets, and all but 1 were in a good nutritional
state. Anticonvulsants used in 16 patients are not
known to affect biogenic amine metabolite levels. Reduced biogenic amine levels could represent secondary
Neopterin, Biopterin, and Dihydropteridine Reductase h e l s in Control Brain and Brain Regions
from a Patient with Rett Syndrome
Brain
Region
Rett syndromea
Cortex
Thalamus
Medulla
Locus ceruleus
Substantia nigra
Caudate nucleus
Controlb
Frontal cortex
Adult control'
Frontal cortex
Temporal cortex
Cerebellum
Brainstem
Neopterin
(pmoVgm)
Biopterin
(PmoVgm)
Dihydropteridine Reductase
(nmol NADH oxidizedmidgm)
237
444
376
195
307
119
45
144
139
700
837
385
622
1,435
1,485
1,150
745
974
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
945
1,285
1,807
1,554
3,731
95
109
117
116
112
178
158
298
1,450
ND
1,500
94 5
"Samples were taken from a 17-year-old Rett syndrome patient at autopsy.
bControl samples were taken from biopsy samples obtained for diagnosis of nondegenerative neurological diseases in children 5 to 15 years old.
'Sample was obtained from an adult at autopsy.
NADH = reduced nicotinamide adenine dinucleotide; N D = not determined.
58 Annals of Neurology
Vol 25
N o 1 January 1989
changes in a disorder affecting the central nervous system diffusely. However, patients with the classical
form of Rett syndrome appeared to have abnormal
levels of CSF biogenic amine metabolites, whereas the
2 atypical patients, who clearly had severe involvement
of the central nervous system, had normal values as
well as normal results on sleep and respiratory polygraphic studies. Another observation is the lack of effect of age or duration of disease on the CSF monoamine metabolites. Finally neuropathological studies
have demonstrated a reduction of brain weight (6070% of the expected weight) in 8 Rett patients, and in
the substantia nigra melanin was mildly to moderately
decreased 1251. Another autopsy study noted extensive dopamine depletion in the corpus striatum and
marked reduction of norepinephrine and serotonin
levels in the hypothalamus and limbic system 1261.
Since all three monoamine metabolites were noted
to be reduced in CSF from our patients and in neuropathological specimens 1261, we postulated a defect
in the concentration of tetrahydrobiopterin, the common cofactor required for the rate-limiting step in synthesis of the three biogenic amines 171. Total CSF
biopterin, which includes tetrahydrobiopterin and dihydrobiopterin, was significantly elevated in patients
compared with controls. An increase in total biopterin
may reflect a deficiency in DHPR, the enzyme that
keeps biopterin in its fully reduced form 1141. In CSF
from 2 Rett syndrome patients, dihydrobiopterin and
tetrahydrobiopterin were individually measured, and
most of the biopterin was in the tetrahydrobiopterin
form. These results, albeit representative of only 2
patients, argue against DHPR deficiency as an underlying defect. Direct measurements of DHPR activity in
several regions of the brain from a Rett syndrome patient indicated normal DHPR activity. Therefore, a
deficiency of DHPR is unlikely to be a contributory
factor to elevated CSF biopterin levels and decreased
neurotransmitter metabolite levels. The abnormal
biopterin elevation appears to be specific to the central
nervous system. Bolthauser and colleagues measured
urine biopterin in 10 patients and blood biopterin in 4,
and found no evidence for generalized tetrahydrobiopterin deficiency in Rett syndrome 1271. Sahot and
colleagues measured serum and urine biopterin levels
and blood DHPR activity in 9 patients and found them
to be normal 128). These data, coupled with evidence
for normal urinary biogenic amine metabolites reported by Riederer and colleagues 1291, suggest that
the abnormality of CSF biogenic amine metabolites
and biopterin is limited to the central nervous system.
The alterations in CSF monoamine metabolites and
biopterin levels may prove useful as biochemical markers in this disorder, and when combined with the
polygraphic studies provide the only laboratory aid for
establishing the diagnosis. In addition, these findings
identify further areas of investigation that could provide insight into the pathogenesis and treatment of this
disorder.
These studies were supported in part by a grant (RR 00188) from
the Clinical Research Center at Texas Children’s Hospital, the
USDNARS Children’s Nutritional Research Center, by funds provided to the Section of Pediatric Neurology by Texas Children’s
Hospital, and by a grant (RR 02558) from the Clinical Research
Center at the University of Texas Medical School at Houston.
The authors wish to thank Nancy Ivy for careful preparation of the
manuscript and Marinell White, BS, for analysis of biogenic amine
metabolites in cerebrospinal fluid. Also, our appreciation is expressed to Dr Richard J. Allen, Department of Pediatric Neurology,
University of Michigan Medical Center, Ann Arbor, MI, for providing the two CSF samples from which tetrahydrobiopterin contents
could be determined.
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