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Children with comorbid attention-deficit-hyperactivity disorder and tic disorder Evidence for additive inhibitory deficits within the motor system.

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Children With Comorbid
Attention-DeficitHyperactivity Disorder and
Tic Disorder: Evidence for
Additive Inhibitory Deficits
Within the Motor System
Gunther H. Moll, MD,1 Hartmut Heinrich, PhD,1
Götz-Erik Trott, MD,2 Sigrun Wirth, MD,2
Nathalie Bock, MD,1 and Aribert Rothenberger, MD1
For children with attention-deficit-hyperactivity disorder
(ADHD) or tic disorder (TD), we recently reported deficient inhibitory mechanisms within the motor system by
using transcranial magnetic stimulation. These deficits—
stated as reduced intracortical inhibition in ADHD and
shortened cortical silent period in TD—could be seen as
neurophysiological correlates of motor hyperactivity and
tics, respectively. To investigate neurophysiological aspects of comorbidity, we measured motor system excitability for the first time also in children with combined
ADHD and TD. The findings of a reduced intracortical
inhibition as well as a shortened cortical silent period in
these comorbid children provide evidence for additive effects at the level of motor system excitability.
Ann Neurol 2001;49:393–396
In attention-deficit-hyperactivity disorder (ADHD),
motoric hyperactivity is usually the striking abnormality to parents and physicians, typically seen in restlessness, fidgeting, and generally unnecessary gross body
movements.1 In tic disorder (TD), fluctuating motor
and phonic tics can be characterized as involuntary,
sudden, abrupt, repetitive movements, gestures, or utterances that may be seen as poorly modulated fragments of normal sensorimotor behavior.2 As a possible
neurophysiological correlate of these hypermotoric
symptoms in children with ADHD or TD, we recently
tested the hypothesis of deficient inhibitory motor control throughout the sensorimotor circuit by investigating motor system excitability using the technique of
focal transcranial magnetic stimulation (TMS) in a
single- and paired-stimulus paradigm.3,4 Whereas children with ADHD showed a reduced intracortical inhibition,5 children with TD had a shortened cortical silent period compared to healthy children.6
Because these two hypermotoric disorders coexist in
up to 50% in clinical samples,7 we investigated neurophysiological correlates of motor system excitability in
a comorbid group (ADHD ⫹ TD) relative to the two
single-disorder groups (ADHD only or TD only) to
test whether a distinct dysfunctional pattern for this
common comorbidity exists.
Patients and Methods
The study was performed on 16 children with ADHD
(ADHD only), 16 children with TD (chronic motor tic disorder/Tourette’s disorder; TD only), 16 children with comorbid ADHD and TD (ADHD ⫹ TD), and 16 healthy
children (controls). The demographic and clinical data are
summarized in Table 1. All children (60 boys, 4 girls) were
right-handed and of at least normal intelligence (IQ ⬎ 80).
The four groups did not differ significantly in mean age
and sex.
All disordered children had to fulfill the diagnostic criteria
for ADHD and/or chronic motor tic disorder/Tourette’s disorder according to the APA’s DSM-IV. None of the ADHD
only, TD only, or ADHD ⫹ TD children fulfilled the criteria for any other psychiatric disorder as stated in a structured clinical interview (DISC). In all of the children with
an ADHD diagnosis, the score of the abbreviated (10 item)
Conners scale was ⱖ15.8 In addition, none of the ADHD
only children had any actual tic symptoms nor any history of
tics. For the TD-only group, a Conners score ⱖ15 was used
as the exclusionary criterion.
Children with an ADHD diagnosis either had never taken
any psychostimulant medication or had been drug-free for at
least 48 hours before TMS investigation (the only drug used
was methylphenidate in a standard formulation). Some of
the children with a TD diagnosis received a neuroleptic medication (Table 1).
The healthy children were drug-free and devoid of any
child psychiatric disorder. The subjects of all four groups
lacked gross neurological or other organic disorders.
After a complete description of the study to the children
and their parents, assent was obtained from the children and
written informed consent from their parents. The study was
conducted according to the declaration of Helsinki and was
approved by the local ethics committee.
From the 1Child and Adolescent Psychiatry Department, University
of Göttingen, Göttingen; and 2Practice of Child and Adolescent
Psychiatry, Aschaffenburg, Germany
Received Sep 1, 2000, and in revised form Oct 13. Accepted for
publication Oct 18, 2000.
Address correspondence to Prof Rothenberger, von-Siebold-Str. 5,
D-37075 Göttingen, Germany. E-mail:
Focal TMS was applied to the hand area of the left motor
cortex (figure-eight magnetic coil with a diameter of one
wing of 70 mm, Magstim 200 HP magnetic stimulator;
Magstim, Whiteland, United Kingdom), and surface electromyography was recorded from the right abductor digiti
minimi muscle. The exact equipment and protocol are reported in detail by Ziemann et al.4
Resting and active motor threshold were expressed as a
percentage of the maximum stimulator output to elicit a mo-
© 2001 Wiley-Liss, Inc.
Table 1. Demographic and Clinical Data
Age (yr; mo)
Mean (⫾SD)
Conners (10 items)
Mean (⫾SD)
TD medication
(n ⫽ 16)
ADHD only
(n ⫽ 16)
TD only
(n ⫽ 16)
(n ⫽ 16)
12;3 (⫾1;8)
14 Boys,
2 Girls
12;0 (⫾1;6)
15 Boys,
1 Girl
12;8 (⫾1;10)
15 Boys,
1 Girl
12;5 (⫾2;1)
16 Boys
4.3 (⫾3.1)
20.5 (⫾3.4)
6.3 (⫾4.1)
Tiapride (n ⫽ 5)
18.9 (⫾5.1)
Tiapride (n ⫽ 6),
tiapride/pimozide (n ⫽ 1)
tor evoked potential. The duration of the cortical silent period (in milliseconds) was investigated at a stimulus intensity
of 40% above active motor threshold (single magnetic stimulation). Intracortical excitability was determined in a conditioning test paired-stimulus paradigm and measured as
changes in the amplitude of the suprathreshold test response
by the conditioning subthreshold shock (expressed as a percentage of the unconditioned mean across all inhibitory (2–5
msec) and facilitatory (7–20 msec) interstimulus intervals
tested). Because activation of the relaxed target muscle may
reduce intracortical inhibition and intracortical facilitation,9
for artefact control, all sweeps were visually inspected offline.
Sweeps showing voluntary activation of the target muscle
were rejected and excluded from further processing.
Statistical Analysis
Unique TMS variables were subjected to a 2 ⫻ 2 factorial
analysis of variance with ADHD (with ADHD vs. without
ADHD) and TD (with TD vs. without TD) as factors using
SPSS for Windows v.10. Possible significant interactions
were planned to be examined with post-hoc Tukey tests.
For all statistical procedures, significance was assumed at
p ⬍ 0.05.
No significant effects were obtained for passive and active motor thresholds. Concerning the cortical silent
period, a main effect of TD was found, indicating that
children with a TD diagnosis (TD only or ADHD ⫹
TD) had a shorter cortical silent period than children
without TD (controls or ADHD only; Table 2 and
Fig). Post-hoc analysis (t test) revealed no significant
cortical silent period difference between medicated
(139.7 ⫾ 37.8 msec) and unmedicated (134.4 ⫾ 44.3
msec) children with TD [t(1,30) ⫽ 0.36, n.s.].
Analysis of intracortical inhibition revealed a main
effect of ADHD; i.e., children with an ADHD diagnosis (ADHD only or ADHD ⫹ TD) had a reduced
intracortical inhibition compared to children without
ADHD (controls or TD only; see Table 2 and Fig).
For intracortical facilitation, no significant effects
were obtained.
Morphometric and neuroimaging studies suggest that
ADHD and TD as well involve neuropathology of the
sensorimotor circuit, responsible for control of sensorimotor behavior.10 To study aspects of comorbidity of
these two hypermotoric disorders (i.e., additive vs.
nonadditive effects), we applied TMS to assess inhibitory mechanisms within the motor system in children
with ADHD, children with TD, and children with comorbid ADHD ⫹ TD in comparison to healthy children by means of a 2 ⫻ 2 factorial design. The finding
Table 2. Summary of TMS Results
Resting motor
threshold (%)
Active motor
threshold (%)
Cortical silent
period (msec)
inhibition (%)
facilitation (%)
F Values and Significance
(n ⫽ 16)
Mean (⫾SD)
ADHD only
(n ⫽ 16)
Mean (⫾SD)
TD only
(n ⫽ 16)
Mean (⫾SD)
(n ⫽ 16)
Mean (⫾SD)
Main Effect
Main Effect
F(1,63) ⫽ 0.16,
F(1,63) ⫽ 2.04,
F(1,63) ⫽ 0.23,
F(1,63) ⫽ 11.1,
p ⫽ 0.002
F(1,63) ⫽ 2.82,
F(1,63) ⫽ 1.11,
F(1,63) ⫽ 0.04,
F(1,63) ⫽ 11.8,
p ⫽ 0.001
F(1,63) ⫽ 0.02,
F(1,63) ⫽ 0.01,
Annals of Neurology
Vol 49
No 3
March 2001
⫽ 0.00,
⫽ 0.17,
⫽ 0.31,
⫽ 0.00,
⫽ 0.04,
Fig. Main effects of tic disorder (TD) and attention-deficithyperactivity disorder (ADHD) on cortical silent period (top)
and intracortical inhibition (bottom). For each group, mean
value ⫾ standard error is depicted. Open boxes represent children without tic disorder; solid boxes represent children with
tic disorder.
of normal motor thresholds in all psychiatric groups
investigated gives no evidence for hyperexcitability at
the neuronal membrane level of cortical neurons
within the motor system11 for children with ADHD,
children with TD, or comorbid children.
Concerning the cortical silent period, children with
a TD diagnosis had a shorter cortical silent period than
children without TD (see Table 2 and Fig). This finding confirms our previously reported TMS data for
children with TD, in which a significantly shorter cortical silent period was found in children with TD compared to healthy children. This result was seen as a
neurophysiological correlate of deficient motor inhibition within the sensorimotor circuit in children with
TD, probably at the level of the basal ganglia, and might
be at least partly due to dopaminergic hyperinnervation
of the striatum or a supersensitivity of striatal D2 recep-
tors.2,6 The normal duration of the cortical silent period
in children with an ADHD only diagnosis does not support the hypothesis of generally deficient inhibitory
mechanisms within their sensorimotor circuit.5
Concerning intracortical inhibition, children with an
ADHD diagnosis had a reduced intracortical inhibition
compared to children without ADHD (see Table 2 and
Fig). This result also confirms our previously reported
TMS data for children with ADHD only, in which a
significantly decreased intracortical inhibition was
found, providing evidence for inhibitory deficits in
ADHD within the motor cortex.5 Owing to the
method used, this inhibitory deficit within the motor
cortex might be only part of a general deficit in behavioral inhibition.12 Whether these discriminative TMS
findings on motor system excitability in children with
ADHD or TD might be related to their different neuromotor functioning at the behavioral level, eg, the
time to complete motor movements,13 remains to be
In children with combined ADHD ⫹ TD, a significantly reduced intracortical inhibition as well as a significantly shortened cortical silent period were found,
suggesting an additive effect concerning motor system
excitability. This may be in line with neuropsychological and behavioral data, especially on neuromotor
functioning, indicating the most severe impairment in
children with ADHD ⫹ TD.13,14 On the other hand,
neuropathological data at the level of the basal ganglia15 and neurophysiological parameters reflecting
task-dependent cognitive information processing do
not fully support the notion of additive deficits and
dysfunctions, respectively.16 –19 Thus, it might be assumed that purely additive neurobiological effects in
ADHD ⫹ TD comorbidity are restricted to abnormalities within the motor system (as measured by TMS in
this study), but this may not hold true for all aspects of
cognitive information processing.
In conclusion, a distinct dysfunctional pattern of deficient inhibitory motor control can be postulated as a
neurobiological correlate of hypermotoric symptoms in
children with ADHD, TD, or ADHD ⫹ TD. These
findings should be verified with different drug treatments for both kinds of inhibitory deficits on motor
control, i.e., methylphenidate in ADHD and probably
neuroleptics in TD.20
The authors thank Susanne Mock and Eva Nobbe.
1. Barkley RA. Attention-deficit-hyperactivity disorder, 2nd edition. New York: Guilford, 1998.
2. Leckman JF, Peterson BS, Anderson GM, et al. Pathogenesis of
Tourette’s Syndrome. J Child Psychol Psychiatr 1997;38:119 –
Brief Communication: Moll et al: Motor System Excitability in ADHD and Tic Disorder
3. Kujirai T, Caramia MD, Rothwell JC, et al. Corticocortical inhibition in human mortor cortex. J Physiol (Lond) 1993;471:
4. Ziemann U, Paulus W, Rothenberger A. Decreased motor inhibition in Tourette syndrome: evidence from transcranial magnetic stimulation. Am J Psychiatr 1997;154:1277–1284.
5. Moll GH, Heinrich H, Trott GE, et al. Deficient intracortical
inhibition in drug-naive children with attention-deficit hyperactivity disorder is enhanced by methylphenidate. Neurosci Lett
6. Moll GH, Wischer S, Heinrich H, et al. Deficient motor control in children with tic disorder: evidence from transcranial
magnetic stimulation. Neurosci Lett 1999;272:37– 40.
7. Spencer T, Biederman J, Wilens T. Attention-deficit/hyperactivity
disorder and comorbidity. Pediatr Clin North Am 1999;46:915–
8. Goyette CH, Conners CK, Ulrich RF. Normative data on revised Conners parent and teacher rating scales. J Abnorm Child
Psychol 1978;6:221–236.
9. Ridding MC, Taylor JL, Rothwell JC. The effect of voluntary
contraction on cortico-cortical inhibition in human motor cortex. J Physiol (Lond) 1995;487:541–548.
10. Sheppard DM, Bradshaw JL, Purcell R, Pantelis C. Tourette’s
and comorbid syndromes: obsessive compulsive and attention
deficit hyperactivity disorder. A common etiology? Clin Psychol
Rev 1999;19:531–552.
11. Ziemann U, Rothwell JC, Ridding MC. Interaction between
intracortical inhibition and facilitation in human motor cortex.
J Physiol (Lond) 1996;496:873– 881.
12. Barkley RA. Behavioral inhibition, sustained attention, and executive functions: constructing a unifying theory of ADHD.
Psychol Bull 1997;121:65–94.
13. Schuerholz LJ, Cutting L, Mazzocco MM, et al. Neuromotor
functioning in children with Tourette syndrome with and without attention deficit hyperactivity disorder. J Child Neurol
1997;12:438 – 442.
14. Harris EL, Schuerholz LJ, Singer HS, et al. Executive function
in children with Tourette syndrome and/or attention deficit hyperactivity disorder. J Int Neuropsychol Soc 1995;1:511–516.
15. Castellanos FX, Giedd JN, Hamburger SD, et al. Brain morphometry in Tourette’s syndrome: the influence of comorbid
attention-deficit/hyperactivity disorder. Neurology 1996;47:
16. Rothenberger A, Banaschewski T, Heinrich H, et al. Comorbidity in ADHD-children: effects of coexisting conduct disorder or tic disorder on event-related brain potentials in an auditory selective-attention task. Eur Arch Psychiatr Clin Neurosci
17. Schuerholz LJ, Baumgardner TL, Singer HS, et al. Neuropsychological status of children with Tourette’s syndrome with and
without attention deficit hyperactivity disorder. Neurology
1996;46:958 –965.
18. Yordanova J, Dumais-Huber C, Rothenberger A. Coexistence of
tics and hyperactivity in children: no additive effect at the psychophysiological level. Int J Psychophysiol 1996;21:121–133.
19. Yordanova J, Dumais-Huber C, Rothenberger A, Woerner W.
Frontocortical activity in children with comorbidity of tic disorder and attention-deficit hyperactivity disorder. Biol Psychiatr
20. Gadow KD, Sverd J, Sprafkin J, et al. Long-term methylphenidate therapy in children with comorbid attention-deficit hyperactivity disorder and chronic multiple tic disorder. Arch Gen
Psychiatr 1999;56:330 –336.
Annals of Neurology
Vol 49
No 3
March 2001
Neurological and
Heterogeneity in Two
Brothers with Cobalamin C
James M. Powers, MD,1 David S. Rosenblatt, MD,2
Robert E. Schmidt, MD, PhD,3 Anne H. Cross, MD,4
Joseph T. Black, MD,4 Ann B. Moser, BS,5
Hugo W. Moser, MD,5 and Daniel J. Morgan, BS6
Two adult brothers, one documented to have methylmalonic acidemia with homocystinuria, or cobalamin C deficiency, after autopsy, displayed severe but divergent
neurological presentations. One exhibited a myelopathy
and the other chronic endocrine problems (Schmidt’s
syndrome) followed by a neuropsychiatric and dementing
disorder owing to cerebral perivascular demyelination.
The recognition of cobalamin C deficiency has practical
implications because it is one of the few inherited diseases of central white matter that is treatable.
Ann Neurol 2001;49:396 – 400
Neurological deficits due to vitamin B12, or cobalamin
(Cbl), deficiency are highly variable and have been seen
most commonly in the setting of pernicious anemia, in
which a myelopathy and peripheral neuropathy predominate. Neuropsychiatric symptoms are seldom
mentioned, and brain involvement is said to occur only
after the spinal cord lesion.1 Likewise, neuropathology
textbooks emphasize the myelopathy almost to the exclusion of other central nervous system (CNS) lesions.2
There also are rare inherited disorders of transport or
intracellular processing that result in Cbl deficiency;3
the latter include methylmalonic acidemia with homocystinuria (cblC; McKusick 277400), in which there is
an early intracellular defect leading to a failure of synthesis of both Cbl cofactors, adenosylcobalamin
From the 1University of Rochester Medical Center, Departments of
Pathology and Neurology, Rochester, NY; 2McGill University
Health Centre, Division of Medical Genetics, Montreal, Canada;
Departments of Pathology and Immunology and 4Washington
University School of Medicine, Departments of Neurology and
Neurosurgery, St. Louis, MO; 5Johns Hopkins Medical Institutes,
Kennedy-Krieger Institute, Baltimore, MD; and 6University of
Rochester Medical Center, Department of Pathology, Rochester,
Received Aug 18, 2000, and in revised form Oct 23. Accepted for
publication Oct 25, 2000.
Address correspondence to Dr Powers, University of Rochester
Medical Center, Department of Pathology, 601 Elmwood Avenue,
Rochester, NY 14642. E-mail:
(AdoCbl) and methylcobalamin (MeCbl). Two adult
brothers, one documented to have cblC after his death,
demonstrated a striking neurological and neuropathologic heterogeneity. Neither displayed subnormal vitamin B12 levels or the expected hematological abnormalities. This report broadens the spectrum of cblC
and reminds us that neuropsychiatric deficits due to
cerebral white matter lesions can dominate in diseases
of Cbl deficiency.
Patients and Methods
Patient 1
In good health until age 32, Patient 1 presented with numbness of his extremities. Within 3 months, leg weakness, incontinence, and Lhermitte’s sign developed. Five months after presentation, neurological examination revealed
bitemporal disk pallor; weakness and diminished sensation to
temperature, pinprick, touch, and vibration of lower extremities; flexor plantar responses; and waddling gait.
Analysis of the cerebrospinal fluid (CSF) revealed a protein level of 91 mg/100 ml, IgG elevation, IgG synthesis of
7.0 mg/24 hour, no oligoclonal bands, and negative results
on the VDRL test. Human immunodeficiency virus (HIV)
test results were unavailable. Mean corpuscular volume,
erythrocyte sedimentation rate (ESR), and results of antinuclear antibodies (ANAs) studies, serum protein electrophoresis, very long–chain fatty acids studies, and thyroid tests were
normal. Mild leukopenia and a slight excess of hypersegmented polymorphonuclear cells (retrospectively) developed
only during his final admission. The vitamin B12 level was
415 mg/dl (normal 100 –700). Nerve conduction in the posterior tibial and sural nerves was mildly slowed. Brainstem
auditory evoked responses were normal. Visual evoked responses were 98 m/sec oculus dexter and 105 m/sec oculus
sinister. Magnetic resonance imaging (MRI) revealed a confluent periventricular white matter process about both trigone regions and the posterior body of the lateral ventricles.
Five months after presentation, prednisone was administered without benefit. By 6 months after onset, the patient
displayed a scissoring gait, by 9 months he needed a wheelchair, and by 16 months he was quadriparetic. Two years
after onset he had dysphagia; paraplegia; upper extremity
weakness; diminished light touch, proprioception, and vibratory sense below the neck; absent pinprick response below
the waist; and urinary retention. The patient developed multiple lung and urinary tract infections, expiring 26 months
after onset. There were “no obvious cognitive problems” at
time of death.
but had no recall at 5 minutes, was unable to spell, and had
difficulty initiating speech. There was right optic disk pallor.
Motor function, sensation, and coordination were normal.
CSF analysis revealed a protein level of 61 mg/100 ml, a
normal CSF IgG index, and no oligoclonal bands. The thyroxine level was elevated (18 mg/dl), and the cortisone level
was normal (9.9 mg/dl). The ESR and levels of ceruloplasmin, vitamin E, vitamin B12, ANAs, arylsulfatase A, and very
long–chain fatty acids were normal. Folate levels were not
determined. HIV and VDRL test results were negative. An
electroencephalogram revealed 3.5-Hz background. Proton
density and T2-weighted MRI demonstrated periventricular
nonenhancing increased signal abnormalities that were confluent in the occipital lobes and patchy in frontal white matter. T1-weighted images were normal (Fig 1). Brain biopsy
revealed some perivascular demyelinative foci and a few
perivascular lymphocytes without specific diagnostic features.
Within 1 year, he developed dysarthria, difficulty walking,
and inability to feed himself. At that time he had flexor plantar reflexes and rigidity. Deep venous thromboses with pulmonary embolism caused his death at age 45.
Family History
The brothers were of Italian-French-Irish descent without evidence of consanguinity. Their father had Grave’s disease,
their mother hypothyroidism. A younger brother, age 39,
had insulin-dependent diabetes mellitus but no neurological
symptoms. The younger brother had normal serum vitamin
Fig 1. Magnetic resonance imaging study of Patient 2. This
T2-weighted (2400/90) axial image at the level of the atria
shows abnormal increased signal in the periventricular occipital white matter bilaterally.
Patient 2
The older brother of Patient 1, Patient 2 was a college professor with hypoadrenalism and hypothyroidism, diagnosed
as Schmidt’s syndrome, since age 16. Medications included
cortisol, synthroid, and beclomethasone. At 44, he developed
social withdrawal and difficulty doing calculations. Over 4
months he deteriorated such that he was unable to interact
normally and became lost going to familiar locations. Results
of a computed tomography scan of the head at that time
were normal. Seven months after presentation he was alert
Brief Communication: Powers et al: cblC Deficiency and PGA-II
B12, folate, methylmalonic acid, methylcitric acid, homocysteine, and cystathionine levels and normal urinary methylmalonic acid and homocysteine levels.
In Patient 1 the postmortem examination was confined to
the CNS. Sections from CNS were stained with Luxol fast
blue–periodic acid–Schiff (LFB-PAS) and modified
Bielschowsky techniques. In addition, plastic-embedded sections of sural nerve from Patient 2 were examined. In view of
the neuropathologic findings suggestive of a methylation defect and because of the family history, cultured fibroblasts
from Patient 2 were analyzed for a possible inherited metabolic disorder.3
The sural nerve of Patient 2 exhibited a loss of large
and, especially, small myelinated axons with ongoing
axonal degeneration. A dorsal root ganglion of Patient
2 contained multifocal clusters of lymphocytes, as did
the testis. There were no distinctive cytosomes, demyelination, “onion bulbs,” or inflammation. In addition
to pulmonary emboli, systemic findings in Patient 2
consisted of (1) no demonstrable adrenal tissues at autopsy (presumably severe primary atrophy), (2) Hashimoto’s thyroiditis, (3) testicular atrophy, Sertoli cell–
only phenotype, and (4) severe macrovesicular hepatic
Fig 2. Patient 2. Perivascular demyelination of white matter
with periodic acid–Schiff–positive macrophages (arrowhead)
can be seen. Luxol fast blue–periodic acid–Schiff, ⫻125.
Fig 3. Patient 1. The thoracic cord is atrophic, with loss of
myelinated fibers, particularly in the posterior and posterolateral funiculi, and vacuolated myelin adjacent to the anterior
horns. Modified Bielschowsky’s stain, ⫻4.25 before 41%
steatosis. The gastric mucosa was unremarkable on gross
examination. Microscopic examination was omitted.
The CNS of both brothers was unremarkable on
gross examination except for the spinal cord of Patient
1, in which there were ill-defined gelatinous areas. The
cerebrum in both cases demonstrated perivascular demyelination (mainly of arterioles and venules) with occasional vacuoles, oligodendrocytic loss, PAS- and
LFB-positive macrophages, reactive astrocytes, and relative sparing of axons with occasional axonal spheroids
(Fig 2). Patient 1 also displayed a severe vacuolar myelopathy typical of subacute combined degeneration
seen in Cbl deficiency,4 resulting in atrophy of the thoracic cord owing to marked and asymmetric loss of
myelinated fibers in the posterior, lateral, and anterior
columns (Fig 3).
Fibroblast analysis revealed impaired synthesis of
both AdoCbl and MeCbl (Tables 1 and 2). Propionate
fixation was not increased when fibroblasts from Patient 2 were fused with two cblC cell lines (Table 3),
establishing the diagnosis of cblC.
Subacute combined degeneration of the spinal cord
and its accompanying clinical myelopathy is dominant
in Cbl deficiency of pernicious anemia, and neuropsychiatric symptoms and supraspinal lesions are seldom
publicized.1–5 However, the classic study of pernicious
anemia by Adams and Kubik describes and illustrates demyelinative lesions in deep cerebral white matter, which
were responsible for neuropsychiatric manifestations.6
In addition to a deficiency in Castle’s intrinsic factor
needed for the absorption of the external factor, Cbl,
Annals of Neurology
Vol 49
No 3
March 2001
Table 1. Propionate and Methyl-THF Uptake
[14C] Propionate (nmol/mg
protein/18 h)
[14C] Methyl-THF (pmol/mg protein/
18 h)
Cell Line
Patient 2
Control subjects
0.76, 0.46a
10.8 ⫾ 3.7b
3.7, 3.1
10.9 ⫾ 3.5
59, 49
225 ⫾ 165
201, 185
305 ⫾ 125
Mean of triplicate values for two separate experiments.
Based on 12 determinations (3 different control subjects).
OHCbl ⫽ hydroxycobalamin; THF ⫽ tetrahydrofolate.
Table 2. Cobalamin Uptake and Distribution
Cbla Distribution (%)
Cell Line
Cbla Uptake
(pg/106 cells)
Patient 2
Control subjects
4.60b ⫾ 2.01
8.4 ⫾ 3.9
11.3 ⫾ 6.9
15.3 ⫾ 4.2
58 ⫾ 6.7
7.2 ⫾ 2.7
a 57
[ Co] CNCbl 25 pg/ml, 4 days incubation.
Based on 12 determinations (3 different control subjects).
AqCbl ⫽ aquacobalamin; AdoCbl ⫽ adenosylcobalamin; Cbl ⫽ cobalamin;
amin; MeCbl ⫽ methylcobalamin.
Table 3. Complementation Analysis
Propionatea Uptake (nmol/mg protein/18 h)
Fusion with Cells from
Patient 2
Cell Line
Patient 2
a 14
[ C] propionate.
mut ⫽ mut0 or mut⫺, the vitamin B12–nonresponsive type of
methylmalonic acidemia; PEG ⫽ polyethylene glycol.
in pernicious anemia,7 deficiencies of Cbl also can be
inherited and due to abnormalities in its transport or
intracellular processing. The latter have been divided
into complementation groups cblA to cblH.3,8 Our patients fall into the cblC complementation group, the
most common, which causes combined methylmalonic
acidemia and homocystinuria. Neither of the highly effective biochemical screening tests for methylmalonic
acidemia and homocystinuria was performed in our patients. It is noteworthy that serum Cbl and folate levels
are usually normal in cblC because there is no abnor-
Co ⫽ radioactive cobalt; CNCbl ⫽ cyanocobal-
mality in the absorption of either vitamin. Clinical heterogeneity in cblC has been previously reported.9 The
major cblC phenotype is that of an infant who demonstrates feeding difficulties, hypotonia, seizures, developmental delay, decreased visual acuity, nystagmus,
and pigmentary retinopathy. Hematologic manifestations usually consist of thrombocytopenia, macrocytic
anemia, megaloblastosis, and hypersegmented neutrophils. Perivascular (arteriole and precapillary) demyelination throughout the centrum semiovale, often associated with fibrinoid degeneration of the vascular wall,
was reported in 1 child with cblC.10 No spinal symptoms or lesions were noted. The later-onset phenotype
has a more benign clinical course, characterized by dementia, myelopathy, and seizures; neuropathologic data
have not been reported. Chronic progressive and
relapsing-remitting courses may occur, and some patients respond to hydroxycobalamin treatment.11–14
One of 2 sisters developed a fatal myelopathy at 13 years
of age, while the other presented with reversible dementia at 10 years of age.13 An abnormality in folate metabolism due to 5, 10-methylenetetrahydrofolate reductase
deficiency15 also may result in subacute combined degeneration and cerebral perivascular demyelination or
clinical combinations of dementia and myelopathy.16,17
The autopsy findings of cerebral perivascular demyelination and subacute combined degeneration in our patients suggested a methylation defect, but biochemical
studies of cultured skin fibroblasts were necessary to
make a specific diagnosis.
Brief Communication: Powers et al: cblC Deficiency and PGA-II
The neuroradiological similarity and neuropathologic identity of the cerebral white matter lesions in
both brothers provide strong evidence that both had
cblC, even though biochemical confirmation could
only be obtained in Patient 2. We cannot relate the
endocrinologic problems of Patient 2 to cblC.3 Our
patients’ family exhibited signs and symptoms of
polyglandular autoimmune syndrome (PGA) type II
(Schmidt’s syndrome). “Pernicious anemia” is rarely
(0.5%) noted in PGA type II but is more commonly
associated with PGA type I.18 To our knowledge, myelopathy or encephalopathy in PGA–“pernicious anemia” has not been reported. The chromosomal localizations of both cblC and PGA type II are unknown;
consequently, we cannot comment on the genetic implications of these two rare diseases in one family. Patient 1 suffered only from cblC, Patient 2 from both
cblC and PGA type II, and the third brother only from
PGA type II.
The precise molecular defect and cellular pathogenesis
of cblC are unknown, although the activity of several
enzymes presumably involved in the reduction of the
central cobalt of Cbl is decreased. The end result is the
failure to retain Cbl within cells and to synthesize the
active cofactors, AdoCbl and MeCbl. AdoCbl catalyzes
the conversion of methylmalonyl CoA to succinyl CoA,
and MeCbl catalyzes the methylation of homocysteine
to methionine. The pathogenesis of the myelin lesion in
Cbl deficiency is also disputable.
Our cblC patients provide three practical lessons.
First, in contrast to most inherited myelin diseases,
cblC is treatable. Second, endogenous defects in Cbl
can occur without low serum vitamin B12 levels and
hematologic abnormalities. Third, the neurologic spectrum of Cbl deficiency includes optic or neuropsychiatric presentations, which may occur without the myelopathy. Hence, cblC should be considered in
enigmatic optic neuropathy, neuropsychiatric disease,
or subacute myelopathy, even with normal serum vitamin B12 or folate levels. Laboratory studies should include serum or urine screening for homocysteine and
methylmalonic acid.
We thank DeWitte T. Cross, III, MD, for his assistance with the
radiological studies and Mrs Tina Blazey for her usual outstanding
secretarial assistance. We also recognize the importance of the
United Leukodystrophy Foundation, because at one of its annual
scientific sessions the primary author was reminded (learned?) of the
association of perivascular demyelination and methylation disorders
by Robert Surtees. Without that encounter, the diagnosis of our
patients probably would still be unknown.
1. Adams RD, Victor M. Principles of neurology, 6th ed. New
York: McGraw-Hill, 2000.
Annals of Neurology
Vol 49
No 3
March 2001
2. Harper C, Butterworth R. Nutritional and metabolic disorders.
In: Graham DI, Lantos PL, eds. Greenfield’s neuropathology,
6th ed. London: Arnold, 1997:621– 624.
3. Fenton WA, Rosenberg LE. Inherited disorders of cobalamin
transport and metabolism. In: Scriver CR, Beaudet AL, Sly WS,
Valle D, eds. The metabolic and molecular bases of inherited
disease, 7th ed. New York: McGraw-Hill, 1995:3129 –3149.
4. Pant SS, Asbury AK, Richardson Jr EP. The myelopathy of
pernicious anemia: a neuropathological reappraisal. 1968;44:8 –
5. Savage DG, Lindenbaum J. Neurological complications of acquired cobalamin deficiency: clinical aspects. In: Wickramasinghe SN, ed. Baillière’s clinical haematology: international
practice and research. Megaloblastic anaemia. London: Bailliére
Tindall, 1995;8:657– 678.
6. Adams RD, Kubik CS. Subacute degeneration of the brain in
pernicious anemia. N Engl J Med 1944;231:1–9.
7. Cooper BA, Castle WB. Sequential mechanisms in the enhanced absorption of vitamin B12 by intrinsic factor in the rat.
J Clin Invest 1960;39:199 –214.
8. Watkins D, Matiaszuk N, Rosenblatt DS. Complementation
studies in the cblA class of inborn error of cobalamin
metabolism: evidence for interallelic complementation and for a
new complementation class (cblH). J Med Genet 2000;37:510 –
9. Rosenblatt DS, Aspler AL, Shevell MI, et al. Clinical heterogeneity and prognosis in combined methylmalonic aciduria and
homocystinuria (cblC). J Inherit Metab Dis 1997;20:528 –538.
10. Dayan AD, Ramsey RB. An inborn error of vitamin B12 metabolism associated with cellular deficiency of coenzyme forms
of the vitamin: pathological and neurochemical findings in one
case. J Neurol Sci 1974;23:117–128.
11. Shinnar S, Singer HS. Cobalamin C mutation (methylmalonic
aciduria and homocystinuria) in adolescence: a treatable cause
of dementia and myelopathy. N Engl J Med 1984;311:451–
12. Gold R, Bogdahn U, Kappos L, Toyka K. Hereditary defect of
cobalamin metabolism (homocystinuria and methylmalonic aciduria) of juvenile onset. J Neurol Neurosurg Psychiatry 1996;
13. Augoustides-Savvopoulou P, Mylonas I, Sewell AC, Rosenblatt
DS. Reversible dementia in an adolescent with cblC disease:
clinical heterogeneity within the same family. J Inherit Metab
Dis 1999;22:756 –758.
14. Enns GM, Barkovich AJ, Rosenblatt DS, et al. Progressive neurological deterioration and MRI changes in cblC methylmalonic
acidaemia treated with hydroxycobalamin. J Inherit Metab Dis
1999;22:599 – 607.
15. Rosenblatt DS. Inherited disorders of folate transport and metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds.
The metabolic and molecular bases of inherited disease, 7th ed.
New York: McGraw-Hill, 1995:3111–3128.
16. Clayton PT, Smith I, Harding B, et al. Subacute combined
degeneration of the cord, dementia and parkinsonism due to an
inborn error of folate metabolism. J Neurol Neurosurg Psychiatry 1986;49:920 –927.
17. Haworth JC, Dilling LA, Surtees RAH, et al. Symptomatic and
asymptomatic methylenetetrahydrofolate reductase deficiency in
two adult brothers. Am J Med Genet 1993;45:572–576.
18. Neufeld M, Maclaren NK, Blizzard RM. Two types of autoimmune Addison’s disease associated with different polyglandular autoimmune (PGA) syndromes. Medicine 1981;60:355–
Irreversible Brain Creatine
Deficiency with Elevated
Serum and Urine Creatine:
A Creatine Transporter
Kim M. Cecil, PhD,1 Gajja S. Salomons, PhD,3
William S. Ball, Jr., MD,1 Brenda Wong, MD,2
Gail Chuck,2 Nanda M. Verhoeven, PhD,3
Cornelis Jakobs, PhD,3 and Ton J. DeGrauw, MD,2
Recent reports highlight the utility of in vivo magnetic
resonance spectroscopy (MRS) techniques to recognize
creatine deficiency syndromes affecting the central nervous system (CNS). Reported cases demonstrate partial
reversibility of neurologic symptoms upon restoration of
CNS creatine levels with the administration of oral creatine. We describe a patient with a brain creatine deficiency syndrome detected by proton MRS that differs
from published reports. Metabolic screening revealed elevated creatine in the serum and urine, with normal levels of guanidino acetic acid. Unlike the case with other
reported creatine deficiency syndromes, treatment with
oral creatine monohydrate demonstrated no observable
increase in brain creatine with proton MRS and no improvement in clinical symptoms. In this study, we report
a novel brain creatine deficiency syndrome most likely
representing a creatine transporter defect.
Ann Neurol 2001;49:401– 404
Creatine (␣-methyl-guanidinoacetic acid) plays an important role in energy metabolism. In humans, creatine
is synthesized in the liver, kidney, and pancreas. Creatine is ultimately transported via the blood to the muscles, heart, and nervous system, which are rich in creatine kinase. Creatine kinase is an essential enzyme to
catalyze phosphorylation of creatine to provide a highenergy phosphate buffer system.
Previous reports describe an inborn error in metabolism representing a severe deficiency of hepatic guanidinoacetate methyltransferase activity [S-adenosylmethionine: guanidinoacetate N-methyltransferase (EC
From the 1Divisions of Radiology, and 2Neurology, Children’s Hospital Medical Center and the University of Cincinnati, Cincinnati,
OH; and 3Metabolic Unit of the Department of Clinical Chemistry, Free University Hospital, Amsterdam, The Netherlands.
Received Jul 17, 2000, and in revised form Nov 3. Accepted for
publication Nov 6, 2000.
Address correspondence to Dr Cecil, Imaging Research Center,
Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail:; GAMT)].1–3 This condition results in an accumulation of guanidinoacetic acid (GAA) and a deficiency of creatine. Neurologic symptoms associated
with creatine deficiency have some variability but
present overall as extrapyramidal movement disorders,
developmental regression, behavioral problems, and intractable epilepsy. Patients with the GAMT defect,
treated with oral creatine, demonstrate improvement of
clinical symptoms and biochemical abnormalities. Residual deficits may arise from neurotoxic GAA accumulation or an imbalance of brain creatine and highenergy phosphates.4 – 6
A technique useful in recognizing creatine deficiencies and other metabolic abnormalities is proton magnetic resonance spectroscopy (MRS). As a complementary technique with magnetic resonance imaging
(MRI), the use of proton MRS in the clinical MR examination provides a noninvasive method for assessing
unique metabolic information for focal and diffuse disorders in vivo.
Creatine signal was absent in multple regions of the
brain of a 6-year-old boy examined with in vivo proton
MRS. Metabolic testing revealed elevated serum and
urine creatine levels but normal plasma and urine GAA
levels. In this study, we report a novel brain creatine
deficiency syndrome hypothesized to be a creatine
transporter defect.
Case Study
A white male was diagnosed at 7 months of age with
mild developmental delay and central hypotonia. Prenatal and perinatal histories were unremarkable. MRI
at 8 months demonstrated prominent extraaxial spaces.
At age 2 years, he was admitted to the hospital in partial status epilepticus. He recovered completely and was
discharged in good condition. An electroencephalograph (EEG) showed multifocal epileptiform discharges. The MRI performed revealed a small focus of
hyperintensity in the right posterior periventricular
white matter on T2-weighted images. A repeat EEG at
age 5 years was unchanged, with no further seizures
reported. A repeat MRI continued to demonstrate a
stable region of T2-weighted hyperintense signal in the
right posterior periventricular white matter. Physical
examination at 6 years of age showed severe delay in
speech and language function. He did not follow commands and spoke only a few single words. Gross and
fine motor functions were normal, but he was mildly
hypotonic. He had a short attention span; however, he
did establish good eye contact. Head circumference increased from the 75th percentile to the 95th percentile,
prompting MRI and proton MRS. He presented with
no other neurological or general physical abnormalities.
The small focus of hyperintense signal in the right posterior periventricular white matter remained unchanged
compared to the previous MRI examinations.
Cecil et al: Creatine Deficiency
Fig 1. (A) T2-weighted image and (B)
initial short (PROBE-PRESS, TE 35
msec, TR 2 sec) and long (PROBEPRESS, TE 272 msec, TR 2 sec) echo
spectroscopy of 6-year-old male patient.
Note the absence of the creatine and
phosphocreatine at 3.0 ppm. The small
resonance at 3.0 ppm most likely represents ␥-aminobutyric acid insofar as the
signal is higher on the short echo scan.
Fig 2. Normal short (PROBE-PRESS, TE
35 msec, TR 2 sec) and long (PROBEPRESS, TE 272 msec, TR 2 sec) echo
spectroscopy of 7-year-old male patient.
Proton MRS was acquired on the patient using
PRESS single voxel sequences [short echo time (TE)
35 msec and long TE 288 msec, repetition time (TR)
2 sec] on 8 cc volumes in the frontal white matter and
basal ganglia. Spectral reconstruction demonstrated an
almost complete loss of creatine and phosphocreatine
signal (Fig 1, compare Fig 2). N-acetyl aspartate and
choline levels were within normal limits for age. Myoinositol levels appeared slightly diminished on comparison with normal control data, with no lactate signal detected in the spectra. The small peak at 3.0 ppm,
where creatine normally appears, was most likely due
to a small concentration of ␥-aminobutyric acid
(GABA). Further off-line processing with custom soft-
Annals of Neurology
Vol 49
No 3
March 2001
ware was performed to determine metabolite concentrations using methods similar to that previously described in the literature.7
The laboratory findings pertaining to creatine and
related metabolites are found in the Table. Creatinine
in blood and amino acid levels in blood and urine were
normal. Other metabolic testing showed serum lactate,
pyruvate, ammonia, electrolytes, glucose, and liver
function tests were normal. Carnitine and acylcarnitine
profiles were also normal. A cytogenetic investigation
of the chromosomes revealed a normal karyotype
(46,XY). With the initial MRS result indicating a creatine deficiency, the patient was put on oral creatine
monohydrate 2,500 mg three times/day (340 mg/kg/day).
Table. Laboratory Results Prior to Oral Creatine Substitution
Serum (␮mol/liter)
Urine (mmol/liter)
Blood (␮mol/liter)
Creatine kinase (U/liter)
Plasma (␮mol/liter)
Urine (mmol/mol creatinine)
A follow-up proton MRS examination performed 4
months later demonstrated a similar absence of creatine. However, creatine in the urine increased tenfold,
and elevated cerebrospinal fluid (CSF) levels were measured [61.7 ␮mol/liter (normal 38 ⫾ 4.8 ␮mol/liter)].
The patient’s clinical symptoms did not improve after 3
months. Oral substitution of creatine was discontinued.
Initial reports of creatine deficiency revealed by proton
MRS of the brain led clinicians to uncover a synthesis
defect in hepatic guanidinoacetate methyltransferase activity.4,8 Three patients with confirmed GAMT defects
demonstrated developmental delay, intractable epilepsy,
and an extrapyramidal movement disorder.1,2,4 Van
der Knaap et al.5 reported GAMT deficiency in two
males without specific neurological findings and a presentation much milder than that of the patients with
the original GAMT defect. Treatment of these patients
with oral creatine substitution resulted in higher concentrations of creatine in the brain as observed by proton MRS. The patients showed marked clinical and
neurologic improvement.
Laboratory results in our patient demonstrate that
the substrates and products involved in creatine synthesis and excretion (glycine, ornithine, GAA, and creatinine) are at normal levels. Supplementation with oral
creatine results in a tenfold increase in urine creatine
concentrations. In an isolated defect in creatine synthesis, treatment with creatine should result in an increased and measurable concentration of creatine in the
brain. Normal adults, who take creatine supplements,
increase their creatine concentration in the CNS as
measured by MRS.9 Therefore, we exclude GAMT deficiency as the source of our patient’s brain creatine
deficiency. Although the main synthesis of creatine
proceeds in liver, it has been reported that rat brain
cells are capable of creatine synthesis.10,11 However,
this work has been demonstrated only in animal cell
cultures. The data from our patient indicates that, if de
novo brain creatine synthesis is occuring, it is not detectable by proton MRS.
We suspected a defect in the creatine transporter of
our patient. Gregor et al12 assigned the human creatine
transporter (CT1) gene (SLC6A8) to chromosome
Xq28. Sora et al.13 demonstrated the presence of a creatine transporter in human brainstem and spinal cord.
The highest levels of creatine transporter mRNA expression are in human skeletal muscle, kidney, and
heart, with lower levels in brain, small and large intestine, epididymis, testis, vas deferens, seminal vesicles,
prostate, and adrenal.14,15 Creatine uptake into tissues
is an active process and has been shown to depend on
sodium and/or chloride channels.16,17 Creatine transport in the brain is hypothesized to be an astroglial
rather than a neuronal function.16,18 Within the brain,
there is a heterogenous, regional distribution of creatine transporter transcripts.15 In situ hybridization
showed high creatine transporter levels in cerebellum
and hippocampus.19 Several neurological, neuromuscular,
and hearing disorders are mapped to Xq28, but the association of the disorder with the creatine transporter is not
known.18 An autosomal, testis-specific form of the human
creatine transporter (CT2) has been assigned to chromosome 16p11.220 but may represent a pseudogene.21
At high serum concentrations, creatine uptake in
some tissues could represent passive diffusion across the
plasma membrane or alternatively transport via other
transporters. The patient’s abilities to include supplemental dietary creatine into the blood stream and to
maintain relatively normal muscle function indicate
some mechanism of transport or compensation.
Two sisters were recently described with a brain creatine deficiency syndrome presumed to be a creatine
tranporter defect in the brain.22 Again, proton MRS of
the brain detected the absence of creatine. Normal creatine and GAA concentrations in the sister’s blood indicated normal creatine synthesis. Mutation detection
in the SLC6A8 gene was not reported. The improvement in clinical symptoms and the increase in CNS
creatine concentration after creatine supplementation
indicate either an X-linked mosaicism with a less severe
mutation in the transporter or a creatine deficiency
syndrome different from that of our patient. Preliminary sequence analysis of amplified cDNA of SLC6A8
skin fibroblasts in our patient indicates a hemizygous
nonsense mutation.
In the GAMT deficiency cases, a mechanism of injury could be inferred from neurotoxic GAA levels.
However, in the cases reported by Bianchi et al.22 and
our patient, a mechanism for injury must arise from
the deficiency of creatine. Unknown is whether another neurotoxin could be responsible for the neurological symptoms.
Our case further demonstrates a utility of MRS in
diagnosing and treating children with neurodevelopmental problems. This patient represents another defect in creatine metabolism of the CNS revealed by
Cecil et al: Creatine Deficiency
MRS. This case also shows the importance of creatine
metabolism for neurological development. Creatine
supplementation may play a role in improving symptoms of neuromuscular disorders.23 Beal and colleagues
report neuroprotective effects of oral supplementation
with creatine in Huntington’s disease, amyotrophic
lateral sclerosis, and other neurodegenerative diseases.24 –27 Creatine may attenuate 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity and
possibly reverse mitochondrial dysfunctions.25 Further
investigations into the metabolism of creatine and
pathophysiology of creatine disorders are necessary.
1. Schulze A, Hess T, Wevers R, et al. Creatine deficiency syndrome caused by guanidinoacetate methyltransferase deficiency:
diagnostic tools for a new inborn error of metabolism [see comments]. J Pediatr 1997;131:626 – 631.
2. Ganesan V, Johnson A, Connelly A, et al. Guanidinoacetate
methyltransferase deficiency: new clinical features. Pediatr Neurol 1997;17:155–157.
3. Stockler S, Isbrandt D, Hanefeld F, et al. Guanidinoacetate
methyltransferase deficiency: the first inborn error of creatine
metabolism in man. Am J Hum Genet 1996;58:914 –922.
4. Stockler S, Holzbach U, Hanefeld F, et al. Creatine deficiency
in the brain: a new, treatable inborn error of metabolism. Pediatr Res 1994;36:409 – 413.
5. van der Knaap MS, Verhoeven NM, Maaswinkel-Mooij P, et
al. Mental retardation and behavioral problems as presenting
signs of a creatine synthesis defect. Ann Neurol 2000;47:540 –
6. Hirayasu Y, Morimoto K, Otsuki S. Increase of methylguanidine and guanidinoacetic acid in the brain of amygdala-kindled
rats. Epilepsia 1991;32:761–766.
7. Kreis R, Ernst T, Ross BD. Absolute concentrations of water
and metabolites in human brain. II Metabolite concentrations.
J Magn Reson Ser B 1993;102:9 –19.
8. Stockler S, Hanefeld F. Guanidinoacetate methyltransferase
deficiency: a newly recognized inborn error of creatine biosynthesis. Wien Klin Wochenschr 1997;109:86 – 88.
9. Dechent P, Pouwels PJ, Wilken B, et al. Increase of total creatine in human brain after oral supplementation of creatinemonohydrate. Am J Physiol 1999;277:R698 –R704.
10. Defalco AJ, Davies RK. The synthesis of creatine by the brain
of the intact rat. J Neurochem 1961;7:308 –312.
11. Dringen R, Verleysdonk S, Hamprecht B, et al. Metabolism of
glycine in primary astroglial cells: synthesis of creatine, serine,
and glutathione. J Neurochem 1998;70:835– 840.
12. Gregor P, Nash SR, Caron MG, et al. Assignment of the creatine transporter gene (SLC6A8) to human chromosome Xq28
telomeric to G6PD. Genomics 1995;25:332,333.
13. Sora I, Richman J, Santoro G, et al. The cloning and expression of a human creatine transporter. Biochem Biophys Res
Commun 1994;204:419 – 427.
14. Nash SR, Giros B, Kingsmore SF, et al. Cloning, pharmacological characterization, and genomic localization of the human
creatine transporter. Recept Channels 1994;2:165–174.
15. Wyss M, Kaddurah-Daouk R. Creatine and creatinine metabolism. Physiol Rev 2000;80:1107–1213.
16. Moller A, Hamprecht B. Creatine transport in cultured cells of
rat and mouse brain. J Neurochem 1989;52:544 –550.
Annals of Neurology
Vol 49
No 3
March 2001
17. Guimbal C, Kilimann MW. A Na(⫹)-dependent creatine transporter in rabbit brain, muscle, heart, and kidney. cDNA cloning and functional expression. J Biol Chem 1993;268:8418 –
18. Hiel H, Happe HK, Warr WB, Morley BJ. Regional distribution of a creatine transporter in rat auditory brainstem: an insitu hybridization study. Hear Res 1996;98:29 –37.
19. Schloss P, Mayser W, Betz H. The putative rat choline transporter CHOT1 transports creatine and is highly expressed in
neural and muscle-rich tissues. Biochem Biophys Res Commun
1994;198:637– 645.
20. Iyer GS, Krahe R, Goodwin LA, et al. Identification of a testisexpressed creatine transporter gene at 16p11.2 and confirmation of the X-linked locus to Xq28. Genomics 1996;34:143–
21. Eichler EE, Lu F, Shen Y, et al. Duplication of a gene-rich
cluster between 16p11.1 and Xq28: a novel pericentromericdirected mechanism for paralogous genome evolution. Hum
Mol Genet 1996;5:899 –912.
22. Bianchi MC, Tosetti M, Fornai F, et al. Reversible brain creatine deficiency in two sisters with normal blood creatine level.
Ann Neurol 2000;47:511–513.
23. Guerrero-Ontiveros ML, Wallimann T. Creatine supplementation in health and disease. Effects of chronic creatine ingestion
in vivo: down-regulation of the expression of creatine transporter isoforms in skeletal muscle. Mol Cell Biochem 1998;
184:427– 437.
24. Klivenyi P, Ferrante RJ, Matthews RT, et al. Neuroprotective
effects of creatine in a transgenic animal model of amyotrophic
lateral sclerosis. Nature Med 1999;5:347–350.
25. Matthews RT, Ferrante RJ, Klivenyi P, et al. Creatine and cyclocreatine attenuate MPTP neurotoxicity. Exp Neurol 1999;
26. Ferrante RJ, Andreassen OA, Jenkins BG, et al. Neuroprotective effects of creatine in a transgenic mouse model of Huntington’s disease. J Neurosci 2000;20:4389 – 4397.
27. Malcon C, Kaddurah-Daouk R, Beal MF. Neuroprotective effects of creatine administration against NMDA and malonate
toxicity. Brain Res 2000;860:195–198.
Reduction of Menkes
mRNA and Copper in
Leukocytes of Patients with
Primary Adult-Onset
Niels Kruse, PhD,1 Daniela Berg, MD,1
Michael J. Francis, DPhil,2 Markus Naumann, MD,1
Wolf-Dieter Rausch, PhD,3 K. Reiners, MD,1
Peter Rieckmann, MD,1 Andreas Weishaupt, PhD,1 and
Georg Becker, MD1
Studies on postmortem tissue of patients with primary
adult-onset dystonia revealed a significant increase in
copper levels and a reduction of copper transporting
Menkes protein of the lentiform nuclei. Here we demonstrate that patients with idiopathic adult-onset cervical
dystonia (n ⴝ 14) have reduced Menkes mRNA copies
and lower copper levels in leukocytes compared to controls (n ⴝ 17; U test, p < 0.05). Changes were less distinct in patients with blepharospasm. Therefore, disturbances of copper metabolism in focal dystonia may not
be restricted to the basal ganglia.
intracellular copper level. This change in copper level
may cause a dysfunction in the basal ganglia loop at
the receptor level.5–7
In this study we evaluated whether disturbances of
copper metabolism are restricted to the basal ganglia or
whether they can also be detected in blood cells from
patients with idiopathic adult-onset dystonia.
Patients and Methods
Twenty-four patients with primary adult-onset focal dystonia
were included in this study. Fourteen patients suffered from
spasmodic torticollis (mean age 51.7 ⫾ 10.6 years, 10 females and 4 males) and 10 from blepharospasm (mean age
71.9 ⫾ 7.7 years, 2 males and 8 females; Table). The mean
duration of the disease was 10.9 ⫾ 8.1 years. The severity of
dystonia in patients with spasmodic torticollis was graduated
according to the Tsui score (mean Tsui score: 13.0 ⫾ 3.2).
All patients with dystonia were treated with botulinum toxin
and responded well to the injections.
For comparison, 17 healthy adults (mean age 42.4 ⫾ 9.5
years, 10 females and 7 males) were included in this study.
Informed consent was obtained from all individuals participating in this study, which was approved by the local ethics
committee. From patients and controls, peripheral blood
mononuclear cells were purified by Lymphoprep gradient
centrifugation (Nycomed, Oslo, Norway) according to the
manufacturer’s instruction and stored at – 80°C until use.
Ann Neurol 2001;49:405– 408
Measurement of Menkes mRNA in Leukocytes
Evidence from recent studies suggests abnormalities in
copper metabolism of patients with idiopathic adultonset dystonia.1,2 These examinations were stimulated
by transcranial sonography (TCS) findings demonstrating abnormal signals at the lentiform nuclei.3,4 Subsequent neurochemical analyses of postmortem tissue of
patients with primary adult-onset dystonia revealed a
significant increase in copper levels of the lentiform
nuclei but not of the thalamus or caudate nucleus.1
One reason for the elevated tissue copper levels may be
a change in the copper-transporting Menkes protein,
which has been found to be reduced in the lentiform
nuclei but not in the thalamus and caudate nucleus.2 A
reduction in Menkes protein expression results in a reduced efflux of copper out of the cell and an increased
From the 1Department of Neurology, Bayerische JuliusMaximilians-Universität, Würzburg, Germany; 2Human Genetics/
Clinical Medicine, Wellcome Trust Center for Human Genetics,
Oxford, United Kingdom; and 3Institute of Medical Chemistry,
University of Veterinary Medicine, Vienna, Austria.
Received Aug 4, 2000, and in revised form Nov 10. Accepted for
publication Nov 11, 2000.
Address correspondence to Dr Becker, Neurologische Klinik und
Poliklinik des Klinikums der Universität Würzburg, JosefSchneider-Str. 11, 97080 Würzburg, Germany.
cytes using the Qiagen RN easy kit (Qiagen GmbH, Hilden,
Germany) according to the manufacturer’s instructions. Purified RNA was eluted with 33 ␮l DEPC-treated water. For
reverse transcription 3 ␮l oligo(dT) (500 ␮g/ml; Pharmacia
Biotech, Freiburg, Germany) were added, incubated for 10
minutes at 70°C, and chilled on ice. After mixing with 12 ␮l
First Strand Buffer and 6 ␮l 0.1M dithiothreitol (DTT; both
from Life Technologies, Karlsruhe, Germany), 3 ␮l 10 mM
dNTPs (Pharmacia Biotech), and 3 ␮l Superscript II reverse
transcriptase (200 U/␮l; Life Technologies), the assays were
incubated for 50 minutes at 42– 45°C. Reverse trancriptase
was denatured by incubation for 10 minutes at 95°C.
chain reaction (PCR) for ␤-actin mRNA quantification was
performed as previously described.8 For quantification of
Menkes mRNA expression, the following oligonucleotides
were used: ATP7A S: 5⬘-CCC GGT TAC CAA TGA GGA
Equivalents of 108 copies of ␤-actin mRNA were used as
templates for semiquantitative PCR of Menkes mRNA. Serial dilutions of standard Menkes cDNA were prepared in 10
mM Tris, pH 8.0, and amplified in parallel with unknown
cDNAs. Each determination was performed in triplicate. Reaction conditions were as described previously.8 All amplifi-
Brief Communication: Kruse et al: Menkes Protein in Dystonia
Table. Characteristics of Patients and Controls, Including Copper and Menkes mRNA Levels in Leukocytes
Focal Dystonia
Total group
Number of subjects
Mean age (years)
Sex (females/males)
Menkes mRNA copies/108 ␤-actin
mRNAs of leukocytesa
Copper level of leukocytes (ng/mg
Serum copper (mg/dl)
59.2 ⫾ 13.9
123.0 (7; 310)b
6.3 (5.7; 7.6)b
111 (98; 123)
51.7 ⫾ 10.6
70.5 (6; 162)b
6.3 (5.6; 6.8)b
111 (89; 125)
71.9 ⫾ 7.7
287 (208; 343)
42.4 ⫾ 9.5
289 (119; 456)
6.9 (6; 8)
7.8 (7; 9.5)
111 (107; 121)
113 (107; 121)
Median values and 25th and 75th quartiles.
Intergroup comparison using the U test revealed a significant difference compared to the control group ( p ⬍ 0.05).
cation reactions were performed on an ABI PRISM 7700
Sequence Detection System.
Determination of Copper Content in Leukocytes
Leukocytes (1.8 ⫻ 107) were suspended in 200 ml
phosphate-buffered saline and 1% Triton X-100 containing
protease inhibitors. Insoluble material was pelleted by centrifugation, and the protein concentration supernatant was
determined by Comassie Protein Assay Reagent (Pierce,
Rockford, IL). Leukocytes suspensions with defined protein
content (2–5 mg/ml) were directly injected into graphite pyrotubes. Following a drying program at 120°C, samples were
ashed at 600°C and measured in duplicate by atomic absorption at 2,700°C using a polarized Zeeman atomic absorption
spectrophotometer (Hitachi Z 8100). Commercial standards
(Merck Titrisol) were diluted and used in a concentration
range of 0 – 8 ␮g/ml. Measurements of copper and Menkes
mRNA were made blindly with respect to the clinical characteristics of the subjects.
Fig 1. Menkes mRNA copies/108 ␤-actin mRNA of leukocytes
from patients with focal dystonia and controls. Differences in
Menkes mRNA copies of patients with focal dystonia (spasmodic torticollis and blepharospasm) and controls were significant (U test, p ⫽ 0.04). In analyzing mRNA levels of both
subgroups of focal dystonia, differences were significant for
patients with torticollis (U test, p ⫽ 0.02) compared to controls but not for patients with blepharospasm (U test, p ⬎
Annals of Neurology
Vol 49
No 3
March 2001
Analyses of leukocyte mRNA revealed a significant decrease of Menkes mRNA in patients with spasmodic
torticollis [70.5 (25th and 27th percentiles, 6; 162)]
compared to controls [289 (25th and 75th percentiles;
119; 456); U test, p ⫽ 0.02]. Menkes mRNA levels of
patients with blepharospasm [287.0 (208; 343)] were
similar to those of controls, reflecting differences in the
severity of these dystonic disorders (Table, Fig 1). In
addition, patients with a more severe spasmodic torticollis (Tsui score ⬎ 13; 9 of the 14 patients) had lower
Menkes mRNA levels [median 22 (2; 92) Menkes
mRNA copies/108 ␤-actin mRNA copies] than less
disabled patients [Tsui score ⱕ 13; 5 of the 14 patients; median 162 (123– 473) Menkes mRNA copies/
108 ␤-actin mRNA copies; U test, p ⫽ 0.04]. No correlation was identified between age and number of
mRNA copies (Spearman’s rank correlation, r ⫽
– 0.07, p ⫽ 0.91), sex and mRNA copies (U test, p ⫽
Fig 2. Leukocytes copper levels of patients with focal dystonia
and healthy controls. Copper levels of patients in both groups
with focal dystonia were significantly reduced compared to
controls (U test, p ⫽ 0.005). Patients with spasmodic torticollis had lower copper levels than those with blepharospasm, and
only for patients with torticollis were differences significant
compared to controls (U test, p ⫽ 0.002).
0.39), or the duration of the disease and number of
mRNA copie (Spearman’s rank correlation, r ⫽ – 0.47,
p ⫽ 0.63).
As with Menkes mRNA, the copper concentrations
of leukocytes in patients with spasmodic torticollis
were lower [6.3 (5.6; 6.8) ng/mg protein] than in patients with blepharospasm [6.9 (6; 8) ng/mg protein]
or controls [7.8 (7.0; 9.5) ng/mg protein] paralleling
changes of Menkes mRNAs (Table, Fig 2). No correlation between Menkes mRNA levels and copper concentration in leukocytes could be detected (Spearman’s
rank correlation, r ⫽ – 0.03, p ⫽ 0.87). Leukocyte
copper levels were not related to age (Spearman’s rank
correlation, r ⫽ – 0.23, p ⫽ 0.14) or sex of patients
and controls (U test, p ⫽ 0.67).
Serum copper levels in patients and controls are outlined in the Table. No differences between patients and
controls and no correlations with other parameters
were obtained.
Our data revealed reduced Menkes mRNA levels in
leukocytes of patients with primary adult-onset cervical
dystonia compared to controls. Menkes mRNA levels
were lower in more severely affected patients. In addition, the copper concentration of leukocytes was lower
in patients with focal dystonia. Reductions of copper
and particularly mRNA levels were less distinct in patients with bleparospasm, which might reflect differences in the pathogenesis or severity of both disorders.
These data support previous findings of a reduced
Menkes protein content in the lentiform nuclei of patients with primary dystonia and indicate that a disturbance in copper metabolism in dystonia is not restricted to the brain. However, we cannot exclude that
Menkes protein synthesis might be influenced by posttranscriptional gene-regulating mechanisms. Menkes
mRNA levels presented in this study represent steadystate mRNA levels, so it is not clear whether they are
the result of decreased levels of gene expression or an
increased rate of mRNA degradation.
Menkes protein is a membrane-bound protein
thought to be involved in both the sequestration of
copper to essential copper requiring enzymes and the
efflux of copper from the cell.9,10 In humans, expression has been detected in all tissues tested except the
liver. Menkes protein transports copper into the lumen
of the Golgi complex. In case of intracellular copper
levels, Menkes protein effluxes copper out of the cell. A
reduction in the levels of Menkes protein results in an
increase in intracellular copper levels. Menkes protein
reduction will mainly affect the intestinum and the
brain, notably the lentiform nuclei; these areas exhibit
the highest copper turnover of Menkes proteinexpressing cells.11 Although Menkes protein reduction
causes accumulation of copper in some tissues,12,13
body copper content decreases as a result of the reduced transportation of copper across the intestinal
cells.14 This “mosaic pattern” of cellular copper concentration typical for patients with Menkes protein deficiency was also observed in patients with primary dystonia: A recent postmortem study revealed an increased
copper level in the lentiform nuclei of patients with
idiopathic dystonia,1 whereas copper levels of leukocytes were found to be reduced in this study.
Changes in tissue copper levels may have a substantial influence on the activity of the basal ganglia loop
neurons; copper is an endogenous modulator of synaptic function and a potent inhibitor of several receptor
types, such as glutamate, opiate, and GABAA receptors,
all of which are highly expressed in the basal ganglia.5–7 Therefore, one may postulate a relation between Menkes protein depletion, increased copper tissue level in the lentiform nuclei, changes in the
neuronal activity of the basal ganglia loop, and subsequently dystonic movements.
In analyzing our data, one may question whether
Menkes mRNA depletion is the central pathogenetic
process in dystonia because of the broad overlap of
Menkes mRNA copies and copper levels in patients
and controls. In addition, Menkes disease, the disorder
associated with Menkes protein depletion, is phenotypically very different from dystonia,14 although one report mentioned extrapyramidal symptoms in milder
forms of Menkes disease.15 Therefore, the reduction in
Menkes mRNA might reflect the consequence of an as yet
unidentified primary pathology.
The authors thank S. Hellmig for her excellent technical support.
We gratefully acknowledge Prof. K. V. Toyka for his continuous
support and most helpful comments on the manuscript.
1. Becker G, Berg D, Rausch WD, et al. Increased tissue copper
and manganese content in the lentiform nucleus in primary
adult-onset dystonia. Ann Neurol 1999;46:260 –263.
2. Berg D, Weishaupt A, Francis MJ, et al. Changes of coppertransporting proteins and ceruloplasmin in the lentiform nuclei
in primary adult-onset dystonia. Ann Neurol 2000;47:827–
3. Naumann M, Becker G, Toyka KV, et al. Lenticular nucleus
lesion in idiopathic dystonia detected by transcranial sonography. Neurology 1996;47:1284 –1290.
4. Becker G, Naumann M, Scheubeck M, et al. Comparison of
transcranial sonography, magnetic resonance imaging, and single photon emission computed tomography findings in idiopathic spasmodic torticollis. Mov Disord 1997;12:79 – 88.
5. Sharonova IN, Vorobjev VS, Haas HL. High-affinity copper
block of GABA(A) receptor-mediated currents in acutely isolated cerebellar Purkinje cells of the rat. Eur J Neurosci 1998;
6. Weiser T, Wienrich M. The effects of copper ions on glutamate
Brief Communication: Kruse et al: Menkes Protein in Dystonia
receptors in cultured rat cortical neurons. Brain Res 1996;742:
Sadee W, Pfeiffer A, Herz A. Opiate receptor: multiple effects
of metal ions. J Neurochem 1982;39:659 – 667.
Kruse N, Pette M, Toyka KV, Rieckmann P. Quantification of
cytokine mRNA expression by RT PCR in samples of previously frozen blood. J Immunol Methods 1997;210:195–203.
La Fontaine SL, Firth SD, Camakaris J, et al. Correction of the
copper transport defect of Menkes patient fibroblasts by expression of the Menkes and Wilson ATPases. J Biol Chem 1998;
Francis MJ, Jones EE, Levy ER, et al. A Golgi localization signal identified in the Menkes recombinant protein. Hum Mol
Genet 1998;7:1245–1252.
Turnlund JR. Human whole-body copper metabolism. Am J
Clin Nutr 1998;67(5 Suppl):960S–964S.
Danks DM, Cartwright E, Stevens BJ, Townley RRW. Menkes’ kinky hair disease: further definition of the defect in copper
transport. Science 1973;179:1140 –1142.
Kodama H, Abe T, Takama M, et al. Histochemical localization of copper in the intestine and kidney of macular mice:
light and electron microscopic study. J Histochem Cytochem
1993;41:1529 –1535.
Menkes JH. Kinky hair disease: twenty five years later. Brain
Dev 1988;10:77–79.
Haas RH, Robinson A, Evans K, et al. An X-linked disease of the
nervous system with disordered copper metabolism and features
differing from Menkes disease. Neurology 1981;31:852– 859.
Anticardiolipin Antibodies
Are Not a Useful Screening
Tool in a Nonselected Large
Group of Patients with
Multiple Sclerosis
Jaume Sastre-Garriga, MD,1 Juan Carlos Reverter, MD,2
Josep Font, MD, PhD,3 Mar Tintoré, MD,1
Gerard Espinosa, MD,3 and Xavier Montalban, MD, PhD1
Recent works claiming that primary antiphospholipid
syndrome (PAPS) cannot be clinically distinguished from
multiple sclerosis (MS) recommend that MS patients be
screened for anticardiolipin antibodies (ACA). In this
study 296 randomly selected patients with MS and clinically isolated syndromes and 51 healthy controls were
analyzed; ACA, anti-␤2-glycoprotein I, or antiprothrombin was found in 6 patients. No predominance of any
kind of clinical manifestations and no cardinal manifestations of PAPS were found in these patients. ACA tests
should be performed only when a suspicion of PAPS is
raised and atypical clinical presentation for MS is found.
Ann Neurol 2001;49:408 – 411
Annals of Neurology
Vol 49
No 4
April 2001
Antiphospholipid antibodies (aPL) are associated with
venous and arterial thrombosis and recurrent spontaneous abortions or fetal losses.1,2 They may occur with
no other manifestation of systemic disease, a scenario
known as primary antiphospolipid syndrome (PAPS) or
Hughes syndrome.3 Because IgG anticardiolipin antibodies (ACA) are also present in healthy populations,
positivity in a person with no signs or symptoms of
PAPS and no associated connective tissue disorder will
lack significance. Recently, ␤2-glycoprotein I (␤2GPI)
has been shown to be the real antigen of ACA.4 – 6 Although several attempts have been made to correlate
ACA with specific neurological complaints, such as migraine, epilepsy, myelitis, optic neuritis, or vascular dementia, most of these associations have never been
clearly established.
Four recent studies have examined the presence of
ACA, stressing the significance in patients with multiple sclerosis (MS).7–10 These studies claim that PAPS
must be considered as an alternative diagnosis to MS,
and even that MS cannot be differentiated from PAPS
according only to clinical and magnetic resonance imaging (MRI) findings, and a recommendation to test
ACA in MS populations routinely is, therefore, made.
To address the question of whether it is necessary to
test MS patients for ACA, we performed tests for ACA,
anti-␤2-glycoprotein I antibodies (a␤2-GPI), and antiprothrombin antibodies (aPT) in a large, unselected
population of MS patients. We also reviewed the medical records of ACA- or a␤2-GPI-positive patients to
determine whether a specific clinical subset of patients
was linked to this serological pattern.
Materials and Methods
Patients and Controls
Samples from 251 patients with MS [109 relapsing-remitting
(RRMS), 67 secondary progressive (SPMS), and 75 transitional and primary progressive (PPMS)] from a cohort of
1,400 MS patients assisted at our outpatient MS unit and
from 45 patients with clinically isolated syndromes (CIS) of
the central nervous system suggestive of demyelinating disease were randomly selected (with a random-number generator) from our serum bank. Demographic features of these
patients were reviewed. The MS patient population was
From the 1Unitat de Neuroimmunologia Clı́nica, H. Universitari
Vall d’Hebron, 2Hemotherapy and Hemostasis Department, Institut
d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clı́nic, and 3Systemic Autoimmune Diseases Unit, IDIBAPS,
Hospital Clı́nic, Barcelona, Spain.
Received Jul 25, 2000, and in revised form Oct 30. Accepted for
publication Nov 22, 2000.
Address correspondence to Xavier Montalban, Unitat de Neuroimmunologia Clı́nica, H. Universitari Vall d’Hebron, Escola
d’Infermeria, 5a planta, Passeig de la Vall d’Hebron 119 –129,
08035 Barcelona, Spain. E-mail:
Table 1. Neurological and PAPS-Related Manifestations in ACA and/or a␤2-GPI-Positive Patients
Patient Number
(age in yr/sex)
1 (23/female)
2 (58/male)
3 (33/female)
4 (48/female)
5 (40/male)
6 (50/male)
Location of Symptomatic
Lesion in Relapses
Spinal cord/brainstem
Optic nerve
Spinal cord
Optic nerve
Myelopathy (no relapses)
and Other
MS Therapy
IFN␤1b vs placebo
RR ⫽ relapsing-remitting; SP ⫽ secondary progressive; PP ⫽ primary progressive; EDSS ⫽ Expanded Disability Status Scale; PAPS ⫽ primary antiphospholipid syndrome;
IFN␤ ⫽ interferon-beta; IVIG ⫽ intravenous immunoglobulin; AZA ⫽ azathioprine; ACA ⫽ anticardiolipin antibody; a␤2-GPI ⫽ anti-␤2/glycoprotein I antibody.
Thrombotic events and/or recurrent miscarriages.
composed of 95 men and 156 women, and in the CIS group
there were 22 men and 23 women. The mean age of both
MS and CIS groups was 38.7 years [standard deviation (SD)
11.7 years]. Fifty-one serum samples from presumed healthy
controls were picked from the blood bank of our hospital;
there were 32 men and 19 women with a mean age of 41.4
years (SD 11.5 years).
ACA, a␤2-GPI, and aPT Tests
ACA were measured using standardized ELISA.11 Results
were expressed in IgG and IgM units (GPL and MPL) and
reported as negative (⬍15 units), low-positive (16 –25 units,
⫹), moderate positive (26 – 40 units, ⫹⫹), and high positive
(⬎40 units, ⫹⫹⫹). Detection of a␤2-GPI was done by a
standardized enzyme-linked immunosorbent assay (ELISA)
previously described12; aPT were measured using an ELISA
kit. Optical density at 492 nm (OD492) was measured for
both aPT and a␤2-GPI assays. OD492 values higher than 5
SD above the mean of negative controls were considered positive, with low positive between 5 and 7 SD (⫹), moderate
between 7 and 9 SD (⫹⫹), and high when above 9 SD
Laboratory Tests
ACA or a␤2-GPI were found in 6 patients (Table 1).
No patients were positive for aPT. No controls had
positive titers for any of the antibodies tested.
Clinical Findings in ACA- and/or a␤2-GPI-Positive
Table 1 summarizes neurological and PAPS-related
manifestations in the 6 positive patients. There was no
predominance of any type of clinical manifestation in
relapses of patients with RRMS or SPMS. None of the
patients presented thrombotic events, recurrent miscarriages, or livedo reticularis. No patient presented valvulopathy, pulmonary hypertension, or chorea, which
are clinical manifestations thought to be related to
Table 2 summarizes paraclinical studies performed in
the 6 positive patients. In all patients, visual, somatosensory, and brainstem auditory evoked potentials
Table 2. Paraclinical Studies in ACA And/or a␤2-GPIab-Positive Patients
Altered Evoked
MRI Suggestive
of MS
Syphilis Reaginic
and BAEP
SSEP and
⫹ ⫽ Positive; ⫺ ⫽ negative; N ⫽ normal; Np ⫽ not performed; SSEP ⫽ somatosensory evoked potentials; VEP ⫽ visual
evoked potentials; BAEP ⫽ brainstem auditory evoked potentials; MRI ⫽ magnetic resonance imaging; MS ⫽ multiple sclerosis;
OCB ⫽ oligoclonal bands; aPTT ⫽ activated partial thromboplastin time; ANA ⫽ antinuclear antibodies.
All three kinds of evoked potentials were performed in all patients.
Brief Communication: Sastre-Garriga et al: MS and Antiphospholipid Antibodies
(VEP, SSEP, and BAEP, respectively) were performed.
All patients had MRI scans that fulfilled Paty criteria
for MS.13 Two patients had antinuclear antibodies
(ANA) in low titers (1/40 for patient 1, 1/80 for patient 6). Patient 1 had false-positive reaginic syphilis
serology and oligoclonal bands (OCB).
Our work shows that there is no higher prevalence of
ACA than expected (based on our own control population) in the largest unselected MS patients population
tested to date. A recent communication by Cuadrado
et al7 supports the idea that it is not possible to distinguish the neurological manifestations of PAPS from
those of MS, and so it is mandatory to test all MS
patients for ACA routinely. This the authors concluded
after examining 27 patients referred to their lupus
clinic because of symptoms suggesting underlying connective tissue disease, MRI findings uncommon for
MS, atypical evolution of MS, or ACA positivity when
a diagnosis of MS had been made by a neurologist. We
agree that in the face of such a clinical setting it is
difficult to be satisfied with a diagnosis of MS; however, this should not be applied to all MS patients.
From the work of Cuadrado et al, it might be expected
that every patient diagnosed with MS would be under
suspicion of being a symptomatic PAPS patient (because, as the authors conclude, it is impossible to distinguish between the two entities). As a consequence,
every patient diagnosed with MS must be tested for
ACA and, when positive, treated. Nevertheless, in their
study, patients were well-grouped, and the suspicion of
PAPS was correct.
In 1998, Karussis et al10 screened a population of 70
classic and 100 “nonclassic” MS patients, labeling as
“nonclassic” those with features unusual for MS. They
found a strikingly significant proportion of ACApositive patients in the nonclassic MS group. Most of
these positive patients in the nonclassic MS group had
a similar clinical manifestations pattern, such as progressive myelopathy (which is really not surprising, insofar as patients were selected for this reason), spinocerebellar syndrome, or neuromyelitis optica. They
concluded that this clinical subset of patients has ACA
positivity as a defining feature and recommended performing this test in patients with MS showing such
clinical features.
On the other hand, D’Olhaberriague et al14 found
no differences in ACA positivity between MS patients
and patients with other neurological diseases. Tourbah
et al15 found a 6.2% prevalence of ACA among 161
MS patients. Cordoliani et al16 found an 8% prevalence among 62 consecutive MS patients (other than
the present study, this is the only study testing for
ACA, a␤2-GPI, and aPT).
Although with some controversy,17 there is wide
Annals of Neurology
Vol 49
No 4
April 2001
agreement for therapeutic abstinence in asymptomatic
patients with ACA antibodies.18 At this point we need
not remind the reader that MS is an exclusion diagnosis or that patients with other neurological conditions
may fulfill criteria for MS19 (for instance, a patient
with two lacunar infarcts). Also, clinical criteria for MS
were designed, as stated by their authors, as a guideline
for research protocols. PAPS has been said to mimic
MS.20 After careful reading of these case reports, it becomes clear that neurologists would never have taken
some of the cases as suspected MS. Otherwise, clinical
criteria for MS are useful when clinical suspicion is
strong enough, and they should not be used to screen
populations with relapsing neurological disturbances.
Therefore, we must be cautious in making such a diagnosis when atypical features are present, and we must
always seek other possible diagnoses, including PAPS.
Now that a new set of diagnostic criteria for MS is
under development, it might well be useful to include
the clinical and paraclinical manifestations of PAPS
(venous and arterial thrombosis and recurrent spontaneous abortions or fetal losses, low platelet counts, positive reaginic serologies, and high activated partial
thromboplastin time ratios) as red flags that should
prompt us to perform ACA and preferably a␤2-GPI
tests. Our results show that it is not useful to test a
large unselected MS population for ACA antibodies.
The manifestations of PAPS are related more to a␤2GPI than to ACA.12 Positivity for a␤2-GPI was found
in 3 patients only. In patient 6 (PPMS) SSEP and
BAEP were pathologic, VEP were normal, and the
MRI showed medullary and supratentorial lesions suggestive of MS; ANA were positive with a titer of 1/80,
and OCB were not detected. The question therefore
arises of whether this patient has actual symptomatic
ACA with neurological manifestations or whether the
patient has a PPMS plus asymptomatic ACA. Patient 1
has RRMS with 5 relapses and MRI highly suggestive
of MS; a false-positive reaginic syphilis test was detected, and ANA were positive (titer 1/40); OCB were
detected. No other relevant findings suggesting PAPS
were encountered. In this case we are prone to think
that we are dealing with RRMS with asymptomatic
ACA. Patient 5 (SPMS) received intravenous immunoglobulin treatment for 6 days before blood was collected for serology, and the results are thus invalidated
for the purpose of this study. We found no relationship between positivity for ACA antibodies and MS
clinical profiles.
In summary, our work, performed on the largest unselected MS population to date, supports the insight
that ACA and, preferably, a␤2-GPI tests should be performed only when a suspicion of PAPS is raised and
atypical clinical presentation for MS is ascertained.
Screening for these antibodies in MS patients with no
clinical or laboratory manifestations of PAPS is not
useful. Furthermore, after careful analysis of the positive patients in our cohort, we believe that this positivity, when found in isolation, could be taken as incidental and so should not be treated.
The authors are indebted to Mr. J. Graells for help with the
1. Hughes GRV, Harris EN, Charavi AE. The anticardiolipin syndrome. J Rheumatol 1986;13:486 – 489.
2. Merkel PA, Chang Y, Pierangeli SS, et al. The prevalence and
clinical associations of anticardiolipin antibodies in a large inception cohort of patients with connective tissue diseases. Am J
Med 1996;101:576 –583.
3. Hughes GRV. Thrombosis, abortion, cerebral disease and the
lupus anticoagulant. Br Med J 1983;287:1088 –1089.
4. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Antiphospholipid antibodies are directed against a complex antigen
that includes a lipid-binding inhibitor of coagulation: beta
2-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA
1990;87:4120 – 4124.
5. Tsusumi A, Matsuura E, Ichikawa K, et al. Antibodies to beta
2-glicoprotein I and clinical manifestations in patients with systemic lupus erythematosus. Arthritis Rheum 1996;39:1466 –
6. Inanç M, Donohoe S, Ravijaran CT, et al. Anti-beta2glycoprotein I, antiprothrombin and anticardiolipin antibodies
in a longitudinal study of patients with systemic lupus erythematosus and the antiphospholipid syndrome. Br J Rheumatol
1998;37:1089 –1094.
7. Cuadrado MJ, Khamashta MA, Ballesteros A, et al. Can neurological manifestations of Hughes (antiphospholipid) syndrome be distinguished from multiple sclerosis? Analysis of 27
patients and review of the literature. Medicine 2000;79:57– 68.
8. Ijdo JW, Conti-Kelly AM, Greco P, et al. Anti-phospholipid
antibodies in patients with multiple sclerosis and MS-like
illnesses: MS or APS? Lupus 1999;8:109 –115.
9. Fukuzawa T, Morikawa F, Mukai M, et al. Anticardiolipin antibodies in Japanese patients with multiple sclerosis. Acta Neurol Scand 1993;88:184 –189.
10. Karussis D, Leker RR, Ashkenazi A, Abramski O. A subgroup
of multiple sclerosis patients with anticardiolipin antibodies and
unusual clinical manifestations: do they represent a new nosological entity? Ann Neurol 1998;44:629 – 634.
11. Cervera R, Font J, López-Soto A. Isotype distribution of anticardiolipin antibodies in systemic lupus erythematosus: prospective analysis of a series of 100 patients. Ann Rheum Dis 1990;
49:109 –113.
12. Teixidó M, Font J, Reverter JC, et al. Anti-␤2glycoprotein I
antibodies: a useful marker for the antiphospholipid syndrome.
Br J Rheumatol 1997;36:113–116.
13. Paty DW, Oger JJ, Kastrukoff LF, et al. MRI in the diagnosis
of MS: a prospective study with comparison of clinical evaluation, evoked potentials, oligoclonal banding, and CT. Neurology 1988;38:180 –185.
14. D’Olhaberriague L, Levine SR, Salowich-Palm L, et al. Specificity, isotype and titer distribution of anticardiolipin antibodies
in CNS diseases. Neurology 1998;51:1376 –1380.
15. Tourbah A, Clapin A, Gout O, et al. Systemic autoimmune
features and multiple sclerosis: a 5-year follow-up study. Arch
Neurol 1998;55:517–521.
16. Cordoliani MA, Michon-Pasturel U, Rerat K, et al. Multiple
sclerosis and antiphospholipid antibodies: study of 62 consecutive patients. Rev Med Intern 1998;19:635– 639.
Martini A, Ravelli A. The clinical significance of antiphospholipid antibodies. Ann Med 1997;29:159 –163.
Ordi-Ros J, Perez-Peman P, Monasterio J. Clinical and therapeutic aspects associated to phospholipid binding antibodies
(lupus anticoagulant and anticardiolipin antibodies). Haemostasis 1994;24:165–174.
Poser CM, Paty DW, Scheinberg L, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols.
Ann Neurol 1983;13:227–231.
Scott TF, Hess D, Brillman J. Antiphospholipid antibody syndrome mimicking multiple sclerosis clinically and by magnetic
resonance imaging. Arch Intern Med 1994;154:917–920.
Low Cerebral Blood Flow
Velocity and Risk of White
Matter Hyperintensities
Christophe Tzourio, MD, PhD,1 Claude Lévy, MD,2
Carole Dufouil, PhD,1 Pierre-Jean Touboul, MD,3
Pierre Ducimetière, PhD,4 and
Annick Alpérovitch, MD, MSc1
Cerebral blood flow velocity (CBF-V) measured by transcranial doppler was assessed in 628 elderly individuals
who had cerebral magnetic resonance imaging performed
as part of a population-based study on vascular aging.
Cerebral white matter hyperintensities (WMHs) were associated with low CBF-V, such as the adjusted odds ratios of severe WMHs from highest (referent) to lowest
quartile of mean CBF-V were 1.0, 1.7, 3.7, and 4.3 ( p ⴝ
0.001). Further, CBF-V was found to be a stronger risk
factor for WMHs than high blood pressure. These findings suggest that the assessment of CBF-V might be a
powerful tool in future studies on WMHs.
Ann Neurol 2001;49:411– 414
White matter
bral magnetic
sociated with
hyperintensities (WMHs) seen on cereresonance imaging (MRI) have been ascognitive decline and dementia,1–3 gait
depression,5 and risk of future stroke.6
From the 1INSERM (National Institute for Health and Medical
Research) U360, Hôpital Pitié-Salpêtrière, Paris; 2Department of
Radiology Institut hospitalier Jacques Cartier, Massy; 3Department
of Neurology Hôpital Lariboisière, Paris; and 4INSERM U258, Hôpital Paul Brousse, Villejuif, France.
Received Jul 1, 2000, and in revised form Nov 11. Accepted for
publication Nov 11, 2000.
Address correspondence to Dr Tzourio, INSERM U360, Hôpital
de la Salpêtrière, 75651 Paris cedex 13, France.
Brief Communication: Tzourio et al: Cerebral Blood Velocity and White Matter Hyperintensities
They have been shown to be related to vascular risk
factors—in particular hypertension—and are generally
interpreted as a marker of cerebral ischemia due to diffuse small vessel disease.7,8 However, despite their importance, little is known about the etiology of WMHs.
It has been suggested that changes in cerebral blood
flow might play a role in the occurrence of WMHs.7,8
Transcranial doppler is an established, non-invasive
method of assessing cerebral hemodynamics through
the measurement of cerebral blood flow velocities
(CBF-V). The aim of the study presented here was to
analyze the relationship between WMHs and CBF-V
in a large sample of elderly individuals from the general
The EVA (Epidemiology of Vascular Aging) study has already been described previously.9 The sample included 834
participants (484 [58%] women, mean age [SD] of 68.9
years [2.9], age range, 63 to 75 years) who participated in the
4-year follow-up of the EVA study and had a cerebral MRI.
Cerebral MRI was performed using a 1.0 T scanner (Siemens, Munich, Germany). WMHs were read on the T2
weighted acquisition, including a fast multislice double echo
T2-weighted axial acquisition (repetition time [TR] ⫽3500
msec, echo time [TE] ⫽ 140 msec) and a proton density
axial acquisition (TR ⫽ 3500 msec, TE ⫽ 85 msec) with 26
slices 5 mm thick. The images were rated visually with respect to the presence of WMHs using a modified version of
the scale of Sheltens et al.10 It provided an overall WMHs
grade ranging from A (no lesion) to D. All the ratings were
done by a trained medical doctor who was blind to any clinical data or diagnoses. After the rater had read all the MRIs,
100 MRIs were randomly selected and reread by the same
rater. The intra-reader kappa coefficient obtained for the
overall grading was 0.76. Because only 12 subjects had no
WMH (grade A), it was decided to pool grade A and B. In the
rest of the paper the three grades will be denominated mild
(grade A and B), moderate (grade C), and severe (grade D).
Transcranial Doppler
Transcranial doppler was accepted by all participants.
Exams were performed by four sonographers with a
2-MHz pulsed wave probe (Transflo, Atys Medical,
France). The study was performed in a quiet room,
with participants lying in a supine position without
any visual or auditory stimulation. Middle cerebral artery was insonated on both sides and the mean of both
CBF-V values was used in the analyses. CBF-V was
missing in 206 participants (24.7%) because of the absence of bilateral temporal window. Participants with
missing CBF-V were older (69.6 [2.9] vs 68.7 [2.9];
p ⬍ 0.001), more frequently women (76.7% vs 51.9%;
p ⫽ 0.006), and had more severe WMHs (23.3% vs
15.0%; p ⫽ 0.006) than the rest of the population.
There were no other significant differences between
both groups concerning body mass index, systolic or
diastolic blood pressure, and hypertension. One of the
sonographers did not report correctly mean velocity
and these values were not taken into account in the
analyses. Common carotid artery intima-media thickness was measured with a 7.5-MHz transducer (SSD650, Aloka, Tokyo, Japan), as previously described.11
Statistical Analysis
The association between WMHs and mean CBF-V was expressed in terms of odds ratio (OR) estimated by quartile of
CBF-V. Multiple logistic regression analysis was used to evaluate the association after adjustment on potential confounders by a backward elimination procedure. The 95% confi-
Table 1. Clinical Characteristics of Participants, According to WMHs Grade
Grade of WMHs
Women (%)
Age, years (SD)
Body mass index, kg/m2 (SD)
Systolic BP, mmHg (SD)
Diastolic BP, mmHg (SD)
Hypertensionb (%)
Tobacco smokingc (%)
Diabetes (%)
Intima-media thicknessd, mm (SD)
Mild (n ⫽ 251)
Moderate (n ⫽ 283)
Severe (n ⫽ 94)
68.3 (3.0)
25.9 (3.6)
133.3 (18.0)
76.6 (10.2)
6.9 (1.1)
68.9 (2.7)
25.6 (3.8)
136.0 (18.0)
77.0 (11.0)
7.0 (1.2)
69.2 (3.1)
25.8 (3.3)
138.1 (19.5)
79.1 (10.5)
7.2 (1.1)
p for trend, obtained by regression analysis for quantitative variables and with Mantel-Haenszel ␹2 statistic for qualitative
Defined as a systolic BP ⱖ 160 mmHg or a diastolic BP ⱖ 95 mmHg or being under antihypertensive treatment.
Present or ex-smokers.
Common carotid artery intima-media thickness measured longitudinally.
WMHs ⫽ white matter hyperintensities; BP ⫽ blood pressure.
Annals of Neurology
Vol 49
No 3
March 2001
Table 2. Cerebral Blood Flow Velocities According to Grade of WMHs
Grade of WMHs
Transcranial doppler
Mild (n ⫽ 251)
Moderate (n ⫽ 283)
Severe (n ⫽ 94)
Systolic velocity, m/s (SD)
Diastolic velocity, m/s (SD)
Mean velocity, m/s (SD)
Resistance index (SD)
Pulsatility index (SD)
69.9 (11.5)
30.7 (8.2)
46.7 (10.4)
0.56 (0.10)
0.86 (0.23)
68.7 (11.8)
28.5 (7.8)
44.1 (9.6)
0.59 (0.10)
0.91 (0.23)
64.2 (10.8)
27.1 (8.0)
41.4 (10.4)
0.58 (0.10)
0.94 (0.30)
p for linear trend.
WMHs ⫽ white matter hyperintensities.
dence interval (CI) are given for each OR, and all p values
are 2-tailed. Statistical analyses were performed using the
SAS 8.0 software package (SAS Inc. Cary, NC).
In Table 1 clinical characteristics of the participants are
indicated by grade of WMHs. There was a strong positive association between age and grade of WMHs. Frequency of hypertension, levels of systolic and diastolic
blood pressure, and common carotid artery intimamedia thickness also increased with increasing grade of
WMHs (Table I).
Table 2 shows the relationship between grade of
WMHs and cerebral blood flow velocities and indexes.
There was a highly significant negative association between velocities and grade of WMHs: the higher the
grade the lower were the velocities. There was also a
significant trend of increasing resistance and pulsatility
indexes with WMHs grade. With regard to potential
confounders, age, hypertension, diabetes, body mass
index, and hematocrit were significantly associated
with lower velocities and were therefore included in
multivariate models (data not shown).
ORs of moderate and severe WMHs by quartiles of
Table 3. Risk of Moderate and Severe WMHs According to
Quartiles of CBF-V
Risk of Moderate
Risk of Severe
of mean
ORb (95% CI) p
ORb (95% CI) p
1.4 (0.8–2.3)
1.6 (0.9–2.7)
2.3 (1.3–4.1)
1.7 (0.7–4.2)
3.7 (1.6–8.8)
4.3 (1.8–10.6)
Decreasing values of mean CBF-V stratified by quartiles; range of
velocities for each quartile are as follows: ⬎51 m/s; 45.5 to 51 m/s;
38.5 to 45 m/s; ⬍38.5 m/s.
Adjusted for age, sex, BMI, hypertension, diabetes, hematocrit,
intima-media thickness.
Reference category.
WMHs ⫽ white matter hyperintensities; CBF-V ⫽ cerebral blood
flow velocity; OR ⫽ odds ratio; BMI ⫽ body mass index.
mean velocity were estimated in multiple logistic regression models adjusting for potential confounders
(Table 3). The risks of moderate and severe WMHs
increased continuously with decreasing mean velocity.
The adjusted ORs of severe WMHs from highest (referent) to lowest quartile of mean CBF-V were 1.0, 1.7,
3.7, and 4.3 ( p ⫽ 0.001).
We then compared the effect of age, systolic blood
pressure, and mean CBF-V on the risk of WMHs by
comparing the ORs of severe WMHs for the highest
versus the lowest quartile for each variable. For each
variable, the ORs estimated in separate models were all
significant: OR ⫽ 2.4 (95% CI, 1.4 – 4.2 ; p ⫽ 0.003)
for age, 2.1 (95% CI, 1.1– 4.1; p ⫽ 0.023) for systolic
blood pressure, and OR ⫽ 4.3 (95% CI, 1.8 –10.6;
p ⫽ 0.001) for mean CBF-V. When age, systolic blood
pressure, and mean CBF-V were included in the same
model, the only OR remaining significant was for
mean CBF-V: OR ⫽ 4.7 (95% CI, 1.7–12.6; p ⫽
0.002). Age and systolic blood pressure were no more
associated with the risk of severe WMHs.
In this population-based study we found that WMHs
were strongly associated with CBF-V. The adjusted
ORs of severe WMHs from highest (referent) to lowest
quartile of mean CBF-V were 1.0, 1.7, 3.7, and 4.3
( p ⫽ 0.001). This relationship was independent of age
and hypertension and seems to explain in part the association between these variables and WMHs.
Previous cross-sectional studies have shown that patients with WMHs had a decreased cerebral blood
flow12 or an impaired cerebral autoregulation.13,14
However, because of the complex methods used (single
photon emission computed tomography or positron
emission tomography scan) these studies were done on
small samples of selected patients. The CBF-V is an
indirect estimate of actual blood flow,15 and the equivalence between both variables, although suggested by
some studies,15–17 have been challenged by others.18,19
It has been shown that, when interindividual variability
of CBF-V was reduced, changes in CBF-V reflect ac-
Brief Communication: Tzourio et al: Cerebral Blood Velocity and White Matter Hyperintensities
curately changes in cerebral blood flow.15,18 It can
therefore be assumed that in a large, population-based
group of individuals with ultrasonic examinations performed in the same conditions, CBF-V is a good surrogate of cerebral blood flow and that the differences
of CBF-V observed between grades of WMHs actually
reflect differences of cerebral blood flow.15
The relationship observed between CBF-V and
WMHs is only an association, the direction of which
remains speculative. The most plausible explanation is
that the hypertension-related small vessel disease causes
a decrease in CBF-V and cerebral blood flow, which
would contribute to the occurrence of WMHs. Other
explanations may however be considered. It could be
suggested that the differences of CBF-V between
grades of WMHs were actually partly due to cognitive
decline. The reduced CBF-V in patients with severe
WMHs would be related to a reduced metabolic demand in subjects having a cognitive deterioration.20
However, when cognitive measures like the MiniMental State Examination, trail making test, or Wechsler test were included in the models the relationship
between CBF-V and WMHs was unchanged (data not
shown). Further, when participants with severe cognitive decline were excluded,9 the adjusted OR of severe
WMHs for the quartile of lowest mean velocity was
3.7 ( p ⫽ 0.005), very similar to what was observed in
the full sample. These data therefore suggest that the
relationship between CBF-V and WMHs is not related
to cognitive decline, although this could be definitively
established only in longitudinal studies.
Apart from pathophysiological considerations, our
study has shown that CBF-V is a stronger risk factor
for WMHs than age and high blood pressure, which
are the most important known risk factors for WMHs.
These results suggest that transcranial doppler may be a
powerful tool to help to identify groups of patients exposed to the risk of WMHs and to the associated risk
of cognitive decline,1–3 and future stroke.6 They also
raise the possibility that therapeutic interventions aiming at reducing the risk of WMHs in hypertensives or
other high-risk groups of individuals could be selected
on their effect on CBF-V. This will be best explored in
longitudinal studies and therapeutic trials.
The EVA study was carried out under an agreement between the
INSERM and the Merck, Sharp and Dhome-Chibret Laboratories
(West-Point, PA). It is also supported by the Eisai Company
The authors are grateful to Dr Pierre Démolis for his helpful comments during the analysis of the data. We thank the ultrasound
physicians, Drs J.M. Fève, C. Leroux, I. Ruelland, and C. Magne,
who performed the examinations, and Dr V. Besançon for help with
the interpretation of the MRIs.
Annals of Neurology
Vol 49
No 4
April 2001
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