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Progressive Loss of Cardiac
Sympathetic Innervation in
Parkinson’s Disease
Sheng-Ting Li, MD, PhD, Raghuveer Dendi, MD,
Courtney Holmes, CMT,
and David S. Goldstein, MD, PhD
This study addressed whether cardiac sympathetic
denervation progresses over time in Parkinson’s disease. In 9 patients without orthostatic hypotension,
6-[18F]fluorodopamine positron emission tomography
scanning was repeated after a mean of 2 years from the
first scan. 6-[18F]fluorodopamine-derived radioactivity
was less in the second scan than in the first scan, by
31% in the left ventricular free wall and 16% in the
septum. In Parkinson’s disease, loss of cardiac sympathetic denervation progresses in a pattern of loss suggesting a dying-back mechanism.
Ann Neurol 2002;52:220 –223
All of at least a dozen studies have agreed that patients
with Parkinson’s disease have a high prevalence of neuroimaging evidence for decreased sympathetic innervation of the heart. Low myocardial concentrations of radioactivity have been noted after injection of the
sympathoneural imaging agents 123I-metaiodobenzylguanidine1–12 and 6-[18F]fluorodopamine.13 Neurochemical assessments during right heart catheterization
have confirmed that low concentrations of radioactivity
result from loss of functional cardiac sympathetic nerve
Although all patients with Parkinson’s disease and
orthostatic hypotension have diffusely decreased sympathetic innervation throughout the left ventricular
myocardium, among patients who do not have orthostatic hypotension, about half have diffusely decreased
innervation and about half have normal or only locally
From the Clinical Neurocardiology Section, National Institute of
Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, MD.
Received Nov 28, 2001, and in revised form Feb 28 and Mar 7,
2002. Accepted for publication Mar 7, 2002.
Published online Jun 23, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10236
Address correspondence to Dr Li, Building 10, Room 6N252, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 10 Center Drive, MSC-1620, Bethesda, MD
20892-1620. E-mail:
This article is a US Government work and, as such, is in the public domain in the United States of America.
Published 2002 by Wiley-Liss, Inc.
decreased innervation.13 The latter finding afforded an
opportunity to determine whether the loss of cardiac
sympathetic innervation in Parkinson’s disease
progresses over time and, if so, with what timing, pattern, and consistency across patients. This report describes the results of retesting such patients with
6-[18F]fluorodopamine positron emission tomography
scanning after an average of 2 years.
Patients and Methods
The study protocol was approved by the Intramural Research
Board of the National Institute of Neurological Disorders
and Stroke. Each patient gave informed, written consent.
Thoracic positron emission tomography scanning was performed after intravenous injection of 6-[18F]fluorodopamine in 9 patients with Parkinson’s disease (age:
mean, 60 years; SEM, 3 years). None of the patients had
orthostatic hypotension, which was defined as a decrease in
systolic blood pressure greater than 20mm Hg and decrease
in diastolic pressure greater than 5mm Hg between the supine position and standing for 5 minutes. Caffeinecontaining beverages, cigarettes, and alcohol were not allowed for at least 24 hours before the scanning. Patients
were allowed to take their usual medications, including
L-dopa, except for medications known to inhibit neuronal
uptake of catecholamines.
Positron Emission Tomography Scanning
6-[18F]fluorodopamine, synthesized as described previously,14
was infused intravenously at a constant rate for 3 minutes.
Tomography images (35 contiguous transaxial slices 4.25mm
apart) were acquired for up to 30 minutes. Threedimensional positron emission tomography scans were obtained with an Advance whole-body scanner (General Electric, Milwaukee, WI). Transmission scans of 2 minutes and
8 minutes, with rotating 68Ge/68Ga pin sources, were obtained for attenuation correction and for confirming proper
positioning in the scanner.
Follow-up scanning was performed 1 to 4 years (mean,
2.0 years; SEM, 0.3 years) after the first scan, with the identical scanning procedure. None of the patients had orthostatic hypotension at the time of either test. Of the 9 patients, 2 had normal 6-[18F]fluorodopamine-derived
radioactivity and 7 had locally decreased 6-[18F]fluorodopamine-derived radioactivity in the left ventricular
myocardium at the time of the first test.
Data Analysis and Statistics
Tomography images were reconstructed after correction for
attenuation and for physical decay of 18F. Cardiac images
were analyzed as described previously.14 Briefly, circular regions of interest approximately half the ventricular wall
thickness were placed on images of the septum, with time-
averaged pictures of a single slice. Left ventricular septal
radioactivity was averaged from two regions of interest for
the 5-minute scanning interval with a midpoint about
8 minutes after initiation of the infusion. The same
time interval was used for radioactivity in the liver and kidney. For radioactivity in structures of the head and neck,
static three-dimensional data were obtained for 10 to 15
minutes. Images of noncardiac structures, including the
liver, spleen, renal cortex, renal pelvis, salivary glands, nasopharyngeal mucosa, and thyroid, were reconstructed and
analyzed by manual drawing of the regions of interest outlining the structures. Radioactivity concentrations were
normalized by correction for the radioactivity concentration
for the administered dose of radioactive drug per unit of
body mass of the subject and were expressed as nCi-kg/ccmCi.14
Mean values for 6-[18F]fluorodopamine-derived radioactivity were compared with paired t tests. Differences between
groups in trends over time of 6-[18F]fluorodopamine-derived
radioactivity were assessed by analyses of variance for repeated measures. A p value of less than 0.05 defined statistical significance.
At the time of the first scan, patients had had Parkinson’s disease for 5.7 ⫾ 1.2 years (range, 0.3–13 years).
Disease severity averaged 2.2 ⫾ 0.2 (range, 1–3) of a
maximum of 5. Heart rate and beat-to-beat systolic
blood pressure changes during phase II of the Valsalva
maneuver averaged 11 ⫾ 2bpm and ⫺43 ⫾ 5mm Hg
for a mean arterial baroreflex-cardiovagal gain of 2.3 ⫾
0.4ms/mm Hg (normal: mean, 8.5ms/mm Hg; SEM,
2.2ms/mm Hg). Plasma catecholamine levels (norepinephrine: mean, 2.34nmol/L; SEM, 0.37nmol/L; epinephrine: mean, 0.23nmol/L; SEM, 10nmol/L) were
approximately normal. Most patients had tremor and
urinary frequency. None of the patients developed orthostatic hypotension between the first and second
At the time of the first scan, 2 patients had normal
myocardial 6-[18F]fluorodopamine-derived radioactivity, and 7 had decreased myocardial 6-[18F]fluorodopamine-derived radioactivity confined to the
lateral wall or apex, so that no patient had
6-[18F]fluorodopamine-derived radioactivity more than
two standard deviations below the normal mean in
both the lateral wall and interventricular septum.
All 9 patients had lower lateral wall concentrations
of 6-[18F]fluorodopamine-derived radioactivity in
the second scan than in the first scan (Fig 1).
Left ventricular myocardial mean concentrations of
6-[18F]fluorodopamine-derived radioactivity decreased
by 23% between the first scans (mean, 5,122nCi-kg/
cc-mCi; SEM, 564nCi-kg/cc-mCi) and second scans
(mean, 6,634nCi-kg/cc-mCi; SEM, 447nCi-kg/cc-mCi;
p ⫽ 0.003). Lateral wall radioactivity decreased by
31% between the first scans (mean, 4,107nCi-kg/
cc-mCi; SEM, 535nCi-kg/cc-mCi) and second scans
(mean, 5,991nCi-kg/cc-mCi; SEM, 537nCi-kg/cc-mCi;
p ⫽ 0.003), and septal radioactivity decreased by 16%
between the first scans (mean, 6,137nCi-kg/cc-mCi;
SEM, 716nCi-kg/cc-mCi) and second scans (mean,
7,278nCi-kg/cc-mCi; SEM, 385nCi-kg/cc-mCi; p ⫽
0.05; Fig 2, Table). In 1 patient, the lateral ventricular
wall was not visualized in the second scan, and the tissue concentration was assumed to be equal to the left
ventricular chamber concentration.
With the exclusion of data from the patient for
whom the left ventricular wall was not visualized in the
second scan, the percentage decrease in lateral wall radioactivity between the first and second scans (mean,
Fig 1. Progressive loss of myocardial 6-[18F]fluorodopamine-derived radioactivity in a patient with Parkinson’s disease.
Li et al: Sympathetic Innervation in Parkinson’s Disease
Fig 2. Concentrations (mean ⫾ standard error of the mean) of
6-[18F]fluorodopamine-derived radioactivity in the (top) lateral
left ventricular wall and (bottom) interventricular septum in
normal control subjects (open circles), patients with Parkinson’s disease at the time of the first scan (filled squares), and
the same patients at the time of the second scan an average of
2 years later (open squares).
32%; SEM, 7%) exceeded the percentage decrease in
septal wall radioactivity (mean, 13%; SEM, 7%; t ⫽
2.5, p ⫽ 0.04).
Tissue concentrations of 6-[18F]fluorodopaminederived radioactivity in the liver, renal cortex, renal pelvis, salivary glands, and thyroid did not change between the two scans (see Table). Radioactivity in the
spleen, however, was lower in the second scan (mean,
5,020nCi-kg/cc-mCi; SEM, 137nCi-kg/cc-mCi) than
in the first scan (mean, 6,495nCi-kg/cc-mCi; SEM,
364nCi-kg/cc-mCi; p ⫽ 0.001).
These findings, based on sympathetic neuroimaging
with 6-[18F]fluorodopamine positron emission tomography scanning, indicate that in patients with Parkinson’s disease who have normal or only locally decreased
cardiac sympathetic innervation, the loss of innervation
progresses over time, especially in the lateral ventricular
wall, in which 6-[18F]fluorodopamine-derived radioactivity decreased by about 30% over an average of 2
Annals of Neurology
Vol 52
No 2
August 2002
years. This rate of loss of sympathetic terminals appears
to be at least as high as the rate of loss of nigrostriatal
dopamine terminals.15
So far in our ongoing series, all patients with Parkinson’s disease and orthostatic hypotension have had
evidence of diffuse loss of cardiac sympathetic innervation at the time of initial testing.13 About half of
patients with Parkinson’s disease without orthostatic
hypotension have also had evidence of diffuse cardiac
sympathetic denervation, and because of the likelihood of a floor effect, data from these patients were
not included in this study. Given these results, and
the present findings, based on the remaining patients
with Parkinson’s disease who did not have either orthostatic hypotension or diffuse cardiac sympathetic
denervation at the time of initial testing, indicate that
progressive loss of cardiac sympathetic innervation
characterizes the disease. As of the time of the second
scan, none of the patients developed orthostatic hypotension.
The sympathetic innervation of the myocardium
travels with the coronary arteries. The finding of more
severely decreased 6-[18F]fluorodopamine-derived radioactivity in the lateral wall than in the interventricular septal wall leads to a suggestion of a dying-back
mechanism for the loss of sympathetic terminals, as
opposed to death of the cell bodies followed by loss of
the terminals (as in Wallerian degeneration). In agreement with this notion, about one half of patients with
Parkinson’s disease who do not have orthostatic hypotension already have decreased concentrations of
6-[18F]fluorodopamine-derived radioactivity throughout
Table. Tissue Concentrations of 6-[18F]FluorodopamineDerived Radioactivity (nCi-kg/cc-mCi) in Patients with
Parkinson’s Disease
First Scan
Left lateral wall
Right myocardium
Right chamber
Left chamber
Renal cortex
Renal pelvis
Submandibular gland
5,991 ⫾ 537
7,278 ⫾ 385
5,889 ⫾ 564
4,799 ⫾ 411
4,971 ⫾ 517
6,897 ⫾ 724
6,495 ⫾ 364
23,564 ⫾ 2,412
27,667 ⫾ 3,874
1,431 ⫾ 162
1,832 ⫾ 170
1,870 ⫾ 239
1,933 ⫾ 187
Significantly different from first scan.
SEM ⫽ standard error of the mean
Second Scan
4,107 ⫾ 535a
6,137 ⫾ 716a
5,390 ⫾ 485
4,240 ⫾ 263
4,725 ⫾ 423
7,165 ⫾ 869
5,020 ⫾ 137a
20,716 ⫾ 2,070
23,278 ⫾ 5,203
1,501 ⫾ 163
1,698 ⫾ 221
1,782 ⫾ 267
1,624 ⫾ 250
the left ventricular myocardium, and of the remaining
half, most have decreased 6-[18F]fluorodopaminederived radioactivity in the lateral ventricular wall or
apex, with relative sparing of the interventricular septum.13
Because the patients did not have a progressive loss
of 6-[18F]fluorodopamine-derived radioactivity in the
liver or renal cortex but did in the heart and spleen,
rates of loss of sympathetic innervation appear to vary
across organs. This fits with the notion of cardioselective sympathetic denervation in Parkinson’s disease.11,12
The results lead to the general inference that Parkinson’s disease features progressive neurodegeneration not
only in the nigrostriatal dopaminergic system but also
in the sympathetic noradrenergic system.
10. Satoh A, Serita T, Seto M, et al. Loss of 123I-MIBG uptake by
the heart in Parkinson’s disease: assessment of cardiac sympathetic denervation and diagnostic value. J Nucl Med 1999;40:
11. Reinhardt MJ, Jungling FD, Krause TM, Braune S. Scintigraphic differentiation between two forms of primary dysautonomia early after onset of autonomic dysfunction: value of cardiac and pulmonary iodine-123 MIBG uptake. Eur J Nucl Med
2000;27:595– 600.
12. Taki J, Nakajima K, Hwang EH, et al. Peripheral sympathetic
dysfunction in patients with Parkinson’s disease without autonomic failure is heart selective and disease specific. Eur J Nucl
Med 2000;27:566 –573.
13. Goldstein DS, Holmes C, Li ST, et al. Cardiac sympathetic
denervation in Parkinson disease. Ann Intern Med 2000;133:
338 –347.
14. Goldstein DS, Eisenhofer G, Dunn BB, et al. Positron emission
tomographic imaging of cardiac sympathetic innervation using
6-[18F]fluorodopamine: initial findings in humans. J Am Coll
Cardiol 1993;22:1961–1971.
15. Poewe WH. The natural history of Parkinson’s disease. Ann
Neurol 1998;44(suppl 1):1–9.
We gratefully acknowledge the assistance of Sandra Brentzel, RN,
and the Positron Emission Tomography Department of the National Institutes of Health.
1. Braune S, Reinhardt M, Bathmann J, et al. Impaired cardiac
uptake of meta-[123I]iodobenzylguanidine in Parkinson’s disease with autonomic failure. Acta Neurol Scand 1998;97:
2. Braune S, Reinhardt M, Schnitzer R, et al. Cardiac uptake of
[123I]MIBG separates Parkinson’s disease from multiple system
atrophy. Neurology 1999;53:1020 –1025.
3. Takatsu H, Nishida H, Matsuo H, et al. Cardiac sympathetic
denervation from the early stage of Parkinson’s disease: clinical
and experimental studies with radiolabeled MIBG. J Nucl Med
4. Takatsu H, Nagashima K, Murase M, et al. Differentiating Parkinson disease from multiple-system atrophy by measuring cardiac iodine-123 metaiodobenzylguanidine accumulation. JAMA
2000;284:44 – 45.
5. Orimo S, Ozawa E, Nakade S, et al. (123)I-metaiodobenzylguanidine myocardial scintigraphy in Parkinson’s disease.
J Neurol Neurosurg Psychiatry 1999;67:189 –194.
6. Yoshita M, Hayashi M, Hirai S. Iodine 123-labeled metaiodobenzylguanidine myocardial scintigraphy in the cases of idiopathic Parkinson’s disease, multiple system atrophy, and progressive supranuclear palsy. Rinsho Shinkeigaku 1997;37:
476 – 482.
7. Yoshita M. Differentiation of idiopathic Parkinson’s disease
from striatonigral degeneration and progressive supranuclear
palsy using iodine-123 meta-iodobenzylguanidine myocardial
scintigraphy. J Neurol Sci 1998;155:60 – 67.
8. Druschky A, Hilz MJ, Platsch G et al. Differentiation of Parkinson’s disease and multiple system atrophy in early disease
stages by means of I-123-MIBG-SPECT. J Neurol Sci 2000;
9. Satoh A, Serita T, Tsujihata M. Total defect of metaiodobenzylguanidine (MIBG) imaging on heart in Parkinson’s disease:
assessment of cardiac sympathetic denervation. Nippon Rinsho
Li et al: Sympathetic Innervation in Parkinson’s Disease
No Mutation in the TRKA
(NTRK1) Gene Encoding a
Receptor Tyrosine Kinase
for Nerve Growth Factor in
a Patient with Hereditary
Sensory and Autonomic
Neuropathy Type V
Ennio Toscano, MD, PhD,1 Alessandro Simonati, MD,2
Yasuhiro Indo, MD, PhD,3 and Generoso Andria, MD1
Hereditary sensory and autonomic neuropathy type IV
(HSAN-IV) and type V (HSAN-V) are autosomal recessive genetic disorders, both characterized by a lack of
pain sensation. We report a girl with clinical and neurophysiological findings consistent with a diagnosis of
HSAN-V. We sequenced her TRKA gene, encoding a receptor tyrosine kinase for nerve growth factor and responsible for HSAN-IV, but we could not detect any mutation. These data indicate that a gene (or genes) other
than TRKA is probably responsible for HSAN-V in some
Ann Neurol 2002;52:224 –227
Hereditary sensory and autonomic neuropathy
(HSAN) are classified into 5 different types according
to Dyck.1 We have demonstrated that the TRKA
(NTRK1) gene, encoding a receptor tyrosine kinase
that is phosphorylated in response to nerve growth factor, is responsible for HSAN-IV.2 HSAN-IV is characterized by febrile episodes, anhidrosis, insensitivity to
pain, self-mutilating behavior, and mental retardation.
Pathological features of HSAN-IV are severe reduction
of small-diameter afferent neurons, which are activated
by tissue-damaging stimuli,3,4 and a loss of sympathetic
neurons innervating eccrine sweat glands.5
HSAN-V also is characterized by absent reaction to
From the 1Department of Pediatrics, “Federico II” University, Naples, Italy; 2Section of Clinical Neurology, Department of Neurological and Visual Sciences, University of Verona, Verona, Italy; and
Department of Pediatrics, Kumamoto University, Honjo, Kumamoto, Japan.
Received Aug 21, 2001, and in revised form March 11, 2002. Accepted for publication March 11, 2002.
Published online Jun 23, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10245
Address correspondence to Dr Andria, Department of Pediatrics,
“Federico II” University, Via S. Pansini 5, I-80131 Naples, Italy.
© 2002 Wiley-Liss, Inc.
noxious stimuli but usually lacks anhidrosis and mental
retardation. Some patients with possible HSAN-V were
described before 1960 (see Landrieu and colleagues6
for references), and 3 families with HSAN-V subsequently were described in which were demonstrated a
selective severe decrease of the small myelinated fibers7,8 and a small reduction in unmyelinated fibers9 of
the sural nerve. HSAN-V is likely an autosomal recessive disorder, but Landrieu and colleagues6 reported in
a family 2 dominantly transmitted cases with normal
nerve biopsy. Therefore, a clinical entity, also called
congenital indifference to pain, is genetically heterogeneous and probably includes HSAN-IV or HSAN-V,
as suggested by Dyck.1
Recently, Houlden and colleagues10 described a boy
with febrile episodes and a lack of pain sensation that
they diagnosed as HSAN-V, according to the typical
findings in a nerve biopsy.9 DNA analysis showed that
he had a homozygous mutation in the TRKA gene.
They stated that HSAN-IV and HSAN-V are likely to
be allelic.
Here we present a girl with clinical features consistent with HSAN-V, namely, a lack of pain sensation,
but no febrile episodes and normal sural nerve biopsy.
We sequenced her TRKA gene and detected no mutation. Therefore, these data indicate that a gene (or
genes) other than TRKA is probably responsible for
HSAN-V in some patients, arguing against a previously
cited report.10 We also discuss the differential diagnosis
of HSAN-IV versus HSAN-V.
Case Report
I.F., a girl, was the second child of consanguineous parents
(first cousins). She was born at term (birth weight, 3,400gm)
with Apgar scores of 9 and 10. She was found to have insensitivity to painful stimuli, and multiple traumatic episodes
were experienced from early life. She bit off the tip of her
tongue, and she did not cry when she fell or during blood
sampling. At the ages of 3, 4, and 5 years, she had episodes
of left hip dislocation without pain perception. At the age of
6 years, she suffered from bilateral osteochondritis of the
feet. Her psychomotor development was apparently normal,
and she never exhibited behavioral problems or attention
deficit problems. She never had thermoregulatory or feeding
problems, vomiting, bowel dysfunction, or other signs or
symptoms of gastrointestinal dysmotility.
Clinical examination of the girl when she was 11 years
old showed reduced response to painful stimuli and
anosmia; thermic sensation appeared to be conserved.
Fungiform papillae of the tongue, corneal reflexes, and
overflow tears were present. Intelligence and deep tendon reflexes were normal. Blood pressure was normal
in both supine and standing positions. An intradermal
injection of histamine evoked axonal reflex as observed
in a normal control. She could control her body tem-
perature well under hot environmental conditions. Pilocarpine ionophoresis and sympathetic skin response
were normal.
A right sural nerve biopsy was performed and processed according to routine procedures for both morphological and morphometrical investigations.11 Features of the nerve fascicle were normal. At the
ultrastructural level, unmyelinated fibers were regularly
detected; neither denervated Schwann cells nor collagen pockets were seen (Fig 1). Myelinated fiber density
was within normal ranges; the shape of the histogram
was bimodal, showing the small-caliber fiber peak (Fig
2). Skin biopsy was examined on plastic sections only.
Glands were present; both myelinated and unmyelinated fibers were present in the intradermal nerve fascicles.
We sequenced all 17 exons of the TRKA gene of the
patient, including their flanking intronic sequences,12
and detected no putative mutation. Furthermore, we
analyzed 8 polymorphic sites in this gene and found
that the patient is heterozygous at the intragenic polymorphic site (c. 1,953 cytosine/thymine).13 This furFig 1. Sural nerve biopsy. (A) Semithin section; toluidine blue
stain. Normal features of the nerve fascicle showing myelinated
fibers of both small and large calibers. Bar ⫽ 12.5␮m. (B)
Thin section; uranyl acetate and lead citrate stain. Representative clusters of normal unmyelinated fibers. Bar ⫽ 3␮m.
Fig 2. Size distribution (X-axis) of the myelinated fibers of the
index case and an age-matched control. Note the normal, bimodal pattern of the histogram; small-caliber fibers are similarly represented in both the patient and the control. Numbers
on the Y-axis refer to the absolute figures of the measured
ther supports that the patient has no mutation in the
TRKA locus because the parents are consanguineous.
The patient reported here probably presents a hereditary autosomal recessive sensory neuropathy with selective loss of pain sensation. Autonomic abnormalities
apparently were not observed. Reduced sweating was
mentioned once, but she can maintain her body temperature under hot environmental conditions. This is
compatible with the findings in her nerve and skin biopsies. These clinical and laboratory data suggest that
the patient suffers from HSAN-V. Similar features, together with apparently intact peripheral nerves, were
described in some classic reports of congenital indifference to pain.6 The patient reported by Low and colleagues,7 presenting indifference to pain and selective
loss of small myelinated fibers without sweating abnormality, was classified as HSAN-V. Dyck and colleagues9 described a patient with indifference to pain,
sweating abnormality, and a severe decrease in smalldiameter myelinated fibers with a mild reduction of
unmyelinated fibers. It is their view that most earlier
cases of indifference to pain may have had either
In contrast, HSAN-IV is a distinct clinical entity
characterized by recurrent episodic fever, anhidrosis,
insensitivity to pain, self-mutilating behavior, and
mental retardation.3,14 Mental retardation is variable,
Toscano et al: No Mutation in TRKA Gene in HSAN-V
from severe to mild, and some patients were apparently
normal, but later mild retardation was showed by a
formal assessment (patients KI-108 and KI-116 reported by Mardy and colleagues12 and Indo and colleagues,15 respectively). Sweating may be variable but
should be evaluated cautiously, as we recently reported.15 We think that a fundamental phenotype of
HSAN-IV consists of insensitivity to pain, anhidrosis,
and mental retardation, each of variable degree. Recurrent hyperpyrexia, self-mutilating behaviors, traumas,
and bone fractures can be devastating and often lead to
crippling or fatal consequences.
Nerve growth factor supports the survival of sympathetic ganglion neurons and nociceptive sensory neurons in dorsal root ganglia and ascending cholinergic
neurons of the basal forebrain.16 Eccrine sweat glands,
innervated by sympathetic cholinergic fibers, are well
developed in humans. Therefore, the nerve growth
factor-TRKA system has a crucial role in the development and function of the nociceptive reception and establishment of thermoregulation via sweating.2 A negative result of the intradermal histamine test, an
important diagnostic criterion in HSAN-IV, probably
also can be explained by defective peripheral sympathetic neurons. Therefore, anhidrosis and associated
failure to maintain body temperature are characteristic
features of HSAN-IV.
Recently, Houlden and colleagues10 described a boy
9 years of age, born to healthy consanguineous parents,
presenting anhidrosis and loss of pain and temperature
sensation. Sural nerve biopsy demonstrated severe reduction in small-caliber myelinated fiber density but
only modest reduction in unmyelinated axons. The authors did not mention either a skin biopsy or a histamine test, which provide important diagnostic criteria
for the group of hereditary peripheral neuropathies.17
They detected a homozygous missense mutation in the
TRKA gene, changing a tyrosine to a cysteine at codon
359. According to this case, they concluded that the 2
disorders, HSAN-IV and HSAN-V, are likely to be allelic. However, we propose an alternative interpretation
of Houlden and colleagues’ report. Their patient may
suffer from HSAN-IV, but not HSAN-V. They argued
for the diagnosis of HSAN-V, mainly basing their argument on the finding of nerve biopsy with modest
reduction of unmyelinated fibers. However, anhidrosis
observed in the patient strongly indicates dysfunction
of the most distal intradermal portions of the sympathetic axons. That also might account for the normal
appearance of the more proximal unmyelinated fibers
observed after sural nerve. Furthermore, it would be
important to confirm the functional significance of the
missense mutation by an expression study, such as that
reported recently.18
We stress the importance of the molecular analysis
of the TRKA gene in all cases with lack of pain sensa-
Annals of Neurology
Vol 52
No 2
August 2002
tion to distinguish overlapping phenotypes of
HSAN-IV and HSAN-V, both characterized by a lack
of pain sensation. Most patients with HSAN-IV probably have mutations in the TRKA gene,2,12,13 although
we cannot rule out the possibility that a mutation (or
mutations) in another gene (or genes) is responsible for
similar clinical phenotypes. The patient presented here
suffers from HSAN-V, but not HSAN-IV, because she
does not show anhidrosis or mental retardation. The
presence of myelinated and unmyelinated fibers in
nerve biopsy and the normal intradermal histamine test
further support this diagnosis. We could not detect any
putative mutation in the TRKA gene, whereas we
found that the patient is heterozygous for this locus,
despite the parental consanguinity. These findings
strongly indicate that defects of a gene (or genes) other
than TRKA is likely responsible for at least some patients with HSAN-V.
It remains unknown whether peripheral nociceptive
transmission or central processing might be involved in
our case and in some cases with similar phenotypes reported as having HSAN-V. Anosmia observed in our
case remains to be examined and may give us some
clue for studying a mechanism underlying the defect in
pain sensation. Some patients diagnosed as having
HSAN-V do not show an apparent abnormality of peripheral nerve fibers. Therefore, their manifestations
might be caused by defects in peripheral nociceptor or
transduction and transmission of pain sensation or
even central processing, such as a lack of concern for a
painful stimulus well received by the peripheral nervous system, according to the classic definition of indifference to pain.19
1. Dyck PJ. Neuronal atrophy and degeneration predominantly affecting peripheral sensory and autonomic neurons. In: Dick PJ,
Thomas PK, Griffin JW, Low PA, Podreslo JC, eds. Peripheral
neuropathy. Philadelphia: Saunders, 1993:1065–1093.
2. Indo Y, Tsuruta M, Hayashida Y, et al. Mutations in the
TRKA/NGF receptor gene in patients with congenital insensitivity to pain with anhidrosis. Nat Genet 1996;13:485– 488.
3. Swanson AG, Buchan GG, Alvord EC Jr, et al. Autonomic
changes in congenital insensitivity to pain: absence of small primary sensory neurons in ganglia, roots and Lissauer’s tract.
Arch Neurol 1965;12:12–18.
4. Rafel E, Alberca R, Bautista J, et al. Congenital insensitivity to
pain with anhidrosis. Muscle Nerve 1980;3:216 –220.
5. Langer J, Goebel HH, Veit S. Eccrine sweat gland are not innervated in hereditary sensory neuropathy type IV: an electronmicroscopic study. Acta Neuropathol (Berl) 1981;54:199 –202.
6. Landrieu P, Said G, Allaire C. Dominantly transmitted congenital indifference to pain. Ann Neurol 1990;27:574 –578.
7. Low PA, Burke WJ, McLeod JG. Congenital sensory neuropathy with selective loss of small myelinated fibers. Ann Neurol
1978;3:179 –182.
8. Donaghy M, Hakin RN, Bamford JM, et al. Hereditary sensory
and autonomic neuropathy with neurotrophic keratitis. Brain
9. Dyck PJ, Mellinger JF, Reagan TJ, et al. Not “indifference to
pain” but varieties of hereditary sensory and autonomic neuropathy. Brain 1983;106:373–390.
10. Houlden H, King RHM, Hashemi-Nejad A, et al. A novel
TRK A (NTRK1) mutation associated with hereditary sensory
and autonomic neuropathy type V. Ann Neurol 2001;49:
11. Simonati A, Fabrizi GM, Pasquinelli A, et al. Congenital hypomyelination neuropathy with Ser72Leu substitution in
PMP22. Neuromuscul Disord 1999;9:257–261.
12. Mardy S, Miura Y, Endo F, et al. Congenital insensitivity to
pain with anhidrosis: novel mutations in the TRKA (NTRK1)
gene encoding a high-affinity receptor for nerve growth factor.
Am J Hum Genet 1999;64:1570 –1579.
13. Miura Y, Mardy S, Awaya Y, et al. Mutation and polymorphism analysis of the TRKA (NTRK1) gene encoding a highaffinity receptor for nerve growth factor in congenital insensitivity to pain with anhidrosis (CIPA) families. Hum Genet
2000;106:116 –124.
14. Swanson AG. Congenital insensitivity to pain with anhidrosis.
Arch Neurol 1963;8:299 –306.
15. Indo Y, Mardy S, Miura Y, et al. Congenital insensitivity to
pain with anhidrosis (CIPA): novel mutations of TRKA
(NTRK1) gene encoding the receptor tyrosine kinase for nerve
growth factor, a putative uniparental disomy and a linkage of
the mutant TRKA and PKLR genes in a family with CIPA and
pyruvate kinase deficiency. Hum Mutat 2001;18:308 –318.
16. Levi Montalcini R. The nerve growth factor: thirty-five years
later. EMBO J 1987;6:1145–1154.
17. Axelrod FB, Pearson J. Congenital sensory neuropathies. Am J
Dis Child 1984;138:947–954.
18. Mardy S, Miura Y, Endo F, et al. Congenital insensitivity to
pain with anhidrosis (CIPA): effect of TRKA (NTRK1) missense mutations on autophosphorylation of the receptor tyrosine kinase for nerve growth factor. Hum Mol Genet 2001;
10:179 –188.
19. Jewesburry ECO. Congenital indifference to pain. In: Vinken
PJ, Bruyn GW, eds. Handbook of clinical neurology. Vol 8.
Amsterdam: Elsevier, 1979:187–204.
X-Linked Creatine
Deficiency Syndrome: A
Novel Mutation in Creatine
Transporter Gene SLC6A8
Alberto Bizzi, MD,1 Marianna Bugiani, MD,2
Gajja S. Salomons, PhD,3 Donald H. Hunneman, PhD,4
Isabella Moroni, MD,2 Margherita Estienne, MD,2
Ugo Danesi, PhD,1 Cornelis Jakobs, PhD,3
and Graziella Uziel, MD2
Among creatine deficiency syndromes, an X-linked condition related to a defective creatine transport into the
central nervous system has been described recently. Hallmarks of the disease are the absence of a creatine signal
at brain spectroscopy, increased creatine levels in blood
and urine, ineffectiveness of oral supplementation, and a
mutation in the SLC6A8 (Online Mendelian Inheritance
in Man [OMIM] 300036) creatine transporter gene. We
report on a patient in whom a novel mutation (12211223delTTC) was identified.
Ann Neurol 2002;52:227–231
Creatine deficiency syndromes are recently identified
inborn errors of metabolism resulting in a progressive
encephalopathy with early onset and mental retardation, extrapyramidal features, and drug-resistant epilepsy.1–3 Symptoms are related to a depletion of the
creatine/phosphocreatine pool within the central nervous system, making this condition easily detectable by
brain spectroscopy. Most of the cases reported so far
were caused by a defect of the second enzyme involved
in creatine biosynthesis: guanidino-acetate methyltransferase (GAMT; OMIM 601240). This defect results in
increased guanidino-acetate (GAA) and reduced creatine levels in blood and urine.4,5 GAMT-deficient patients benefit from oral creatine monohydrate supplementation, which helps to control movement disorders
and epilepsy, partially recovers mental impairment, and
restores neurological development over time.6 The ab-
From the 1Departments of Neuroradiology and 2Child Neurology,
Istituto Nazionale Neurologico “C. Besta,” Milano, Italy; 3Metabolic Unit, VU Medical Center, Amsterdam, The Netherlands; and
University Kinderklinik, Gottingen, Germany.
Received Nov 6, 2001, and in revised form Mar 11, 2002. Accepted
for publication Mar 11, 2002.
Published online Jun 21, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10246
Address correspondence to Dr Uziel, Department of Child Neurology, Istituto Nazionale Neurologico “C. Besta,” Via Celoria 11,
20133 Milano, Italy. E-mail:
© 2002 Wiley-Liss, Inc.
sence of complete recovery can be explained by neurotoxic GAA accumulation or by an imbalance of brain
creatine and high-energy phosphates.7,8 Very recently,
Item and colleagues9,10 described 2 sisters with mental
impairment, low GAA levels in urine, and undetectable
activity of arginine to glycine amidinotransferase
(AGAT; OMIM 602360), the first enzyme involved in
creatine biosynthesis. A homozygous nonsense mutation in AGAT gene was detected. Clinical symptoms
and brain creatine deficiency were partially recovered
by means of creatine supplementation. A different disease due to impairment of creatine transport into the
brain was reported by Salomons and colleagues11,12 in
a boy with language delay, short attention span, epilepsy, and increased creatine levels in blood and urine.
A nonsense mutation in the X-linked creatine transporter gene SLC6A8 was demonstrated. Creatine supplementation in this boy was totally ineffective. This
article reports on a second case caused by defective creatine transport into the central nervous system.
Case Report
The patient was the second-born son of healthy, nonconsanguineous parents. The child’s prenatal and perinatal history
was unremarkable. Since the first months of life, he presented with motor delay, reduced interest in surroundings,
and no language acquisition. At 8 months, he was admitted
to the hospital after a febrile seizure. An electroencephalogram recording and a magnetic resonance imaging scan were
normal. Routine blood and urine analysis and investigation
for infectious (screening for rubella, cytomegalovirus, toxoplasma, herpes simplex, Coxsackie virus, and Mycoplasma
pneumoniae) and neurometabolic disorders (plasma lactate
and metabolic screening of a 24-hour urine sample with an
assessment of amino acids, organic acids, and mucopolysaccharides) were normal. Since 16 months of age, he experienced complex partial seizures with secondary generalization,
responding to sodium valproate. Serial electroencephalogram
tracings showed a progressive instability of background activity with abundant fast activity and epileptic discharges from
frontal and temporal leads during sleep. Physical examination
at 3 years and 9 months showed a severe delay in speech and
language functions with behavioral disturbances in agreement
with an autistic disorder. He did not follow commands or
speak, he presented with stereotypical motor behaviors, and
he could not engage in any structured play. Gross and fine
motor functions were normal.
A second magnetic resonance imaging showed mild atrophy with signal abnormality in the right hippocampus, suggesting mesial temporal sclerosis. Proton magnetic resonance
spectroscopic imaging (H-MRSI) was performed as part of
the diagnostic workup for mesial temporal sclerosis conducted at our institution and showed a normal N-acetylaspartate/choline ratio in both hippocampi without abnormality in the asymmetry index. The surprising feature was the
absence of the creatine peak in the whole brain (Fig), indi-
Fig. Proton magnetic resonance spectroscopic imaging shows a complete absence of the creatine peak in both white and gray matter
(1A, 2A, 3A, 4A). The position of selected voxels is indicated on the T2-weighted magnetic resonance image at the level of the centrum semiovale. There was no restoration of the creatine pool after 3 months of oral creatine monohydrate supplementation at
400mg/kg/day (1B) and after 8 months of supplementation at 700mg/kg/day (1C). There were no significant changes in choline
and N-acetylaspartate levels. A normal spectrum from an age-matched control is given for comparison: the peaks of choline (Cho;
3.2ppm), creatine (Cr; 3.02ppm), and N-acetylaspartate (NAA; 2.02ppm) are indicated.
Annals of Neurology
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August 2002
cating a creatine deficiency syndrome, without any abnormal
peak in the expected frequency of GAA (3.8ppm). Creatine
and GAA, therefore, were measured in plasma and urine,
and oral creatine monohydrate supplementation (400mg/kg/
day) was started. Because H-MRSI showed no creatine recovery after 3 months of therapy, supplementation was increased to 700mg/kg/day. Eight months later, a follow-up
H-MRSI examination confirmed no appearance of brain creatine. By this time, clinical symptoms were not improved,
and therapy was discontinued. Currently, the patient is 5
years old, and his clinical symptoms are grossly unchanged.
The very recent identification of the first patient with a creatine transporter defect11,12 suggested that our patient suffered from a creatine uptake defect as well, which was in line
with the ineffectiveness of creatine supplementation and with
biochemical data. Mutational analysis of the SLC6A8 gene,
therefore, was performed.
Before therapy, H-MRSI (two-dimensional phase encoding
point resolved spectroscopy repetition (PRESS) technique:
recovery time/echo time, 1,500/136msec; field of view,
160mm; matrix, 16 ⫻ 16; 20mm slice thickness) was performed from 3 separate sections at the level of the centrum
semiovale, basal nuclei, and hippocampi. During therapy,
H-MRSI was performed at the level of the centrum semiovale. Single-voxel spectra (PRESS: recovery time/echo delay
time, 1,500/34msec) were acquired from a 30ml volume in
the frontoparietal parasagittal cortical gray matter before and
during therapy. Raw data were transferred to a SUN workstation and reconstructed with custom-made software.13
Mutational analysis complementary DNA–based sequence
analysis was performed according to methods described elsewhere.12 DNA was isolated from blood cells with a QIAamp
blood kit (Qiagen, Chatsworth, CA) for conformation of the
mutation at the genomic level. Primers specific for exon 8 of
the SLC6A8 gene were designed: forward 5⬘TCCCAGCCCCTGCCGCAC and reverse 5⬘TACAAACTGTGGCCAGGGC.
Creatine and GAA levels before, during, and after the
withdrawal of creatine supplementation are shown in
Table 1. Sequence analysis of the creatine transporter
gene SLC6A8 identified an hemizygous 3bp deletion in
exon 8 involving nucleotides TTC in position 12211223 (1221-1223delTTC; GenBank accession number,
NM 005629). This mutation resulted in the deletion
of a single phenylalanine at residue 408 of the protein
(delF408). The patient’s mother was heterozygous for
the mutation.
The failure of creatine supplementation to restore brain
creatine by H-MRSI and to improve clinical symptoms
and normal plasma and urine GAA levels ruled out a
defect of creatine biosynthesis and prompted a search
for molecular defects in the SLC6A8 gene.12,14 A novel
hemizygous deletion located in a short repeat of 3 phenylalanines in exon 8 was detected. The repeat is part
of transmembrane domain VIII, which is a very conserved region among the Na⫹- and Cl⫺-dependent
neurotransmitter family.15 The mutation, resulting in a
phenylalanine deletion at position 408, most likely
causes a partial or even complete loss of creatine transport function. The activity of the transporter could not
be tested in fibroblasts because the parents refused consent to a skin biopsy.
To our knowledge, this is the first creatine deficiency
case studied with the multivoxel H-MRSI technique,
which has better spatial resolution and allows an evaluation of lesion heterogeneity in the shortest amount of
time. We did not find any significant difference in creatine levels across the brain regions examined, suggesting
that the transporter defect does not spare any brain area.
Symptoms of creatine deficiency have been related
previously to a defect in creatine transport by
Salomons and colleagues,11,12 who described a male
patient with mental retardation and severe delay in expressive speech and language function presenting a
hemizygous nonsense mutation in the SLC6A8 gene.
As in our case, a lack of creatine transport into the
central nervous system resulted in the failure of creatine supplementation to reverse clinical symptoms and
brain spectroscopy abnormality. Very recently, another
unrelated family with a creatine transporter defect was
recognized by the same authors.16 The biochemical
profile of our patient showed a massive loss of creatine
in urine, but in contrast with the first reported index
case, creatine levels in plasma were normal even during
creatine supplementation.
Table 1. Laboratory Results before, during, and after Withdrawal of Oral Creatine Monohydrate Supplementation (700mg/kg/day)
Normal values
(mmol/mol creatinine)
(mmol/mol creatinine)
GAA ⫽ guanidino-acetate; ND ⫽ not determined.
Bizzi et al: Creatine Transporter Defect
Table 2. Creatine and GAA Levels in GAMT and AGAT Defects and in Creatine Transporter Gene (SLC6A8) Mutations
Defect or mutation
AGAT ⫽ arginine to glycine amidinotransferase; GAA ⫽ guanidino-acetate; GAMT ⫽ guanidino-acetate methyltransferase; ND ⫽ not determined.
The mother underwent a brain H-MRSI that demonstrated a mildly reduced creatine signal compared
with that of age-matched controls. Moreover, the results showed that she was heterozygous for the mutation identified in her son. However, no learning disabilities were reported for the patient’s female relatives,
in contrast with what was encountered in 2 of 3 female
carriers in the first family described.12 These data agree
with skewed X inactivation (mosaic expression of mutant and wild-type alleles), resulting in a variably favorable mosaic expression of the wild-type allele.
The 3 creatine deficiency syndromes described so far
(ie, GAMT and AGAT defects and impairment of creatine transport) share overlapping symptoms of mental
retardation with severe language impairment, autistic behavior, and epilepsy. Movement disorders have been reported only in patients with GAMT deficiency, suggesting that extrapyramidal features may result from
neurotoxic GAA accumulation rather than from reduced
creatine availability in brain. The major involvement of
higher cortical functions and the frequent finding of epilepsy suggest that the cerebral cortex may be selectively
vulnerable to creatine deficiency. How creatine deficiency adversely affects cortical functions is still to be
established. It is conceivable that creatine plays a role in
the latest stages of cortical organization, including synaptogenesis, a process that continues after birth. This
could explain the homogeneity of clinical presentation
and age at onset in the 3 creatine deficiency syndromes,
even though in children with biosynthesis defects, creatine is supplied through the placenta during fetal life,
whereas patients with a transporter defect suffer from
creatine depletion already in utero.
Spectroscopy alone cannot always distinguish between
synthesis and transport defects. In GAMT deficiency,
brain spectroscopy at a short echo time can identify an
abnormal peak that is assigned to GAA (3.8ppm), but
this elevation may be subtle, and it has been reported
only in a few cases. Therefore, all patients in whom a
diagnosis of creatine deficiency is reached should undergo a careful biochemical evaluation to assess creatine
and GAA levels in blood and urine (Table 2). Brain
spectroscopy is becoming more available; a quick automated spectrum acquisition can be performed at the
Annals of Neurology
Vol 52
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August 2002
time of conventional magnetic resonance imaging and
may practically disclose creatine or other metabolites depletion: just recently, the first case of N-acetylaspartate
brain deficiency was reported in a patient with mental
retardation and severe language impairment.17
Clinical features of mental impairment, language delay, and autistic behavior with epilepsy frequently are
encountered in infancy. Conceivably, creatine deficiency could be underdiagnosed, and if brain spectroscopy cannot be easily achieved, an assessment of blood
and urinary creatine should always be performed.
We thank Dr S. J. M. van Dooren and D. Brunea for excellent technical support to Dr G. S. Salomons.
Electronic Database Information: OMIM.
omim; GenBank.
1. 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.
2. Stockler S, Marescau B, De Deyn PP, et al. Guanidinoacetate
methyltransferase deficiency, a new inborn error of creatine synthesis. Metabolism 1997;46:1189 –1193.
3. Ganesan V, Johnson A, Connelly A, et al. Guanidinoacetate
methyltransferase deficiency: new clinical features. Pediatr Neurol 1997;17:155–157.
4. 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. J Pediatr
1997;131:626 – 631.
5. Ilas J, Muhl A, Stockler-Ipsiroglu S. Guanidinoacetate methyltransferase deficiency: non-invasive enzymatic diagnosis of a
newly recognized inborn error of metabolism. Clin Chim Acta
2000;290:179 –188.
6. Stockler S, Hanefeld F, Frahm J. Creatine replacement therapy
in guanidinoacetate methyltransferase deficiency, a novel inborn
error of metabolism. Lancet 1996;348:789 –790.
7. van der Knaap MS, Verhoeven NM, Maaswinkel-Mooij P, et al.
Mental retardation and behavioural problems as presenting signs
of a creatine synthesis defect. Ann Neurol 2000;47:540 –543.
8. Leuzzi V, Bianchi MC, Tosetti M, et al. Brain creatine
depletion: guanidinoacetate methyltransferase deficiency (improving with creatine supplementation). Neurology 2000;55:
9. 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.
10. Item CB, Stockler-Ipsiroglu S, Stromberger C, et al. Arginine:
glycine amidinotransferase deficiency: the third inborn error of
creatine metabolism in humans. Am J Hum Genet 2001;69:
11. Cecil KM, Salomons GS, Ball WS, et al. Irreversible brain creatine deficiency with elevated serum and urine creatine: a creatine transporter defect? Ann Neurol 2001;49:401– 404.
12. Salomons GS, van Dooren SJM, Verhoeven NM, et al.
X-linked creatine-transporter gene (SLC6A8) defect: a new
creatine-deficiency syndrome. Am J Hum Genet 2001;68:
13. Soher BJ, van Zijl PCM, Duyn JH, et al. Quantitative proton
spectroscopic imaging of the human brain. Magn Reson Med
1996;35:356 –363.
14. 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.
15. Nash SR, Giros B, Kingsmore SF, et al. Cloning, pharmacological characterization and genomic localization of the human creatine transporter. Receptors Channels 1994;2:165–174.
16. Salomons GS, Dooren SJ, Verhoeven NM, et al. X-linked creatine transporter defect: the second family. J Inherit Metab Dis
2001;24(suppl 1):119.
17. Martin E, Capone A, Schneider J, et al. Absence of
N-acetylaspartate in the human brain: impact on neurospectroscopy? Ann Neurol 2001;49:518 –521.
Vertebrobasilar Stroke as a
Late Complication of a
Blalock-Taussig Shunt
Philippe Gailloud, MD,1 Argye Hillis, MD,2
Bruce Perler, MD,3 and Kieran J. Murphy, MD1
We describe a 39-year-old patient with a cerebellar infarct and a history of a tetralogy of Fallot corrected during childhood. This is the first documented case of vertebrobasilar stroke occurring as a late complication of a
Blalock-Taussig shunt followed by total cardiac repair.
Ann Neurol 2002;52:231–234
From the Department of 1Radiology and Radiological Sciences,
Neurology, and 3Vascular Surgery, Johns Hopkins Hospital, Baltimore, MD.
Received May 15, 2000, and in revised form Feb 28, 2002. Accepted for publication Mar 12, 2002.
Published online Jun 21, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10249
Address correspondence to Dr Gailloud, Department of Radiology,
Johns Hopkins University School of Medicine, 600 N. Wolfe
Street, Radiology B-100, Baltimore, MD 21287.
The Blalock-Taussig shunt, first performed in 1944 at
the Johns Hopkins Hospital,1 is a surgical anastomosis
established between a subclavian artery and the ipsilateral pulmonary artery. In patients with a tetralogy of
Fallot, this procedure allows deferment of the definitive
cardiac repair to a more robust stage of life by bypassing the pulmonary artery stenosis. The Blalock-Taussig
shunt, however, creates the permanent anatomical condition of a subclavian steal phenomenon (Fig 1). The
subclavian steal phenomenon, initially described by
Reivich and colleagues in 1961,2 is related to a proximal subclavian artery stenosis or occlusion responsible
for the occurrence of retrograde collateral flow in the
ipsilateral vertebral artery. A few reports have suggested
a possible link between a Blalock-Taussig shunt and
the late occurrence of cerebrovascular disease. We report the first documented case of vertebrobasilar stroke
occurring as a late complication of a Blalock-Taussig
shunt followed by total cardiac repair during childhood.
Case Report
A 39-year-old man was admitted to our hospital because of
an acute episode of nausea without vomiting, vertigo, and
decreased hearing on the right side, rapidly followed by right
facial numbness as well as weakness and decreased sensation
in the left arm. The patient also described a visual disturbance consistent with nystagmus. These symptoms appeared
without an identifiable precipitating factor and lasted for approximately 20 to 30 minutes. The patient was known to
have tetralogy of Fallot treated by a Blalock-Taussig shunt at
the age of 2 years followed by definitive surgical repair at the
age of 9 years. He had been in good health since then. The
review of risk factors for cerebrovascular disease, including
obesity, tobacco use, hyperlipidemia, and hypertension, was
negative. The social and familial histories showed only hypertension and hyperlipidemia in both parents.
On admission, the general and neurological examinations
were normal except for asymmetrical upper extremity blood
pressure measurements (125/80mm Hg on the right, 146/
95mm Hg on the left) and unpalpable distal arterial pulses
in the right arm. According to the patient, this discrepancy
had been known since his second cardiac surgery. The laboratory values were unremarkable; in particular, the lipid
profile was normal, and there was no evidence of a hypercoagulable state (the protein C and S antigens, antithrombin III activity, dilute Russell’s viper venom time test, and
homocysteine level were within normal ranges; the factor V
Leiden mutation was negative). Computed tomography of
the brain was normal. Magnetic resonance imaging showed
signal anomalies in the inferior aspect of the right cerebellar
hemisphere consistent with an acute ischemic lesion in the
right posterior inferior cerebellar artery territory (Fig 2A).
Magnetic resonance imaging incidentally showed inflammatory changes in the maxillary sinus and ethmoid cells bilaterally, whereas the sphenoid and frontal sinuses were
unremarkable. The patient was not treated for this asymptomatic inflammatory sinus disease. A transesophageal
echocardiogram, including a bubble study performed to
© 2002 Wiley-Liss, Inc.
tery branches, including the right vertebral artery and the
right ascending and deep cervical arteries (see Fig 2C). The
cerebral vasculature was otherwise unremarkable. There was
in particular no evidence of intracranial or extracranial atheromatous disease and no arterial or venous anomalies potentially associated with stroke.
The patient was discharged without sequelae from his
stroke. However, he continued to report frequent episodes
of dizziness, associated once with facial numbness and diplopia, despite being anticoagulated with warfarin at a therapeutic level. At this stage, surgical correction of his vascular anomaly was advised to the patient, who wished to
proceed with this option after the exposition of its potential
risks and benefits. Interposition of a synthetic graft
(Hemoshield Dacron Graft, Boston Scientific Corporation,
Natick, MA) between the right common carotid and subclavian arteries was performed uneventfully, allowing antegrade flow to be reestablished in the right vertebral artery.
After surgery, his episodic symptoms attributable to brainstem ischemia resolved. He has remained asymptomatic for
more than 1 year.
Fig 1. Schematic representation of the morphological changes
associated with a Blalock-Taussig shunt. The pulmonary trunk
and arteries are represented in light gray; the aortic arch (1)
and supra-aortic trunks are in dark gray. The Blalock-Taussig
shunt is established between the proximal right subclavian
artery (6) and the right pulmonary artery (8) by interposition
of a synthetic graft (7). The subclavian steal phenomenon,
that is, antegrade flow in the left vertebral artery and retrograde flow in the right vertebral artery (4), provides blood
supply to the right arm (direction of blood flow as indicated
by the arrows). The figure also shows the right common carotid artery (2), the right carotid bifurcation (3), and the
basilar artery (5).
rule out potential cardioembolic sources, was unremarkable
except for a mildly dilated right ventricular cavity. Reverse
flow in the right vertebral artery was by documented transcranial Doppler. The transcranial Doppler bubble test was
negative for embolic events at rest and during Valsalva maneuver and coughing. Cerebral digital subtraction angiography then was performed. The aortic arch study showed
interruption of the right subclavian artery at its origin (see
Fig 2B). Selective injections of the left vertebral artery confirmed the presence of a left-to-right subclavian steal phenomenon. Revascularization of the right upper extremity
was provided by retrograde filling of several subclavian ar-
Annals of Neurology
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August 2002
We report the case of a 39-year-old man presenting
with a vertebrobasilar stroke in the absence of identifiable cerebrovascular risk factors. The symptoms described by our patient pointed to a lesion in the territory of the right posterior inferior cerebellar artery,
which was confirmed by neuroimaging. In our patient,
the co-occurrence of a vertebrobasilar infarct and a
subclavian steal phenomenon strongly suggest a causative relationship between the 2 events, that is, a subclavian steal syndrome secondary to the cardiac surgery
undergone during childhood. Although an embolic
mechanism cannot be completely excluded because of
normal transesophageal echocardiogram and transcranial Doppler, both with negative bubble tests, that the
involved branch (the right posterior inferior cerebellar
artery) originates from the vertebral artery with reverse
flow renders this explanation very unlikely. Conversely,
this anatomical situation is consistent with an increased
risk of preferential hypoperfusion in the right posterior
inferior cerebellar artery territory.
The possible association between a Blalock-Taussig
shunt and a subclavian steal phenomenon initially was
proposed by Folger and Shah in 1965.3 By reviewing
123 cardiac angiograms performed on patients with a
Blalock-Taussig shunt, these authors could identify late
opacification of a subclavian artery suggestive of a subclavian steal phenomenon in 12 instances. The steal
phenomenon was associated with dizziness in 3 patients and with visual disturbances in 2 patients. However, in those patients and in other case reports describing early cerebrovascular events after a BlalockTaussig shunt,4,5 the vertebrobasilar manifestations
occurred after the creation of the shunt but before definitive repair of the tetralogy of Fallot. In these pa-
Fig 2. (A) Magnetic resonance imaging study of the brain. This
axial T2-weighted image shows increased signal in the lower
portion of the right cerebellar hemisphere associated with minimal mass effect on the right posterior-lateral aspect of the brainstem and subtle leftward midline shift. A small area of hypersignal also is seen in the right side of the myelencephalon
(arrowhead). This pattern of abnormal signal is consistent with
an ischemic lesion in the territory of the right posterior-inferior
cerebellar artery. Note the incidental finding of chronic maxillary sinus inflammation. (B) Digital subtraction angiography of
the aortic arch, left anterior oblique view. The aortic arch, the
right common carotid artery (RCC), the left common carotid
artery (LCC), and the left subclavian artery (LSC) are unremarkable. The right subclavian artery is interrupted close to its
origin from the innominate artery (arrow). (C) Selective digital
subtraction angiography of the left vertebral artery, anteroposterior view. The catheter tip (arrowhead) is placed at the origin
at the left vertebral artery (LV). Retrograde flow in the right
vertebral artery (RV) and in several cervical branches allows for
late opacification of the right subclavian artery (arrow).
tients, contributing factors related to cardiac dysfunction, such as hypoxia, polycythemia, bacterial
endocarditis, and mural or valvular thrombosis, have to
be considered.6 The association of a Blalock-Taussig
shunt with delayed ischemic complications occurring
after total cardiac repair of the tetralogy of Fallot was
suggested in 1984 by Kurlan and colleagues.6 These
authors described a 38-year-old patient developing
transient vertebrobasilar ischemia 31 years after a
Blalock-Taussig shunt and 4 years after total repair of
Gailloud et al: Blalock-Taussig Shunt and Stroke
his tetralogy of Fallot. Our 39-year-old patient presented with a cerebellar infarct 37 years after the creation of the shunt and 30 years after total cardiac repair. This observation, the first report of a documented
stroke after Blalock-Taussig shunt and total cardiac repair, appears to confirm that patients who underwent a
Blalock-Taussig procedure in childhood have an increased risk of vertebrobasilar insufficiency later in life.
The few cases reported so far that have attributed a late
cerebrovascular event to a Blalock-Taussig shunt might
reflect only the relatively young age of the concerned
population. Blalock-Taussig shunts have been performed on infants and small children for approximately
50 years, so much of that population is now approaching middle age. It is conceivable that the morphological risk factor constituted by the surgically created subclavian steal phenomenon generally is not significant
enough to become symptomatic in isolation, as appears
to have been the case for our patient. However, the
association of the Blalock-Taussig anatomical anomalies with age-related processes such as hypertension and
atheromatous disease might predispose these patients to
more precocious development of cerebrovascular diseases. If this assumption is correct, patients with a history of Blalock-Taussig shunt constitute an overlooked
group at risk for cerebrovascular disease. Furthermore,
these patients are now in good general health; most of
them have lost contact with their cardiologist and are
not followed up on a systematic basis.
In view of the potentially devastating consequences
of a vertebrobasilar stroke, patients who underwent a
Blalock-Taussig shunt may benefit from prophylactic
measures, such as the administration of platelet antiaggregant agents. More aggressive treatments may
be necessary for patients who remain symptomatic
under conservative management, such as the carotidsubclavian bypass performed in our patient. Follow-up
studies of patients with a Blalock-Taussig shunt have
been conducted mainly from a cardiological perspective.7 There is, as far as we know, no published
long-term evaluation of this population from a specific neurological viewpoint. Such a study is required
to better define the late cerebrovascular risk potentially associated with a history of Blalock-Taussig
1. Blalock A, Taussig HB. The surgical treatment of the heart in
which there is pulmonary stenosis or pulmonary atresia. JAMA
1945;128:189 –202.
2. Reivich M, Holling HE, Roberts B, Toole JF. Reversal of blood
flow through the vertebral artery and its effect on cerebral circulation. N Engl J Med 1961;265:878 – 885.
3. Folger GM, Shah KD. Subclavian steal in patients with BlalockTaussig anastomosis. Circulation 1965;31:241–248.
4. Naito H, Kurokawa K, Kanno T, et al. Status epilepticus and
cortical blindness due to subclavian steal syndrome in a girl with
Blalock’s operation. Surg Neurol 1973;1:46 – 49.
© 2002 Wiley-Liss, Inc.
5. Sokol S, Narkiewicz M, Billewicz O. Subclavian steal syndrome
after Blalock-Taussig anastomoses. J Cardiovasc Surg (Torino)
1969;10:350 –354.
6. Kurlan R, Krall RL, Deweese JA. Vertebrobasilar ischemia after
total repair of tetralogy of Fallot: significance of subclavian steal
created by Blalock-Taussig anastomosis. Stroke 1984;15:
359 –362.
7. Murphy JG, Gersh BJ, Mair DD, et al. Long-term outcome in
patients undergoing surgical repair of tetralogy of Fallot. N Engl
J Med 1993;329:593–599.
Cerebral X-Linked
in a Girl with
Xq27-Ter Deletion
Eli Hershkovitz, MD,1 Ginat Narkis, BA,2
Zamir Shorer, MD,1 Ann B. Moser, BA,3
Paul A. Watkins, MD, PhD,3 Hugo W. Moser, MD,3
and Esther Manor, PhD2
An 8.5-year-old girl with a pathogenic mutation
(515insC) of the ATP-binding cassette, subfamily D,
member 1 gene (ABCD1) on her maternally derived X
chromosome showed clinical, biochemical, and magnetic
resonance imaging abnormalities similar to those in affected males. Cytogenetic studies led to the surprise finding of a de novo deletion of Xq27 on the paternally derived X chromosome. A bone marrow transplant had an
apparently favorable effect. Cytogenetic studies should be
performed in all severely symptomatic X-linked adrenoleukodystrophy heterozygotes.
Ann Neurol 2002;52:234 –237
X-linked adrenoleukodystrophy (X-ALD) is a progressive
disorder that affects myelin because of a defect in the
gene for the adenosine triphosphate (ATP)-binding cassette, subfamily D, member 1 (ABCD1).1 ABCD1 is located on Xq28. It codes for ALD protein (ALDP),2 a
From the 1Pediatric Department and 2Genetic Institute, Soroka
University Medical Centre, Faculty of Health Sciences, Ben-Gurion
University of the Negev, Beer Sheva, Israel, and 3Kennedy Krieger
Institute, Johns Hopkins University, Baltimore, MD.
Received Dec 14, 2001, and in revised form Mar 12, 2002. Accepted for publication Mar 12, 2002.
Published online Jun 23, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10248
Address correspondence to Dr Hershkovitz, Pediatric Department,
Soroka University Medical Centre, POB 151, Beer Sheva 84101,
Israel. E-mail:
peroxisomal membrane protein that belongs to the ATPbinding cassette superfamily of membrane transport proteins.3 X-ALD is associated with the accumulation of
saturated, very long chain fatty acid in tissues, cultured
skin fibroblasts,4 and plasma.5 In males, phenotypic expression ranges from the severe childhood or adolescent
cerebral forms, which are associated with a white matter
inflammatory reaction,6 to slowly progressive adrenomyeloneuropathy, which manifests most commonly in
young adults and mainly affects the long tracts in the
spinal cord, with the inflammatory reaction mild or absent.7 Approximately 70% of affected males have primary adrenocortical insufficiency. Adrenal steroid therapy is effective for the endocrine dysfunction but does
not appear to alter neurological progression. Bone marrow transplantation (BMT) can be of long-term benefit
to boys and adolescents with the cerebral forms of the
disease when provided at a time when brain involvement
is still relatively mild.8 There is no specific therapy for
Approximately 50% of women who are heterozygous
for X-ALD develop a progressive syndrome that resembles adrenomyeloneuropathy but is milder and usually
does not manifest until 35 years of age or later. Adrenocortical insufficiency is rare. Approximately 1% of
heterozygous women have a more severe phenotype, in
which there is progressive cerebral involvement, adrenocortical insufficiency, and earlier onset9 and which
resembles what is seen in boys or adolescents with the
cerebral phenotypes (H.W.M., unpublished observation).
The cause of the severe manifestations in this small
proportion of heterozygous women has not been determined definitively. Skewed inactivation of the normal
X chromosome has been considered to be the most
likely explanation.9,10 X-inactivation patterns in cultured skin fibroblasts or white blood cells can be assessed by an examination of the expression of ALDP by
immunocytochemical techniques11 in women who are
members of families in which affected males are known
to lack immunoreactive material. In 15 heterozygous
women, 23 to 86% of cells expressed ALDP.12 In contrast, ALDP was expressed in only 0 to 2% of cells
from 2 women who had the adolescent cerebral phenotype,10 suggesting that in these patients the normal
X chromosome failed to exert its protective effect. This
conclusion must be considered tentative because findings in fibroblasts may not reflect those in the nervous
system. We now report a patient with the severe phenotype who was known to be heterozygous for X-ALD
because of a pathogenic mutation in the maternal cell
line but who had in addition a deletion of Xq27-ter in
the paternal chromosome, thereby rendering her totally
deficient in ALDP.
Case Report
The patient is the first child of unrelated parents. Two maternal uncles have X-ALD, 1 with the adolescent cerebral
phenotype and the other with the adrenomyeloneuropathy
phenotype. Pregnancy and early development were normal.
She started to walk at 14 months and to speak at 1 year.
Several behavioral problems were noted in childhood. These
included separation anxiety, with refusal to stay alone in the
kindergarten and school; oversensitivity to noises; and isolation from peers and kindergarten staff. She continued to develop neurologically in agreement with her age, and she
started to read at 5 to 6 years of age. At that time, she was
referred to formal psychological assessment because of
“school phobia,” but no diagnosis was made at that time.
The deterioration of her academic performance and behavior
was noted at 8.5 years. Physical and neurological examinations were normal. Formal psychological assessment (Wechsler intelligence scale for children-revised) showed that she
functioned at the low normal range. The verbal intelligence
quotient was 87, the performance intelligence quotient was
83, and the total intelligence quotient was 83. Her achievements were adversely affected by easy distractibility and
visuospatial defects. Cortisol response to 1␮g of intravenous
adrenocorticotropic hormone was normal at 30 minutes
(26.2␮g/dl), ruling out clinically significant adrenal insufficiency. Brain magnetic resonance imaging demonstrated diffuse white matter involvement that was most prominent in
the frontal regions.
Biochemical and genetic studies were performed after
informed consent had been obtained from the parents.
Levels of very long chain fatty acid in plasma and cultured skin fibroblasts were measured by capillary gasliquid chromatography4,5 and found to be elevated
(Table 1). DNA analysis of the patient’s white blood
cells and her mother’s white blood cells showed a cytosine insertion in codon 515 (515insC) resulting in a
frameshift after amino acid 171 (tyrosine). Immunocytochemical studies of the cultured skin fibroblasts11
showed that ALDP-reactive material was lacking in
99% of the patient’s cells (100 cells counted) and in all
of the cells of the affected maternal uncles. Cytogenetic
analyses showed a deletion at Xq27.23ter in the patient’s peripheral blood lymphocytes. Her karyotype
was 46X;Xdel(q27-tel) (data not shown). Both parents’
karyotype analyses were normal. A deletion of the distal part of Xq, including the telomeric region in the
patient’s fibroblasts, also was observed by fluorescence
in situ hybridization with a specific Xq telomeric
probe. This region was present in the X chromosomes
of both parents (data not shown). DNA analysis with
23 polymorphic markers spanning the q26-28 region
of the patient’s and her parents’ X chromosomes was
performed to define the extent of the deletion, using
methods that have been described previously.13 An
analysis of 13 informative markers showed that a de
novo deletion had occurred in the paternal chromo-
Hershkovitz et al: Cerebral X-ALD
Table 1. Very Long Chain Fatty Acid Levels in Patient’s Plasma and Cultured Skin Fibroblasts
X-ALD Male
X-ALD Heterozygote
0.68 ⫾ 0.15
0.018 ⫾ 0.009
0.64 ⫾ 0.32
0.41 ⫾ 0.15
0.69 ⫾ 0.19
0.72 ⫾ 0.26
0.27 ⫾ 0.17
0.40 ⫾ 0.23
0.90 ⫾ 0.50
0.09 ⫾ 0.07
0.08 ⫾ 0.03
Cultured skin fibroblastsb
The plasma assays were performed at the Sharee Zedek Hospital in Jerusalem (Dr Orly Elpeleg) and those in fibroblasts at the Kennedy Krieger
Institute. The C26:0 levels in fibroblasts are similar to those of X-ALD male, and the C26:0/C22:0 ratios in fibroblasts are intermediate
between those in male and heterozygous X-ALD patients and compatible with either genetic status.
Expressed as micrograms per milliliter of plasma.
Expressed as micrograms per milligram of protein.
ND ⫽ not done; X-ALD ⫽ X-linked adrenoleukodystrophy.
some. The deleted region is distal to marker DXS1227,
which maps to Xq27.2 (Table 2). Therefore, the deleted segment spans part of Xq27.2 and all of Xq27.3
and Xq28.
At 8.9 years of age, she received a bone marrow
transplant. Her human leukocyte antigen–identical
normal younger sister was the donor. The procedure
was tolerated well. Full engraftment was documented.
Although some deterioration in concentration and
short-term memory were noticed during the early period after transplantation, her neurological and neuropsychological status were stable 18 months after BMT.
The abnormal magnetic resonance imaging results and
moderately abnormal neuropsychological dysfunction
in this 8.5-year-old girl known to be heterozygous for
X-ALD on the basis of family history, mutation analysis, and very long chain fatty acid studies indicate that
she is among the few heterozygotes with a severe phenotype similar to that in boys with the cerebral forms
of the disease. Mutation analysis indicates that the maternal side of her family has a mutation that abolishes
the expression of ALDP. Studies in other heterozygotes
have led to the hypothesis that severe disability, such as
cerebral involvement in childhood, is caused by skewed
X inactivation,9,10 and the absence of ALDP immunoreactive material would be compatible with this. However, cytogenetic studies led to a different conclusion,
namely, the unexpected demonstration of a de novo
deletion in her paternal X chromosome that involves
all of Xq28 and part of Xq27. Combined with the abnormality on the maternal X chromosome, this leads to
failure to express functional ALDP, similar to what occurs in affected males. Cytogenetic studies are performed only rarely in patients with X-ALD. To our
knowledge, this type of abnormality has not been reported before in women heterozygous for X-ALD. The
Annals of Neurology
Vol 52
No 2
August 2002
finding is of practical significance because under these
circumstances therapeutic approaches are similar to
those that would be used in affected males. Given this
reasoning and because the patient met current criteria
for BMT in affected males,8 we decided to perform the
transplant. Her condition was stable 18 months later,
and we hope that her long-term outcome will be favorable, as has been the case for male patients.8
We recommend that cytogenetic studies be performed in the approximately 1% of heterozygotes who
show evidence of cerebral involvement by magnetic resonance imaging. A karyotype study should be the first
step. A normal result should be followed by a search
for microdeletions in chromosome X with specific
DNA markers spanning the ALD gene region. This information is clinically significant because patients with
cerebral involvement may be candidates for BMT. At
this time, BMT is considered only for patients who
have evidence of cerebral involvement.8 It is never recommended for males or females who have spinal cord
Table 2. Haplotype DNA Analysis of Xq26-28 Using
Informative DNA Markers
involvement only. For female patients with cerebral involvement, BMT would be recommended only for
those in whom both alleles are involved, thereby making them equivalent to hemizygotes. We do not believe
that BMT would be indicated for women who do have
1 normal allele, where the pathogenesis of brain involvement is unknown (although skewed inactivation is
the most likely cause), and we do not believe that in
such patients the risk-benefit ratio would warrant
BMT. Although it might be of some interest to do
cytogenetic studies on all heterozygotes, the added expense is not warranted in women without cerebral involvement.
1. Moser HW, Smith KD, Watkins PA, et al. X-linked adrenoleukodystrophy. In: Scriver CR, Beaudet AL, Sly WS, Valle D,
eds. The metabolic and molecular bases of inherited disease. 8th
ed. New York: McGraw-Hill, 2001:3257–3301.
2. Mosser J, Douar AM, Sarde CO, et al. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC
transporters. Nature 1993;361:726 –730.
3. Higgins CF, Gallagher MP, Mimmack ML, Pearce SR. A family of closely related ATP-binding subunits from prokaryotic
and eukaryotic cells. Bioessays 1988;8:111–116. fs
4. Moser HW, Moser AB, Kawamura N, et al. Adrenoleukodystrophy: elevated C26 fatty acid in cultured skin fibroblasts.
Ann Neurol 1980;7:542–549.
5. Moser AB, Kreiter N, Bezman L, et al. Plasma very long chain
fatty acids in 3,000 peroxisome disease patients and 29,000
controls. Ann Neurol 1999;45:100 –110.
6. Powers JM, Liu Y, Moser AB, Moser HW. The inflammatory
myelinopathy of adreno-leukodystrophy: cells, effector molecules, and pathogenetic implications. J Neuropathol Exp Neurol 1992;51:630 – 643.
7. Powers JM, DeCiero DP, Ito M, et al. Adrenomyeloneuropathy: a neuropathologic review featuring its noninflammatory myelopathy. J Neuropathol Exp Neurol 2000;59:89 –102.
8. Shapiro E, Krivit W, Lockman L, et al. Long-term beneficial
effect of bone marrow transplantation for childhood onset cerebral X-linked adrenoleukodystrophy. Lancet 2000;356:
9. Heffungs W, Hameister H, Ropers HH. Addison disease and
cerebral sclerosis in an apparently heterozygous girl: evidence
for inactivation of the adrenoleukodystrophy locus. Clin Genet
1980;18:184 –188.
10. Naidu S, Washington C, Thirumalai S, et al. X-chromosome
inactivation in symptomatic heterozygotes in X-linked adrenoleukodystrophy. Ann Neurol 1997;42:498 (Abstract).
11. Watkins PA, Gould SJ, Smith MA, et al. Altered expression of
ALDP in X-linked adrenoleukodystrophy. Am J Hum Genet
12. Feigenbaum V, Lombard-Platet G, Guidoux S, et al. Mutational and protein analysis of patients and heterozygous women
with X-linked adrenoleukodystrophy. Am J Hum Genet 1996;
13. Parvari R, Mumm S, Galil A, et al. Deletion of 8.5 Mb, including the FMR1 gene, in a male with the fragile X syndrome
phenotype and overgrowth. Am J Med Genet 1999;83:
A Novel Mutation in the
Deoxyguanosine Kinase
Gene Causing Depletion of
Mitochondrial DNA
Jan-Willem Taanman, PhD,1 Ihab Kateeb, MD,2
Ania C. Muntau, MD,3 Michaela Jaksch, MD,4
Nadine Cohen, MD,2 and Hanna Mandel, MD5
Recently, a homozygous single-nucleotide deletion in
exon 2 of the deoxyguanosine kinase gene (DGUOK) was
identified as the disease-causing mutation in 3 apparently
unrelated Israeli-Druze families with depleted hepatocerebral mitochondrial DNA. We have discovered a novel
homozygous nonsense mutation in exon 3 of DGUOK
(313C3 T) from a patient born to nonconsanguineous
German parents. This finding shows that mutations in
DGUOK causing mitochondrial DNA depletion are not
confined to a single ethnic group.
Ann Neurol 2002;52:237–239
In 1991, Moraes and colleagues1 identified a group of
infants with marked depletion of mitochondrial DNA
(mtDNA) in association with defective mitochondrial
respiratory chain function. This condition, often called
mtDNA depletion syndrome (Online Mendelian Inheritance in Man [OMIM] 251880), now has been described for more than 50 patients;2–11 this suggests that
it may be an important cause of mitochondrial dysfunction in neonates and infants. Most of the reported
patients present in the neonatal period with muscle
weakness, liver failure, and neurological abnormalities
associated with lactic acidemia and die before 12
From the 1University Department of Clinical Neurosciences, Royal
Free and University College Medical School, University College
London, London, United Kingdom; 2Department of Genetics,
Tamkin Human Molecular Genetics Research Facility, TechnionIsrael Institute of Technology, Bruce Rappaport Faculty of Medicine, Haifa, Israel; 3Dr von Hauner Children’s Hospital, LudwigMaximilians-Universität, Munich, Germany; 4Stoffwechselzentrum
und Institut für Klinische-Chemie, Krankenhaus MünchenSchwabing, Munich, Germany; and 5Metabolic Disease Unit, Department of Pediatrics, Rambam Medical Center, Technion-Israel
Institute of Technology, Bruce Rappaport Faculty of Medicine,
Haifa, Israel.
Received Dec 13, 2001, and in revised form Mar 12, 2002. Accepted for publication Mar 13, 2002.
Published online Jun 23, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10247
Address correspondence to Dr Taanman, University Department of
Clinical Neurosciences, Royal Free and University College Medical
School, University College London, Rowland Hill Street, London
NW3 2PF, United Kingdom. E-mail:
© 2002 Wiley-Liss, Inc.
months of age. Others present in infancy with isolated
myopathy associated with motor regression or a slowly
progressive encephalomyopathy. Inheritance appears to
be autosomal recessive, suggesting that nuclear gene defects are responsible for mtDNA depletion. In agreement with this notion, cybrid studies have shown that
the defect is not transmitted by patient mtDNA,3,12
and partial sequencing of patient mtDNA has failed to
identify mutations.1,4
Recently, homozygosity mapping followed by candidate gene sequence analysis in 3 kindreds of IsraeliDruze origin with the early-onset hepatocerebral variant of the disease showed a homozygous mutation in
the gene for deoxyguanosine kinase (DGUOK) on
chromosome 2p13.13 In addition, mutations in the
gene for mitochondrial thymidine kinase (TK2) on
chromosome 16q22 were identified in patients with
the late-onset muscle-specific variant of the disease.14
Deoxyguanosine kinase is responsible for phosphorylation of purine deoxyribonucleosides in the mitochondrial matrix compartment.15 The 3 apparently unrelated Israeli-Druze families carried the same singlenucleotide deletion in exon 2 of DGUOK.13 This
mutation is expected to result in premature termination of translation. To investigate whether mutations
in DGUOK are also present in patients from a different
ethnic origin, we sequenced the exons and intron/exon
boundaries of DGUOK from a German patient with
the hepatocerebral form of the disease. The patient was
found to harbor a novel homozygous nonsense mutation in DGUOK. This shows that mutations in
DGUOK causing mtDNA depletion are not restricted
to patients belonging to one particular ethnic group.
Case Report
The clinical details of the patient and histochemical findings
are documented in detail elsewhere.11 In brief, the patient
was the first child of healthy, nonconsanguineous German
parents (birth weight, 2,570gm). The boy presented with lactic acidosis, hepatomegaly, hypoglycemia, and jaundice
shortly after birth. He had a severe encephalopathy, characterized by marked muscle hypotonia, hyperreflexia, irritability, and pendular horizontal nystagmus. At the age of 2
months, neonatal giant cell hepatitis was diagnosed by light
microscopy. Electron microscopy of the liver showed an accumulation of abnormal mitochondria and steatosis. Histochemistry and immunohistochemistry for cytochrome-c oxidase demonstrated a mosaic pattern of normal and deficient
hepatocytes. Skeletal muscle was normal on both light and
electron microscopy, but biochemical assays showed a minor
cytochrome-c oxidase deficiency. Southern blot analysis of
liver biopsies, taken at 2 and 3 months of age, showed that
mtDNA was of normal size, but mtDNA levels were only 17
and 18%, respectively, of the mean of 6 age-matched control
specimens (range controls, 66 –140%).11 The patient died of
hepatic failure at the age of 5 months. A younger brother
shows similar symptoms.
Annals of Neurology
Vol 52
No 2
August 2002
After informed parental consent, in accordance with the
guidelines of the local institution, DNA was extracted from
liver of the proband, blood of the parents, and cultured fibroblasts of the second child.6 All exons of DGUOK were
amplified by polymerase chain reaction, purified, and sequenced exactly as described earlier.13 Sequencing was performed in both directions. The human DGUOK messenger
RNA sequence, with accession number U41668, and the human chromosome 2 working draft sequence segment, with
accession number NT025651, were used to determine the
intron/exon structure of DGUOK.
Sequencing of the 7 exons and the intron/exon boundaries of the DGUOK gene from the proband showed a
homozygous C3 T transition at nucleotide position
313 (numbering according to Johansson and Karlsson16) of exon 3. The same homozygous mutation was
found in the clinically affected sibling. Both parents
were heterozygous for the mutation (Fig). The mutation changes the arginine CGA codon 105 into a TGA
stop codon (see Fig). This base change is predicted to
result in a 173–amino acid residue truncation at the C
terminus of the DGUOK protein product. The mutation was not found in 15 control subjects of European
In an earlier study, we identified an infant, born to
healthy, nonconsanguineous German parents, with
early-onset encephalopathy and rapidly progressing
liver failure associated with severely depleted levels of
mtDNA.11 The recent discovery of a single-nucleotide
deletion in exon 2 of DGUOK in 3 unrelated IsraeliDruze families with depleted hepatocerebral mtDNA
prompted us to screen the German patient for mutations in the gene. We found a homozygous nonsense
mutation in exon 3 of the proband and his affected
younger brother, whereas both parents were carriers.
The mutation will lead to a shortening of the DGUOK
protein product by more than half. The truncation includes 3 domains that are evolutionarily conserved between nucleoside kinases and are thought to be essential for catalysis.16 It is, therefore, highly unlikely that
the mutated DGUOK gene is still functionally active in
the patient. The absence of the mutation in control
subjects of European origin further supports the pathogenicity of the mutation.
The mutation in the DGUOK gene that we identified in a German family indicates that mutations in
DGUOK causing mtDNA depletion are not limited to
Israeli-Druze patients. However, mutation screening of
DGUOK in 11 additional families with early-onset encephalopathy, liver failure, and mtDNA depletion of
British (5 patients), Greek-Cypriot (3 patients), German (1 patient), French (1 patient), and Turkish (1
This study was supported by the Wellcome Trust (grant 048410,
J.W.T.), the DFG (grant Ja 802/2-1, M.J.), and the Joseph Elias
Fund/Technion VPR Fund (grant 181-421, H.M.).
We thank Dr A. H. V. Schapira and Dr J. V. Leonard for helpful
Fig. Electropherograms showing part of the exon 3 sequences of
the deoxyguanosine kinase gene (DGUOK) of a control, both
parents, the proband (1st child), and his brother (2nd child).
The position of the mutation is indicated with an arrow.
Numbering is according to Johansson and Karlsson.16
patient) origin, including the 6 families described by us
previously,3,6,8 did not show any mutations in the 7
exons and the intron/exon boundaries of DGUOK (not
shown). The clinical, biochemical, and molecular findings of these patients were not markedly different from
the case described here. These results suggest that mutations in the coding region of DGUOK are not common in patients with hepatocerebral mtDNA depletion. Although the disease may be genetically
heterogeneous, our results do not rule out that these 11
families carry mutations elsewhere in DGUOK that affect gene expression. This possibility is likely in 2 additional families of Druze and Moroccan origin, respectively, in which the disease has been linked to
DGUOK but in which mutations in the coding region
of the gene could not be identified.13 More detailed
mutation analysis and expression studies of DGUOK,
therefore, are necessary to investigate the genetic heterogeneity of hepatocerebral mtDNA depletion.
1. Moraes CT, Shanske S, Trischler H-J, et al. mtDNA depletion
with variable tissue expression: a novel genetic abnormality in
mitochondrial diseases. Am J Hum Genet 1991;48:492–501.
2. Tritschler H-J, Andreetta F, Moraes CT, et al. Mitochondrial
myopathy of childhood associated with depletion of mitochondrial DNA. Neurology 1992;42:209 –217.
3. Bodnar AG, Cooper JM, Holt IJ, et al. Nuclear complementation restores mtDNA levels in cultured cells from a patient with
mtDNA depletion. Am J Hum Genet 1993;53:663– 669.
4. Mariotti C, Uziel G, Carrara F, et al. Early-onset encephalomyopathy associated with tissue-specific mitochondrial DNA
depletion: a morphological, biochemical and molecular-genetic
study. J Neurol 1995;242:547–556.
5. Macmillan CJ, Shoubridge EA. Mitochondrial DNA depletion:
prevalence in a pediatric population referred for neurologic
evaluation. Pediatr Neurol 1996;14:203–210.
6. Morris AAM, Taanman J-W, Blake J, et al. Liver failure associated with mitochondrial DNA depletion. J Hepatol 1998;28:
556 –563.
7. Vu TH, Sciacco M, Tanji K, et al. Clinical manifestations of
mitochondrial DNA depletion. Neurology 1998;50:1783–1790.
8. Blake JC, Taanman J-W, Morris AMM, et al. Mitochondrial
DNA depletion syndrome is expressed in amniotic fluid cell
cultures. Am J Pathol 1999;155:67–70.
9. Barthélé my C, Ogier de Baulny H, Diaz J, et al. Late-onset
mitochondrial DNA depletion: DNA copy number, multiple
deletions, and compensation. Ann Neurol 2001;49:607– 617.
10. Mandel H, Hartman C, Berkowitz D, et al. The hepatic mitochondrial DNA depletion syndrome: ultrastructural changes in
liver biopsies. Hepatology 2001;34:776 –784.
11. Müller-Höcker J, Muntau AC, Schäfer S, et al. Depletion of
mitochondrial DNA in the liver of an infant with neonatal giant cell hepatitis. Hum Pathol 2002;33:247–253.
12. Taanman J-W, Bodnar AG, Cooper JM, et al. Molecular mechanisms in mitochondrial DNA depletion syndrome. Hum Mol
Genet 1997;6:935–942.
13. Mandel H, Szargel R, Labay V, et al. The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA. Nat Genet 2001;29:337–341.
14. Saada A, Shaag A, Mandel H, et al. Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat
Genet 2001;29:342–344.
15. Jüllig, M, Eriksson S. Mitochondrial and submitochondrial localization of human deoxyguanosine kinase. Eur J Biochem
2000;267:5466 –5472.
16. Johansson M, Karlsson A. Cloning and expression of human
deoxyguanosine kinase cDNA. Proc Natl Acad Sci U S A 1996;
93:7258 –7262.
Taanman et al: A Novel Mutation Depleting mtDNA
Dyskinesias and Grip
Control in Parkinson’s
Disease Are Normalized by
Chronic Stimulation of the
Subthalamic Nucleus
Roland Wenzelburger, MD,1 Bao-Rong Zhang, MD,1
Meike Poepping, MD,1 Bettina Schrader, MD,2
Dieter Müller, PhD,3 Florian Kopper, MD,1
Urban Fietzek, MD,1 Hubertus M. Mehdorn, PhD,2
Günther Deuschl, PhD,1 and Paul Krack, PhD1
Deep-brain stimulation of the subthalamic nucleus appears to reduce levodopa-induced dyskinesias, but
whether this effect is caused by the reduction of the total
levodopa ingestion or represents a direct effect on the
motor system is unknown. Precision grip force of grasping movements and levodopa-induced dyskinesias was
analyzed in 10 parkinsonian patients before and after 3
months of deep-brain stimulation of the subthalamic nucleus. Peak grip force was abnormally increased before
surgery in the off-drug state and, particularly, in the ondrug state (sensitization). This grip force upregulation
normalized with chronic deep-brain stimulation in both
conditions (desensitization). Peak-dose dyskinesias also
improved, and off-dystonia was completely abolished.
Mean dosage of dopaminergic drugs was reduced, but
force overflow and dyskinesias were equally improved in
2 patients without a reduction. Despite the same single
levodopa test dose, force excess and levodopa-induced
dyskinesias were drastically reduced after 3 months of
deep-brain stimulation of the subthalamic nucleus. This
indicates that direct effects of deep-brain stimulation of
the subthalamic nucleus on levodopa-induced dyskinesias
are likely to occur. Grip force overflow is a promising
parameter to study the desensitizing effect of chronic
deep-brain stimulation on levodopa-induced dyskinesias.
Ann Neurol 2002;52:240 –243
From the Departments of 1Neurology and 2Neurosurgery, Christian
Albrechts University, Kiel; and the 3Department of Neurosurgery,
University Hamburg, Hamburg, Germany.
Received Dec 27, 2001, and in revised form Mar 14, 2002. Accepted for publication Mar 23, 2002.
Published online Jun 23, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10254
Dr Zhang’s current address is the Department of Neuology, Second
Affiliated Hospital, Zhejiang University, Hangzhou, P.R. China.
Address correspondence to Prof Dr Deuschl, Neurologische Klinik
of the University Hospital Kiel, Niemannsweg 147, 24105 Kiel,
Germany. E-mail:
© 2002 Wiley-Liss, Inc.
dyskinesias (LIDs) and motor fluctuations are major complications of therapy in longstanding Parkinson’s disease (PD). Once a patient develops LID, the sensitization to dopaminergic
treatment cannot be reversed by drugs.1 Deep-brain
stimulation of the subthalamic nucleus (STN DBS) is
an effective treatment for both fluctuations and dyskinesias in L-dopa–responsive PD.2,3 Its beneficial effects
on akinesia, rigidity, and tremor resemble that of
L-dopa, except for LIDs, which are promoted by acute
STN stimulation, whereas they improve with chronic
stimulation.5 This substantial improvement of LID by
STN DBS even in the on-drug state (desensitization) is
not fully understood.2,3,6 It has been ascribed to a reduction of dopaminergic dosage often allowed by the
motor benefits of the procedure, but its pathophysiology has remained unclear. We followed the hypothesis
that an inhibition of the excess of command sent to
the muscle could underlie the antidyskinetic effects of
chronic STN DBS and therefore measured the calibration of force in the precision grip, a function governed
mainly by the motor cortex.7,8 An overshooting of grip
force in late-stage PD has been described in this condition. Patients often apply excessive force in the ondrug state when grasping to lift a small object.9 –11
This force excess is closely related to the severity of
LID in PD12 and therefore is suited to objectively
study the beneficial effect of STN DBS on a surrogate
parameter for LID. We also tried to elucidate if direct
(related to stimulation) or indirect (related to reduction
of dopaminergic drugs) effects account for therapeutic
Patients and Methods
We studied a series of 10 advanced-stage patients with PD
who underwent STN DBS. All suffered from severe motor
fluctuations and LID (Table). Written informed consent was
given by all patients. Ten age-matched healthy controls also
were included in the study, which was approved by the local
ethics committee. The assessment was conducted after a 12hour overnight withdrawal of dopaminergic drugs. Patients
were assessed in 2 treatment conditions before (off-drug and
on-drug) and in 4 conditions 3 months after surgery (offdrug/off-stimulation, on-drug/off-stimulation, off-drug/onstimulation, and on-drug/on-stimulation). A suprathreshold
dose of L-dopa was applied.4 The motor score of the Unified
Parkinson’s Disease Rating Scale (UPDRS) and a dyskinesia
score of 7 body regions (score range 0 –28) were rated in all
conditions.5 The amplitude of motor fluctuations was defined as the difference between UPDRS motor score off-drug
and on-drug both before surgery and after surgery (onstimulation). The L-dopa equivalent daily dose (LEDD) was
calculated as described previously.4 The subjects grasped an
object (220gm, equipped with 3-dimensional force sensors)
between the thumb and index finger and lifted it 15 times at
a natural pace. We focused on the peak grip force (GFPEAK),
a measure with a proven sensitivity for LID.12 Mean values
Table. Mean Scores of the UPDRS Motor Score and the Dyskinesias Score Before and 3 Months after Surgery
Before surgery
After surgery
Motor Score ( ⫾ SD)
Dyskinesia Score ( ⫾ SD)
Off drug
On drug
⌬ Off-on
Off drug/off stim
On drug/off stim
Off drug/on stim
On drug/on stim
⌬ Off-on drug
50.3 ⫾ 12.8
20.4 ⫾ 6.3
29.9 ⫾ 14.4
42 ⫾ 17.2
17.7 ⫾ 7.1
19.9 ⫾ 10.6b
13.8 ⫾ 5.7c
6.1 ⫾ 7.4d
3.4 ⫾ 5.4
9.1 ⫾ 5.6
2.9 ⫾ 2.9c
2.9 ⫾ 1.9
4.9 ⫾ 4.2c
p ⬍ 0.001, bp ⬍ 0.01, compared with presurgery off drug condition.
p ⬍ 0.01 compared with presurgery drug condition.
p ⬍ 0.01 compared with amplitude of motor fluctuations before surgery (⌬ off-on drug, on stim).
SD ⫽ standard deviation; stim ⫽ stimulation.
of GFPEAK of the thumb were calculated from the lifting
trials 6 to 15 of both sides (Fig 1).
Grip force was compared between groups and treatment
conditions by a general linear model (SPSS 10). Significance
was assumed for p values less than 0.05 after Bonferroni correction. Clinical scores and LEDD were compared using the
Wilcoxon test with a p level of less than 0.01.
The LEDD was reduced in all but 2 patients, on average by 45% ( p ⬍ 0.01). The UPDRS motor score
was improved by ⫺60% off-drug and by ⫺32% ondrug. The amplitude of motor fluctuations decreased
by ⫺80% (see Table).
Off-dystonia was abolished in all 9 patients involved.
On-phase dyskinesias of the total body were completely
abolished in 1 patient, and the score was reduced in all
the other patients, on average by ⫺67%. Dyskinesias
tended to increase on combined challenge with L-dopa
and STN DBS, but the mean score was still ⫺44%
lower than it was preoperatively.
Peak grip force (GFPEAK) was significantly influ-
enced by treatment. After 3 months of chronic stimulation, the force overflow vanished. No excess of
GFPEAK was observed any longer regardless of the state
of drug or stimulation, and the grip force was equivalent to controls (see Figs 1 and 2). The presurgical excess of GFPEAK in the off-drug condition was abolished. In the on-drug state GFPEAK decreased by
⫺110%. In the combined treatment state GFPEAK was
still significantly lower than before surgery (see Fig 2).
A reduction of initially exaggerated GFPEAK was observed not only in the 8 patients with a reduced LEDD
after surgery, but also in the 2 patients on-stimulation
whose LEDD was not reduced because of remaining
minor fluctuations and positive effects on mood. In
on-drug condition, the peak grip force decreased by
⫺51% and by ⫺48% in the first and second patient,
respectively. The same was true in the off-drug state
(GFPEAK, ⫺45% and ⫺25%). Thus, grip force excess
resolved, although LEDD was not decreased in either
of the patients. Furthermore, GFPEAK was not correlated significantly with LEDD, neither before nor after
Fig 1. Peak grip force (GFPEAK
in Newtons) in a healthy control
subject and a representative patient who suffered from severe
L-Dopa–induced dyskinesia.
Force overshooting in on-state
was abolished after chronic stimulation. (thick lines) Mean;
(thin lines) standard error of
mean from 10 trials. Scale is
identical for all conditions.
PD ⫽ Parkinson’s disease; N ⫽
Newton; S ⫽ seconds.
Wenzelburger et al: Desensitization by DBS of the STN in PD
Fig 2. Mean peak grip force
(GFPEAK ⫹ SD) before and 3
months after surgery. The overshooting of force was abolished
in all conditions after surgery.
(single dagger) p ⬍ 0.05,
(double dagger) p ⬍ 0.01 compared with on-drug state before
surgery. (asterisk) p ⬍ 0.05
compared with presurgical offdrug state. (single circle) p ⬍
0.05, (double circle) p ⬍ 0.01
compared with controls.
surgery (Spearman coefficient, ⬍ 0.4; p ⬎ 0.05). The
reduction of GFPEAK closely matched the effect of
STN DBS on peak-dose dyskinesias.
We observed a remarkable normalization of force calibration in dexterous movements induced by chronic
STN DBS which was found both during on-state and
off-state. The preoperative L-dopa challenge caused an
overshooting of grip force in line with previous findings,11,12 but this was no longer the case in the postoperative challenge with the same dosage. The initial
grip force excess vanished almost completely under
chronic STN DBS. In parallel with the reduction of
grip force overshoot, the LID score during a suprathreshold L-dopa challenge was reduced substantially,
which was within the range expected from recent reports.2,3,5,6 These findings extend our earlier observation that grip force excess in on-state is found in parkinsonian patients with motor fluctuations only, and
overshooting of force correlates with the severity of
LID.12 The close relationship between LID and force
regulation is now supported by similar benefits of STN
DBS on both.
The reduction of LID in patients with STN DBS
has been ascribed to a reduction of dopaminergic dosage,3,5,6 and a direct effect of DBS has been discussed
mainly for off-dystonia.5 These observations make this
view unlikely. We observed substantial benefits on LID
in all patients, regardless whether the dopaminergic
drugs were reduced. Furthermore, the LID-suppressing
effect of STN is not simply part of a general effect on
Annals of Neurology
Vol 52
No 2
August 2002
all the parkinsonian symptoms because switching off
the stimulator led to a reoccurrence of severe akinesia
and rigidity but left LID and force excess unchanged.
Therefore, the LID-suppressing effect is more likely to
reflect a desensitizing long-term effect of chronic STN
DBS on LID and force regulation that outlasts even a
temporary interruption of stimulation. The development of response fluctuations is believed to be caused
by the discontinuous pharmacological stimulation of
the dopamine-receptors by drug treatment. In contrast,
STN DBS is continuously stimulating the motor system. We propose that such continuous stimulation
may explain desensitization of both LID and grip force
excess. This desensitization of involuntary motor activity might be explained by plastic changes in the motor
system directly related to chronic STN DBS. It is not
yet clear if this desensitization takes place within the
basal ganglia or the cortex. The profound changes of
cortical metabolism when comparing the on-state and
the off-state with functional imaging might support the
latter possibility but is far from proving this interpretation.
This research was supported by the Deutsche Forschungsgemeinsschaft (01KO9811/7, R.W.) and the Kompetenznetzwerk Parkinson.
B.-R.Z. was on sabbatical leave from the Department of Neurology,
Second Affiliated Hospital, Zhejiang University, Hangzhou, P.R.
China, and was supported by the Kiel University.
We thank Mrs Witt for the excellent support of this investigation,
Dr Johansson and Mr Bäckström for advice concerning the grip
paradigm and the SC/ZOOM software, and Dr Pohl for valuable
help with the statistics.
1. Nutt JG. Clinical pharmacology of levodopa-induced dyskinesia. Ann Neurol 2000;47(suppl 1):S160 –S164; discussion,
S164 –S166.
2. Limousin P, Krack P, Pollak P, et al. Electrical stimulation of
the subthalamic nucleus in advanced Parkinson’s disease.
N Engl J Med 1998;339:1105–1111.
3. Krack P, Limousin P, Benabid AL, Pollak P. Chronic stimulation of subthalamic nucleus improves levodopa-induced dyskinesias in Parkinson’s disease. Lancet 1997;350:1676.
4. Krack P, Pollak P, Limousin P, et al. Subthalamic nucleus or
internal pallidal stimulation in young onset Parkinson’s disease.
Brain 1998;121:451– 457.
5. Krack P, Pollak P, Limousin P, et al. From off-period dystonia
to peak-dose chorea. The clinical spectrum of varying subthalamic nucleus activity. Brain 1999;122:1133–1146.
6. Bejjani BP, Arnulf I, Demeret S, et al. Levodopa-induced dyskinesias in Parkinson’s disease: is sensitization reversible? Ann
Neurol 2000;47:655– 658.
7. Jeannerod M. The formation of finger grip during prehension.
A cortically mediated visuomotor pattern. Behav Brain Res
1986;19:99 –116.
8. Lemon RN, Johansson RS, Westling G. Corticospinal control
during reach, grasp, and precision lift in man. J Neurosci 1995;
15:6145– 6156.
9. Gordon AM, Ingvarsson PE, Forssberg H. Anticipatory control
of manipulative forces in Parkinson’s disease. Exp Neurol 1997;
145:477– 488.
10. Fellows SJ, Noth J, Schwarz M. Precision grip and Parkinson’s
disease. Brain 1998;12:1771–1784.
11. Gordon AM, Reilmann R. Getting a grasp on research: does
treatment taint testing of parkinsonian patients? Brain 1999;
12. Wenzelburger R, Zhang BR, Pohle S, et al. Force overflow and
levodopa-induced dyskinesias in Parkinson’s disease. Brain
2002;125:871– 879.
Demonstration of Acute
Ischemic Lesions in the Fetal
Brain by Diffusion Magnetic
Resonance Imaging
Cristina Baldoli, MD,1 Andrea Righini, MD,2
Cecilia Parazzini, MD,2 Giuseppe Scotti, MD,1
and Fabio Triulzi, MD2
The possibility of detecting acute hypoxic-ischemic brain
lesions by prenatal magnetic resonance imaging or ultrasound is low. We present a case of a fetus with a vein of
Galen arteriovenous malformation in whom prenatal
diffusion-weighted magnetic resonance imaging at 33
weeks of gestation clearly detected cerebral acute ischemic lesions, associated with remarkable decrease of the
average apparent diffusion coefficient, whereas T2weighted imaging was still not informative.
Ann Neurol 2002;52:243–246
Prenatal magnetic resonance imaging (MRI) is widely
used to confirm ultrasound findings in cases of complex fetal brain malformations.1 Prenatal MRI and ultrasound also can evaluate the end-stage sequelae of
hypoxic-ischemic fetal brain damage2; however, the
possibility of detecting these lesions by prenatal MRI
or ultrasound in the acute phase is low.3 Diffusionweighted MRI (DWI) has been shown to be very sensitive in detecting hyperacute and acute hypoxicischemic brain damage in adults4 and in premature5 or
term6 –10 newborns. We present the case of a fetus with
a vein of Galen arteriovenous malformation (VGAM),
in whom prenatal DWI at 33 weeks of gestation clearly
detected acute ischemic lesions within the brain,
whereas T2-weighted imaging was still not informative.
Case Report
A 27-year-old pregnant woman was referred to our institution at 33 weeks of gestation because of suspected fetal
VGAM at Doppler ultrasonography. A prenatal MRI study
at 1.5T (Eclipse; Marconi, Cleveland, OH) using the flex
From the 1Neuroradiology Departments, Università Salute e Vita
IRCCS–San Raffaele and 2Istituti Clinici di Perfezionamento, Milan, Italy.
Received Jan 25, 2002, and in revised form Mar 20. Accepted for
publication Mar 23, 2002.
Published online Jun 23, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10255
Address correspondence to Dr Righini, Radiologia e Neuroradiologia, Ospedale dei Bambini V. Buzzi, Via castelvetro 32, 20154 Milan, Italy. E-mail:
© 2002 Wiley-Liss, Inc.
body surface coil and T2-weighted 5mm-thick single-shot
fast spin-echo sequences was performed.
T2-weighted images confirmed the presence of the
VGAM, which appeared as an interhemispheric flow
void mass in the region of the vein of Galen, connected to the torcular. Some irregular and prominent
vessels were noted around the third ventricle; they were
compatible with choroidal arteries feeding the malformation. The brain parenchyma did not exhibit clear
signal alterations, and there was no ventricular enlargement (Fig 1). Because hypoxic-ischemic brain lesions
can be associated with VGAM, a DWI acquisition was
performed in the same occasion. The DWI study was
based on an echo-planar 3-axis diffusion sensitized sequence, and 5mm-thick slices were acquired at fetal
brain level (matrix, 128 ⫻ 128; field of view, 30cm;
b-factor, 0 –1,000sec/mm2). DWI showed areas of hyperintense abnormal signal within the parietal and occipital lobe of the right hemisphere, and there were
similar findings in the left temporal lobe; these foci of
high signal encompassed not only the cortex, but also
part of the adjacent subcortical and periventricular
white matter (see Fig 1); in these areas, the rotationally
averaged apparent diffusion coefficient (trace-ADC)
was remarkably reduced (average value, 0.97; standard
deviation [SD], 0.038␮m2/msec) both for apparently
normal frontal lobe areas (average value, 1.83; SD,
0.18␮m2/msec) or for literature data on ADC in premature neonates.11,12
At 38 weeks of gestation, a cesarean section was performed, and a baby girl was delivered. She presented
with severe muscular hypotonia and signs of moderate
left ventricle failure at Doppler ultrasonography
(23mm end diastolic diameter and 41% ejection fraction), whereas she did not show significant polypnea or
pulmonary edema at chest x-rays. Four days after birth,
an MRI study showed severe diffuse atrophy of both
cerebral hemispheres with drastic enlargement of cortical liquoral spaces and ex vacuo dilatation of lateral
ventricles; MRI showed also severe general cortical necrosis, most of the cortex being reduced to a thin rim,
and diffuse loss of white matter, which presented several areas of high T1 signal. These were probably a sign
of diffuse calcification, as they were too confluent to be
hemorrhagic (Fig 2). No DWI or fluid-attenuated inversion recovery sequences were performed on this occasion. These findings were compatible with the endstage sequelae of severe ischemic insults.
This report shows that DWI studies of fetal brain are
feasible. This case highlights the potential of DWI in
detecting acute fetal hypoxic-ischemic brain damage,
Fig 1. (Top row) Single-shot fast spin-echo T2-weighted axial sections from the prenatal magnetic resonance imaging study at 33
weeks of gestation, showing no clear signal alterations within brain parenchima. The presence of the a vein of Galen arteriovenous
malformation (arrowheads) and of possible abnormal choroidal arteries (arrow) is clearly noticeable. (Middle row) Diffusionweighted axial sections at similar levels depicting areas of markedly abnormal hyperintense signal in the occipital and parietal lobe
of the right hemisphere (arrows). Some abnormal hyperintensity is visible also in the left temporal lobe (curved arrow). Both cortical and adjacent white matter areas are affected. (Bottom row) Corresponding trace–apparent diffusion coefficient (ADC) maps
showing a clear ADC decrease in the same areas (arrows) of diffusion-weighted magnetic resonance imaging signal alterations.
Annals of Neurology
Vol 52
No 2
August 2002
Fig 2. (Top row) Two T2-weighted axial
sections (top left and top middle) and 1
axial T1-weighted section (top right) from
the postnatal magnetic resonance imaging
(MRI) study showing diffuse severe atrophy
of both hemispheres, lateral ventricles ex
vacuo dilatation, extensive white matter
loss, and large areas of T1 hyperintensity
within it, compatible with diffuse calcifications (arrows). (Bottom row) Two sagittal T1-weighted sections from the postnatal
MRI study showing diffuse drastic enlargement of cortical liquoral spaces and thinning of corpus callosum. The presence of
abnormal choroidal arteries feeding the
VGAM is confirmed (arrows).
confirming the well-known high accuracy of DWI in
acute stroke diagnosis both in adults and in children.
Although we cannot provide a direct demonstration of
the ischemic nature of the lesions we detected, previous
reports13–15 have extensively documented that ischemic
and malacic lesions are the main complications that occur in subjects with VGAM, in both the prenatal and
postnatal period. Moreover, the remarkable reduction in
ADC that we noticed in the lesions has been reported
only in a few other brain diseases, such as encephalitis,
mature abscess, and status epilepticus16; in our case, the
location of the lesions, their evolution, and the clinical
course were poorly compatible with these conditions.
Three mechanisms have been postulated for the
pathogenesis of the hypoxic-ischemic brain injuries detectable in VGAM patients15: a steal phenomenon
caused by blood shunting from the normal parenchymal arteries into those feeding the malformation; parenchymal hypoperfusion secondary to intracranial venous hypertension and congestion; and multiple organ
hypoperfusion and hypoxia due to congestive heart failure. One or more of these mechanisms, acting together, might have produced the brain lesions that we
observed in the fetus. We believe that the lesions were
caused first by a steal phenomenon and second by a
venous congestion mechanism, because the degree of
cardiac failure was not so clinically important after
birth. For these reasons, it is likely that the lesions of
the cortex and white matter were mainly ischemic (because of reduce blood flow) rather than hypoxic (because of arrival of less oxygenated blood). The postnatal MRI showed diffuse cortical atrophy and
leukomalacia, well beyond the areas of initial gray and
white matter DWI alteration. A possible explanation
for this discrepancy is that episodes of more extensive
brain hypoperfusion repeatedly occurred after the prenatal MRI. The cerebral hemodynamics of our fetus
was probably unstable, and the intracranial blood flow
autoregulation mechanism was weak, so the prenatal
MRI provided a snapshot of still evolving and spreading damage. The end stage of the damage included
signs of calcific degeneration of the residual white matter at postnatal MRI; such calcifications are known to
develop within the brain of newborns who have suffered hypoxic-ischemic insults.17
The explanation for the ADC decrease observed in
our case resembles what is commonly reported for
acute ischemia in adult humans or animal models18,19;
it is based on energy failure of the cell membrane, with
development of cytotoxic edema and consequent relative decrease of the extracellular water and increase of
the intracellular one, which is more restricted and less
diffusible. However, water diffusion in the brain differs
according to maturity: the normal averaged ADC values in the white matter of premature neonates are
much higher (range, approximately 1.8 –1.9␮m2/
msec)11,12 than those of adults (range, approximately
0.7– 0.9␮m2/msec)12,20; this finding could be related
to the higher water content, to the lower macromolecules concentrations, or to the poorer neuronal and
glial “packing” in developing and premyelinated brain
tissue.12,21 This difference might account also for the
abnormal ADC variations that were observed in our
case. We noticed an ADC decrease to an average value
of 0.97␮m2/msec (SD, 0.038␮m2/msec); similar ADC
reductions have been reported recently in the acute
Baldoli et al: Diffusion MRI of Fetal Brain Ischemia
stage of periventricular leukomalacia affecting premature babies.5 These values are almost double those
commonly reached by mean diffusivity decrease in
acute ischemic lesions of adults.20 However, in this
case and in those of premature babies, the approximately 50% reduction in ADC for normal areas was
similar to what has been observed with ischemia in
adults. We hypothesize that the same tissular features
(eg, water content, macromolecules concentrations,
neuronal and glial “packing”), which produce the differences in ADC between the normal developing brain
and adult brain, can play a role also in producing different ADC value decreases in pathological conditions,
such as ischemia. To better understand these aspects,
we need to study more cases of acute fetal or premature
neonatal brain ischemia by using DWI, possibly correlating the results with neuropathological findings. Previous work8,9 on timing of the ADC modifications after acute perinatal hypoxic-ischemic damage suggests
that a first ADC reduction can be noticed within 6 to
72 hours after the acute event, with a possible delayed
and more widespread reduction in the following days,
when conventional MRI also shows signal alterations
due to developed vasogenic edema. Although in our
case, we present only 1 time point of the ADC change
time course, we hypothesize that we imaged mostly lesions associated with the first ADC decrease, because
prenatal T2-weighted images did not show significant
signal alterations.
Although the prenatal detection of VGAM is rare,
the early diagnosis by DWI of any associated destructive brain lesions can be important in therapeutic decision making, for example, in the decision to perform
therapeutic intravascular procedures. Fetal brain
hypoxic-ischemic damage also can occur in several
other conditions, such as twin-to-twin transfusion syndrome, abruptio placentae, preeclampsia, severe maternal anemia, and severe hypovolemia. Under these circumstances, prenatal DWI is a promising technique
that can detect acute hypoxic-ischemic complications,
which could influence clinical and parental decisions.
1. Girard N, Raybaud C, Gambarelli D, et al. Fetal brain MR
imaging. Magn Reson Imaging Clin N Am 2001;9:19 –56.
2. De Laveaucoupet J, Audibert F, Guis F, et al. Fetal magnetic
resonance imaging (MRI) of ischemic brain injury. Prenat Diagn 2001;21:729 –736.
3. Langer B, Boudier E, Gasser B, et al. Antenatal diagnosis of
brain damage in the survivor after second trimester death of a
monochorionic monoamniotic co-twin. Fetal Diagn Ther 1997;
12:286 –291.
4. Warach S, Chien D, Li W, et al. Fast magnetic resonance
diffusion-weighted imaging of acute human stroke. Neurology
5. Inder T, Huppi PS, Zientara GP, et al. Early detection of
periventricular leukomalacia by diffusion-weighted magnetic
resonance imaging techniques. J Pediatr 1999;134:631– 634.
Annals of Neurology
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No 2
August 2002
6. Cowan FM, Pennock JM, Hanrahan JD, et al. Early detection
of cerebral infarction and hypoxic-ischemic encephalopathy in
neonates using diffusion-weighted magnetic resonance imaging.
Neuropediatrics 1994;25:172–175.
7. Johnson AJ, Lee BC, Lin WL. Echoplanar diffusion-weighted
imaging in neonates and infants with suspected hypoxicischemic injury: correlation with patient outcome. Am J Roentgenol 1999;172:219 –226.
8. Robertson RL, Ben-Sira L, Barnes PD, et al. MR line scan diffusion weighted imaging of term neonates with perinatal brain
ischemia. AJNR Am J Neuroradiol 1999;20:1658 –1670.
9. Soul JS, Robertson RL, Tzika AA, et al. Time course of changes
in diffusion-weighted magnetic resonance imaging in a case of
neonatal encephalopathy with defined onset and duration of
hypoxic-ischemic insult. Pediatrics 2001;108:1211–1214.
10. Barkovich AJ, Westmark KD, Bedi HS, et al. Proton spectroscopy and diffusion imaging of the first day of life after perinatal
asphyxia: preliminary report. AJNR Am J Neuroradiol 2001;22:
1786 –1794.
11. Huppi PS, Maier SE, Peled S, et al. Microstructural development of human newborn cerebral white matter assessed in vivo
by diffusion tensor magnetic resonance imaging. Pediatr Res
1998;44:584 –590.
12. Tanner SF, Ramenghi LA, Ridgway JP, et al. Quantitative
comparison of intrabrain diffusion in adults and preterm and
term neonates and infants. AJR Am J Roentgenol 2000;174:
13. De Koning TJ, Gooskens R, Veenhoven R, et al. Arteriovenous
malformation of vein of Galen in three neonates: emphasis on
associated early ischaemic brain damage. Eur J Pediatr 1997;
156:228 –229.
14. Baeziger O, Martin E, Willi U, et al. Prenatal brain atrophy
due to a giant vein of Galen malformation. Neuroradiology
15. Brunelle F. Arteriovenous malformation of the vein of Galen in
children. Pediatr Radiol 1997;27:501–513.
16. Righini A, Pierpaoli C, Alger JR, Di Chiro G. Brain parenchyma apparent diffusion coefficient alterations associated with
experimental complex partial staus epilepticus. Magn Reson Imaging 1994;12:865– 871.
17. Ansari MQ, Chincanchan CA, Armstrong DL. Brain calcification in hypoxic-ischemic lesions: an autopsy review. Pediatr
Neurol 1990;6:94 –101.
18. Pierpaoli C, Righini A, Linfante I, et al. Histopathologic correlates of abnormal water diffusion in cerebral ischemia:
diffusion-weighted MR imaging and light and electron microsopic study. Radiology 1993;189:439 – 448.
19. Lutsep HL, Albers GW, DeCrespigny A, et al. Clinical utility
of diffusion weighted magnetic resonance imaging in the assessment of ischemic stroke. Ann Neurol 1997;41:574 –580.
20. Warach S, Gaa J, Siewert B, et al. Acute human stroke studied
by whole brain echo planar diffusion-weighted magnetic resonance imaging. Ann Neurol 1995;37:231–241.
21. Baratti C, Barnett AS, Pierpaoli C. Comparative MR imaging
study of brain maturation in kittens with T1, T2, and the trace
of the diffusion tensor. Radiology 1999;210:133–142.
Selective Loss of Cholinergic
Sudomotor Fibers Causes
Anhidrosis in Ross
Claudia Sommer, MD,1 Thies Lindenlaub, MD,1
Detlef Zillikens, MD,2 Klaus V. Toyka, MD,1
and Markus Naumann, MD1
Ross syndrome consists of segmental hyperhidrosis with
widespread anhidrosis, Adie syndrome, and areflexia. The
cause of this disorder is unknown. Selective degeneration
of cholinergic fibers or of neural crest–derived structures
has been suggested. We present clinical and skin biopsy
data of 4 patients, providing evidence of reduced cholinergic sweat gland innervation in hypohidrotic skin by
morphometric analysis. These findings indicate a selective
degenerative process of the cholinergic sudomotor neurons.
Ann Neurol 2002;52:247–250
The pathogenesis of Ross syndrome1 (tonic pupils,
areflexia, and segmental hyperhidrosis) is as yet unknown. Wide overlap between Ross syndrome, Adie
syndrome, Harlequin syndrome (isolated progressive
segmental hypohidrosis), and a more widespread autonomic disease has been suggested.2 Here, we present
findings from 4 patients with anhidrosis and segmental
hyperhidrosis and variable expression of Adie’s pupils,
hyporeflexia, and pathological neurophysiological findings. Importantly, the underlying pathology was the
same in all patients, with reduction of cholinergic
sweat gland innervation and normal epidermal and
subepidermal nerve fibers.
Patients and Methods
Skin biopsies were obtained from a hyperhidrotic and an anhidrotic area of the patients’ back after informed consent.
After fixation in 4% paraformaldehyde, 40␮m frozen sections were stained with antibodies to the panneuronal
From the Departments of 1Neurology and 2Dermatology, University of Würzburg, Würzburg, Germany.
Received Feb 21, 2002, and in revised form Mar 18. Accepted for
publication Mar 23, 2002.
Published online Jun 23, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10256
Current address for Dr Lindenlaub: Neurologische Klinik, Universitätskliniken des Saarlandes, 66424 Homburg, Germany.
Address correspondence to Dr Sommer, Neurologische Universitätsklinik, Josef-Schneider-Strasse 11, D-97080 Würzburg, Germany.
marker protein gene product 9.5 (PGP 9.5, 1:800; Ultraclone, Wellow, UK) and to vasoactive intestinal peptide
(VIP, 1:500; Peninsula, San Carlos, CA). Immunofluorescence was performed using Cy3 or Cy2-labeled secondary
antibodies (1:100; Amersham, Arlington Heights, IL). An
ABC system (Vector, Burlingame, CA) was used with the
same primary antibodies to verify the staining. For analysis
of skin morphology (hematoxylin and eosin stain) and immune mediators, 6␮m-thick frozen sections were reacted
with primary antibodies to T cells (CD3, CD4, CD8,
CD45RO), B cells (CD20), and macrophages (CD68) or
fluorescein isothiocyanate–labeled antibodies to human immunoglobulin (Ig) G, IgM, IgA, and complement component C3 (Dako, Hamburg, Germany). The sections were
viewed with a Zeiss Axiophot 2 (Zeiss, Göttingen, Germany)
microscope equipped with internal z-focus and a motorized
scanning table by Märzhäuser, Wetzlar, Germany. Using Image Pro Plus 4.0 software, we generated 3-dimensional reconstructions of the epidermis and the sweat glands. Epidermal innervation was quantified by counting the nerve
endings per millimeter of epidermal length and by determination of the epidermal nerve area with optical densitometry.
Nerve endings were required to have a visible length greater
than 20␮m to be considered. The subepidermal nerve plexus
was measured in an area of 100␮m ⫻ 0.3mm along the
subepidermal/epidermal border. Sweat gland innervation was
quantified by measuring the complete nerve fiber area associated with sweat glands. Values from the ipsilateral and contralateral side were compared using Student t test; p values of
less than 0.05 were considered significant.
Neurophysiological Studies and Sudomotor Testing
Nerve conduction studies and somatosensory and magneticevoked potentials were performed using our standard neurophysiological techniques.3 Hyperhidrotic and anhidrotic areas were visualized using Minor’s iodine starch test.
Quantitative sensory testing (QST) was performed using a
Peltier device (Medoc thermal analyzer) and the methods of
limits.4 Heart rate variability was measured as described previously.5
Case 1
A 50-year-old man reported increased sweating on the
right side of the trunk and on the left face for 18 years.
Both hands and feet were dry. Physical examination
showed hyperhidrosis in the right axilla, in the dermatomes T6 to T10 on the right and T11 to T12 on
the left, and on the left face and neck. Ankle jerks were
absent. Nerve conduction studies were normal. The
H-reflex and the sympathetic skin response (SSR) on
both feet were absent. QST showed normal thresholds
in hyperhidrotic and anhidrotic areas of the back and
on both hands, but increased warm thresholds on the
dorsum of the right foot. The patient was treated with
200 units of botulinum toxin (Botox威) in the hyperhidrotic area in T6 to T10, with a 50% reduction in
sweat secretion within 4 days. Treatment was repeated
© 2002 Wiley-Liss, Inc.
3 months later, with a good effect lasting for 4 months
(Table 1).
Case 2
A 49-year-old man had hyperhidrosis in the right T5
to T7 dermatomes and in both knees for 7 years.
Hands and feet did not sweat. The left pupil was
wider, unreactive to light but had a tonic reaction to
convergence, consistent with Adie’s pupil. Tendon
jerks were elicitable only after reinforcement; right ankle jerk was absent. Latencies of somatosensory-evoked
potentials of both tibial nerves were delayed, and the
amplitude of the sural nerve action potential was
slightly reduced. Further nerve conduction studies and
SSRs (palms) were normal. The patient was injected
with 1,000 units of botulinum toxin (Dysport) in the
hyperhidrotic area of the trunk with marked reduction
of sweating.
Case 3
A 41-year-old woman had Adie’s pupils, areflexia, and
hyperhidrosis in the left lower thoracic and gluteal
area, and on the right ventral thigh and knee. The rest
of the body was anhidrotic. Hypoesthesia was present
on both lower legs; vibration thresholds were reduced
at the ankles and knees. Heart rate variability was at
the lower limit of normal. Nerve conduction studies
showed delayed latencies of somatosensory-evoked potentials of the right tibial nerve. Sensory nerve potentials of the sural nerve could not be elicited. SSR of the
right hand was absent. The patient opted for local
treatment with aluminum chloride hexahydrate, which
yielded satisfactory results.
Case 4
A 30-year-old man reported anisocoria since age 17
years, hyperhidrosis on the right side of the trunk and
the left temporal region since age 23 years, and heat
intolerance. He had Adie’s pupils, normal tendon jerks,
and hyperhidrosis of the dermatomes T4 to T6 on the
right and in the inguinal area on the left. Palms and
soles were dry. The SSR was absent. The patient was
treated with 1,000 units of botulinum toxin (Dysport)
in the hyperhidrotic area of the trunk. The area of excessive sweating decreased by approximately 60% for 6
Skin Biopsy Results
No inflammatory infiltrates and no deposition of IgG
or C3 were observed around sweat glands, which themselves appeared normal. Using PGP 9.5 immunohistochemistry, we found that epidermal innervation and
the subepidermal plexus were normal, whereas sweat
gland innervation was reduced in the anhidrotic areas
(Fig, a–d). Immunoreactivity for VIP was present in
the fibers innervating the sweat glands, but not in the
epidermal fibers, and was markedly reduced around
sweat glands of the anhidrotic side (Fig, e and f).
Morphometry showed normal epidermal and subepidermal innervation density in hyperhidrotic and anhidrotic regions. Sweat gland innervation was reduced
significantly in the anhidrotic compared with the hyperhidrotic regions ( p ⬍ 0.005; Table 2).
Here, we show selective loss of cholinergic sweat gland
innervation in 4 patients with Ross syndrome by using
the panneuronal marker PGP 9.5 and a marker for
cholinergic fibers, VIP.6 Because downregulation of
neuropeptides such as VIP may occur in nerve lesions
before degeneration of nerve fibers occurs, we used
PGP 9.5 for quantification of nerve fibers. Interestingly, epidermal innervation remained intact, well in
accordance with normal QST. Denervation as the
cause of anhidrosis in patients with Ross syndrome has
hitherto been suggested only on theoretical grounds.1
We previously presented qualitative data in another patient with Ross syndrome, also in whom a selective reduction of PGP 9.5 immunoreactive fibers around
sweat glands could be found.7 Earlier skin biopsies
Table 1. Clinical and Electrophysiological Data of 4 Patients with Ross Syndrome
Case Age
no. (yr) Gender
Duration of
Hyperhidrosis Pupils
T6–T10 R
T11 L
T5–T7 R
Gluteal L
Knee L
Ankle jerk Tibial nerve: H-reflex
Ankle jerk Sural nerve: A reduced Normal
tSEP: A reduced
Areflexia Sural nerve: no response Absent
tSEP: Lat delayed
A ⫽ amplitude; Lat ⫽ latency; L ⫽ left; NCS ⫽ nerve conduction studies; ND ⫽ not done; QST ⫽ quantitative sensory testing; R ⫽ right;
SSR ⫽ sympathetic skin response; tSEP ⫽ somatosensory-evoked potentials of the tibial nerves.
Annals of Neurology
Vol 52
No 2
August 2002
Fig. Forty-micrometer-thick frozen sections
from skin biopsies of Patient 2, immunofluorescence, and secondary antibody Cy3.
Sections from the hyperhidrotic side are on
the left (a, c, e) and from the anhidrotic
side on the right (b, d, f). (a, b) Immunoreaction with antibodies to the panneuronal marker protein gene 9.5 (PGP 9.5)
shows normal epidermal innervation in
the hyperhidrotic (a) and in the anhidrotic
(b) skin. (c, d) PGP 9.5–immunoreactive
fibers innervating sweat glands are abundant on the hyperhidrotic side (c), but
depleted on the anhidrotic side (d). (e, f)
Immunohistochemistry for vasoactive intestinal peptide shows normal cholinergic
sweat gland innervation on the hyperhidrotic side (e) and loss of this innervation on the anhidrotic side (f). Bar ⫽
from Ross syndrome patients were reported as normal;
however, immunohistochemical methods to visualize
axons were not used.8,9
Segmental anhidrosis and hyperhidrosis are often the
presenting symptoms in patients with Ross syndrome.
Of our 4 patients with these symptoms, 3 had addi-
tional Adie’s pupils and 3 also had areflexia or hyporeflexia. Nerve conduction studies were abnormal in 2
patients, and SSR was absent in 3 patients. Heart rate
variability was normal in 3 and at the lower level of
normal in 1 patient. QST was normal in the 2 patients
in whom it was performed. Our observations are con-
Table 2. Morphometric Analysis of Skin Biopsies
Epidermal Nerve Fibers
per mm
Case no.
Mean ⫾ SD
Subepidemal Nerve Fiber Area
per mm2
Area of Fibers Innervating
Sweat Glands per 1,000␮m2
sweat gland area
17.0 ⫾ 1.0
18.1 ⫾ 1.2
1249 ⫾ 82.4
1312.3 ⫾ 169.4
99.2 ⫾ 24.5
42.9a⫾ 5.5
Significant difference from contralateral side, p ⬍ 0.005.
SD ⫽ standard deviation.
Sommer et al: Sudomotor Fiber Loss in Ross Syndrome
sistent with previous reports suggesting that segmental
anhidrosis is part of the spectrum of distinct autonomic disorders due to a generalized injury to autonomic and dorsal root ganglia neurons.2,10 –12 It is unknown why these particular structures are involved.
Both the ciliary nerves and the sympathetic innervation
of sweat glands are cholinergic. However, a selective
degeneration of cholinergic fibers does not explain
areflexia, which has been suggested to be caused by loss
of large diameter afferent fibers.13 Shin and colleagues
suggested a combined lesion in these structures due to
their derivation from the neural crest.2 Pigment cells
and Schwann cells also are derived from the neural
crest. No Schwann cell pathology has been described in
Ross syndrome so far to our knowledge, but we previously described depigmentation in a patient with Ross
syndrome.7 The sympathetic innervation of sweat
glands is unusual, because it is initially catecholaminergic but becomes cholinergic after interactions with
the target tissue. Catecholamines are necessary to induce secretory responsiveness of the sweat glands.14
The reason for the segmental hyperhidrosis in patients with Ross syndrome has not been discovered yet.
The hyperhidrosis is not likely to be efficient as a thermoregulatory measure, and during treatment symptoms of heat intolerance did not occur to any greater
degree than before. The area of hyperhidrosis was always localized in the lower thoracic to upper lumbar
region. Why these ganglia/fibers are spared from the
degenerative process is unclear. In sudomotor fibers of
the rat, muscarinic M2 receptors are inhibitory presynaptic autoreceptors.15 The same function has been postulated for these receptors in human skin.16 We thus
speculate that the cholinergic fibers innervating sweat
glands first lose their M2 autoreceptors and that hyperhidrosis is caused by diminished presynaptic inhibition,
before further degeneration leads to anhidrosis.
This research was supported by research funds of the University of
We thank Dr C. Rose for evaluating skin pathology and B. Dekant
for excellent technical help.
1. Ross AT. Progressive selective sudomotor denervation. Neurology 1958;8:809 – 817.
2. Shin RK, Galetta SL, Ting TY, et al. Ross syndrome plus: beyond horner, Holmes-Adie, and harlequin. Neurology 2000;55:
3. Naumann M, Schalke B, Klopstock T, et al. Neurological multisystem manifestation in multiple symmetric lipomatosis: a
clinical and electrophysiological study. Muscle Nerve 1995;18:
693– 698.
4. Claus D, Hilz MJ, Hummer I, Neundorfer B. Methods of measurement of thermal thresholds. Acta Neurol Scand 1987;76:
288 –296.
Annals of Neurology
Vol 52
No 2
August 2002
5. Flachenecker P, Wermuth P, Hartung H-P, Reiners K. Quantitative assessment of cardiovascular autonomic function in
Guillain-Barré syndrome. Ann Neurol 1997;42:171–179.
6. Schütz B, Schäfer MK, Eiden LE, Weihe E. VIP and NPY expression during differentiation of cholinergic and noradrenergic
sympathetic neurons. Ann N Y Acad Sci 1998;865:537–541.
7. Bergmann I, Dauphin M, Naumann M, et al. Selective degeneration of sudomotor fibers in Ross syndrome and successful
treatment of compensatory hyperhidrosis with botulinum toxin.
Muscle Nerve 1998;21:1790 –1793.
8. Bartin RH, Schmutz JL, Cuny JF, et al. Le syndrome de Ross.
A propos d’une observation. Ann Dermatol Venereol 1990;117:
9. Caparros-Lefebvre D, Hache JC, Hurtevent JF, et al. Unilateral
loss of facial flushing and sweating with contralateral anhidrosis:
harlequin syndrome or Adie’s syndrome? Clin Auton Res 1993;
3:239 –241.
10. Jacobson DM, Hiner BC. Asymptomatic autonomic and sweat
dysfunction in patients with Adie’s syndrome. J Neuroophthalmol 1998;18:143–147.
11. Bacon PJ, Smith SE. Cardiovascular and sweating dysfunction
in patients with Holmes-Adie syndrome. J Neurol Neurosurg
Psychiatry 1993;56:1096 –1102.
12. Drummond PD, Lance JW. Site of autonomic deficit in harlequin syndrome: local autonomic failure affecting the arm and
the face. Ann Neurol 1993;34:814 – 819.
13. Pavesi G, Macaluso GM, Medici D, et al. On the cause of
tendon areflexia in the Holmes-Adie syndrome. Electromyogr
Clin Neurophysiol 1994;34:111–115.
14. Tian H, Habecker B, Guidry G, et al. Catecholamines are required for the acquisition of secretory responsiveness by sweat
glands. J Neurosci 2000;20:7362–7369.
15. Haberberger RV, Bodenbenner M. Immunohistochemical localization of muscarinic receptors (M2) in the rat skin. Cell Tissue
Res 2000;300:389 –396.
16. Cavanah DK, Casale TB. Cutaneous responses to
anticholinergics: evidence for muscarinic receptor subtype participation. J Allergy Clin Immunol 1991;87:971–976.
Normokalemic Periodic
Paralysis Revisited: Does
It Exist?
Patrick F. Chinnery, PhD, MRCP,1
Timothy J. Walls, MD, FRCP,1
Michael G. Hanna, MD, MRCP,2
David Bates, MA, FRCP,1
and Peter R. W. Fawcett, BSc, FRCP3
of normoKPP confirmed earlier suspicions that these
families actually had a variant of hyperKPP due to a
SCN4A gene mutation.1,5 However, normoKPP remains enshrined in standard medical textbooks largely
because the original detailed clinical description of the
disorder by Poskanzer and Kerr2 in a family from the
northeast of England remains unchallenged.6 This family recently became the subject of study again when a
member of the next generation developed periodic paralysis, allowing us to revisit the original diagnosis.
Case Report
Normokalemic periodic paralysis (normoKPP) is well established in the literature, but there are doubts as to
whether it exists as a discrete entity. Retrospective clinical
and molecular analysis has confirmed suspicions that
most normoKPP families actually have a variant of hyperkalemic periodic paralysis (hyperKPP) due to a mutation of the muscle-specific sodium channel gene
(SCN4A). However, the original normoKPP family described by Poskanzer and Kerr (Poskanzer DC, Kerr
DNS. A third type of periodic paralysis, with normokalemia and favourable response to sodium chloride. Am J
Med 1961;31:328 –342) has remained unchallenged. We
identified the Met1592Val mutation of SCN4A in an affected descendent of this original normoKPP family. This
is the final piece in the puzzle: normoKPP is actually a
variant of hyperKPP and is not a distinct disorder.
Ann Neurol 2002;52:251–252
A 22-year-old man presented to the neurology department of
Royal Victoria Infirmary, Newcastle Upon Tyne, because of
an increase in the frequency and severity of his periodic muscle weakness. His symptoms began at 2 years of age. He experienced episodes of muscle weakness in all four limbs,
sometimes associated with painful muscle stiffness. The episodes typically were precipitated by cold and damp weather
and would begin during a period of rest after a period of
strenuous exercise. There was no relationship with fasting or
food intake. A severe attack typically would last for several
days, followed by a prolonged recovery phase taking days to
weeks. Two attacks had been so severe that he remained in
bed and could only lift his head off the pillow. Although he
occasionally experienced dysphagia during an attack, there had
never been any respiratory symptoms, and he had not noticed
any pigmenturia. His medical history included a camptodactyly correction to both hands but was otherwise unremarkable.
There was no history to suggest cardiac dysrhythmias.
Traditionally, three categories of periodic paralysis have
been described: hypokalemic (hypoKPP), hyperkalemic
(hyperKPP), and normokalemic (normoKPP).1 The
last decade has seen major advances in our understanding of the molecular and electrophysiological basis of 2
of these groups. Three point mutations in the skeletal
muscle dihydropyridine sensitive calcium channel
(CACLN1A3) are responsible for most cases of hypoKPP, and mutations in the muscle-specific sodium
channel gene (SCN4A) have been identified in a spectrum of disorders that includes hyperKPP, paramyotonia congenita, and potassium aggravated myotonia.1
Despite its prominence in standard texts, normoKPP
has been reported only in a few families.2– 4 Clinical
and molecular reanalysis of some of the original cases
There were no abnormal findings on systemic or neurological examination. Routine hematological and biochemical tests were normal (including serum electrolytes and thyroid function tests), with the exception of
an elevated alanine transaminase (53 U/L, normal
⬍45) and an elevated creatine kinase (807 U/L, normal ⬍190). A 12-lead electrocardiogram and chest
x-ray were normal. A neurophysiological study showed
normal peripheral nerve function, but several abnormalities on concentric needle electromyography. The
most striking feature was the frequent myotonic discharges seen in every muscle sampled (Fig). There was
also excess insertional activity and spontaneous activity
in the form of fibrillation potentials and positive sharp
waves recorded from biceps brachii, indicating minimal
muscle fiber degeneration and early myopathic changes.
Quantitative multi–motor unit potential analysis of the
motor unit potentials in biceps showed normal motor
unit potential durations, amplitudes, and number.
From the 1Department of Neurology, University of Newcastle Upon
Tyne, Newcastle Upon Tyne; 2Department of Neurology, Institute of
Neurology, London; and 3Department of Clinical Neurophysiology,
Royal Victoria Infirmary, Newcastle Upon Tyne, United Kingdom.
Received Feb 15, 2002, and in revised form Mar 19, 2002. Accepted for publication Mar 25, 2002.
Published online Jun 23, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10257
Address correspondence to Dr Chinnery, Department of Neurology,
The Medical School, Framlington Place, Newcastle Upon Tyne,
NE2 4HH, United Kingdom. E-mail:
Family History
The patient in this index case is the son of case V:10
who was studied in detail by Poskanzer and Kerr.2
Molecular Genetic Studies
The typical pattern of the episodes of weakness, the
muscle stiffness, and the prominent myotonic dis-
© 2002 Wiley-Liss, Inc.
Fig. Continuous raster display of the concentric needle electromyography findings in the right biceps brachii of the index
case. E ⫽ end of the myotonic discharge; I ⫽ insertion activity; O ⫽ onset of a myotonic discharge. Scale bar ⫽ 0.1mV
per 100 milliseconds (ms).
charges on electromyography pointed toward a muscle
sodium channel disorder.1 Molecular genetic analysis
by a standard polymerase chain reaction/restriction enzyme analysis identified the Met1592Val mutation of
SCN4A in the case described above.
We have shown that the original normoKPP family
studied by Poskanzer and Kerr2 harbor a point mutation in SCN4A that is found in approximately 30% of
families with hyperKPP.7 Molecular analysis of other
reported normoKPP families also has shown mutations
in SCN4A (Thr704Met).5 Based on these findings, our
conclusion is that it is highly likely that normoKPP is
actually a variant of hyperKPP, and it should not be
considered as a distinct disease entity.
In retrospect, the clinical and biochemical features of
the original family of Poskanzer and Kerr are consistent
with a diagnosis of hyperKPP.2 Twenty-one individuals
were reported in that study, each developing symptoms
in their first decade. The individuals experienced attacks of weakness every 1 to 3 months, each lasting
from 2 days to 3 weeks. The attacks were provoked by
rest after exertion, cold and damp conditions, and alcohol, particularly the local beer, which contained a
high concentration of potassium. Oral potassium chloride exacerbated the attacks of muscle weakness in 4
members of the original family. Potassium sensitivity,
the hallmark of muscle sodium channel disorders,1
therefore was clearly described in the original article.2
Although no significant change in serum potassium
was ever documented during numerous spontaneous or
precipitated attacks of weakness, it is now well recognized that this can occur in patients with hyperKPP.1,7
Perhaps the most interesting feature of this family is
that before they received medical attention, they realized that a high intake of table salt reduced the frequency and severity of the attacks of muscle weakness.
Annals of Neurology
Vol 52
No 2
August 2002
The muscle weakness in patients with hyperKPP is
caused by muscle depolarization. In families with
Met1592Val, this arises through impaired fast inactivation and slow inactivation of the sodium channel.8,9
Poskanzer and Kerr2 documented an objective improvement in muscle strength in 5 individuals who were given
up to 1,750ml of triple normal saline over a 12-hour
period. The mechanism responsible for this improvement is unclear, and neither the weakness nor the infusions were associated with a change in serum Na⫹ or
K⫹ levels. Although no control experiments were conducted, this observation requires further investigation
because of the possibility that an increased sodium load
may also benefit other patients with hyperKPP.
The issue of treatment is highly pertinent to our index case. Our patient’s father (V:10 in the original
study2) now has generalized muscle wasting and a severe fixed vacuolar myopathy limiting normal daily activities. His son is currently not weak between attacks
but has myopathic features on electromyography. Our
objective is to prevent clinical progression in our new
patient, and, although unsubstantiated at present, it is
logical to take steps to reduce the frequency and severity of attacks. This could be achieved by alterations in
his behavior and diet, or using a carbonic anhydrase
inhibitor such as dichlorphenamide.10
We are very grateful to Dr D. N. S. Kerr for his comments on the
original study and on this article.
1. Ptacek LJ, Bendahhou S. Ion channel disorders of muscle. In:
Karpati G, Hilton-Jones D, Griggs RC, eds. Disorders of voluntary muscle. 7th ed. Cambridge: Cambridge University Press,
2001:604 – 635.
2. Poskanzer DC, Kerr DNS. A third type of periodic paralysis,
with normokalemia and favourable response to sodium chloride. Am J Med 1961;31:328 –342.
3. Mayers KR, Gilden DH, Rinaldi CF, Hansen JL. Periodic muscle weakness, normokalemia, and tubular aggregates. Neurology
1972;22:269 –279.
4. Danowski TS, Fisher ER, Vidalon C, et al. Clinical and ultrastructural observations in a kindred with normo-hyperkalemic
periodic paralysis. J Med Genet 1975;12:20 –28.
5. Lehmann-Horn F, Rudel R, Ricker K. Workshop report. Nondystrophic myotonias and periodic paralyses. Neuromuscul Disord 1993;3:161–168.
6. Rudel R, Lehmann-Horn F. Muscle sodium channel and chloride channel diseases. In: Lane RJ, ed. Handbook of muscle
disease. New York: Marcel Dekker, 1996:348.
7. Cannon SC. From mutation to myotonia in sodium channel
disorders. Neuromuscul Disord 1997;7:241–249.
8. Cannon SC, Brown RH Jr, Corey DP. A sodium channel defect in hyperkalemic periodic paralysis: potassium-induced failure of inactivation. Neuron 1991;6:619 – 626.
9. Hayward LJ, Sandoval GM, Cannon SC. Defective slow inactivation of sodium channels contributes to familial periodic paralysis. Neurology 1999;52:1447–1453.
10. Tawil R, McDermott MP, Brown R Jr, et al. Randomized trails
of dichlorphenamide in the periodic paralyses. Ann Neurol
2000;47:46 –53.
Human Antibodies against
Amyloid ␤ Peptide:
A Potential Treatment for
Alzheimer’s Disease
Richard Dodel, MD,1 Harald Hampel, MD,2
Candan Depboylu, MD,1 Suizhen Lin, MD,3
Feng Gao, MD,3 Sabine Schock, MD,1 Steffi Jäckel, MD,1
Xing Wei, MD,3 Katharina Buerger, MD,2
Christine Höft, BSc, 1 Bernhard Hemmer, MD,1
Hans-Jürgen Möller, MD,2 Martin Farlow, MD,3
Wolfgang H. Oertel, MD,1 Norbert Sommer, MD,1
and Yansheng Du, PhD3
Naturally occurring antibodies directed against
␤-amyloid (A␤) were detected in intravenous immunoglobulin preparations. After intravenous immunoglobulin
treatment in patients with different neurological diseases,
total A␤ and A␤1-42 in the cerebrospinal fluid was reduced significantly compared with baseline values. In the
serum, total A␤ levels increased after intravenous immunoglobulin treatment, whereas no significant change was
observed in A␤1-42 levels. Antibodies against A␤ were
found to be increased in the serum and cerebrospinal
fluid after intravenous immunoglobulin treatment. This
study provides evidence that intravenous immunoglobulin or purified A␤ antibodies may modify A␤ and A␤1-42
levels, suggesting potential utility as a therapy for Alzheimer disease.
Ann Neurol 2002;52:253–256
The pathological hallmarks of Alzheimer’s disease (AD)
are the occurrence of plaques in the neural parenchyma
and the formation of neuronal tangles.1 ␤-Amyloid
(A␤), a heterogenous 39 to 42–amino acid peptide, is
the main constituent of senile plaques and cerebrovascular amyloid deposits. The origin of the A␤ deposited
in vasculature and human brain is uncertain. According to the neuronal theory, A␤ is locally produced in
the brain.2 In contrast, the vascular theory proposes
that A␤ originates from the circulation, and that circulating soluble A␤ could contribute to neurotoxicity if
it crosses the blood-brain barrier.3 Production of A␤
via amyloid precursor protein (APP) processing, however, is not the only factor that can influence the probability of A␤ brain deposition. Evidence has accumulated indicating that factors influencing A␤ catabolism,
clearance4 and aggregation,5 are also critical in regulating A␤ metabolism.6
Recent data from transgenic mouse models of AD
suggest that clearance via immune-mediated pathways
may have a major impact on the development of
plaques.7,8 Immunization against A␤ has prevented
subsequent deposition of amyloid plaques. Furthermore, supportive data have shown that passive immunization with antibodies directed against A␤ also prevents amyloid deposition, ameliorates behavioral
deterioration, and may even clear existing plaques.9,10
We hypothesized that if these results derived from
animal experiments are transferable to humans, an
immune-mediated A␤ degrading pathway may be
physiologically present and its actions clinically significant in humans. Recently, we detected naturally occurring human antibodies against A␤ in both the cerebrospinal fluid (CSF) and serum of healthy subjects.11
These antibodies specifically recognize A␤. Furthermore, we detected significantly lower CSF titers of
these anti–A␤ antibodies in AD patients compared
with controls.
Following these results, we hypothesized that a treatment using these naturally occurring antibodies might
be beneficial as a therapeutic strategy for AD patients.
Because these antibodies are predominantly present in
the immunoglobulin G (IgG) fraction, we investigated
whether they are detectable in commercially available
IgG products. In addition, we investigated whether the
administration of a selected IgG product (intravenous
immunoglobulin [IVIG]) affects human CSF and serum A␤ levels.
Patients and Methods
From the 1Department of Neurology, Philipps University Marburg;
Dementia Research Section and Memory Clinic, Geriatric Psychiatry Brand and Alzheimer Memorial Center, Ludwig-Maximilian
University, Munich, Germany; and 3Department of Neurology, Indiana University School of Medicine, Indianapolis, IN.
Received Dec 11, 2001, and in revised form Mar 19, 2002. Accepted for publication Mar 23, 2002.
Published online Jun 23, 2002, in Wiley InterScience
( DOI: 10.1002/ana.10253
Address correspondence to Dr Dodel, Department of Neurology,
Philipps University, Rudolf-Bultmann Strasse 8, 35039 Marburg,
Germany. E-mail: or Dr Du, Department of Neurology, Indiana University School of Medicine, Indianapolis, IN 46202. E-mail:
Patients treated with IVIG were recruited at the Department
of Neurology, Philipps University, Marburg. Each patient received IVIG (Octagam; Octapharma, Langenfeld, Germany)
at a total dose of 0.4gm/kg body weight on 3 consecutive
days. Seven patients (4 male, 3 female; mean age, 62.7 ⫾ 7.0
years) were included. During their regular evaluation and
treatment, CSF and blood samples were withdrawn before
infusion of IVIG and at the indicated times after the infusions. We took CSF samples in the morning at the L3/L4 or
L4/L5 interspace by lumbar puncture after obtaining appropriate informed consent. We determined CSF leukocyte
count and protein levels by standard methods. No contamination by erythrocytes was seen in any of the samples. Aliquots then were stored at ⫺80°C until biochemical analysis.
Albumin and IgG concentrations were determined in the se-
© 2002 Wiley-Liss, Inc.
rum, and the CSF by immunoprecipitation nephelometry.
Only patients with an intact blood-brain barrier and regular
CSF protein concentrations according to our laboratory reference were included (Table 1).
The A␤ antibody enzyme-linked immunoadsorbent assay
(ELISA), the purification of A␤ antibodies, and immunoprecipitation of A␤ were performed as described previously.11
The concentration of A␤ antibodies in commercially available IVIG Flebogamma, (Grifols, Langen), Germany; and
Octagam; Octapharma) was determined using the A␤ antibody ELISA.11
Mean levels of anti-A␤ titers were compared by 2-way
analysis of variance. A log transformation was conducted before statistical analysis to reduce skewness within each patient
group.12 The mean log antibody titers for each patient group
were back-transformed to give geometric mean and standard
We purified the anti–A␤ antibodies from IVIG preparations using affinity chromatography as previously described.11 A marked decrease in anti–A␤ antibody titer
from the IgG fraction was observed when the flowthrough was tested in the ELISA assay (data not
shown). We confirmed the ability of the affinitypurified anti–A␤ antibody from plasma to bind A␤ by
immunoprecipitation of synthetic A␤ peptide (Fig 1A).
Moreover, the affinity-purified anti–A␤ antibody from
human IVIG readily detected a 4kDa A␤ peptide on
Western blots prepared from PDAPP mouse13 hippocampal homogenates but not from cerebellar homogenates (see Fig 1B).
In the next set of experiments, we incubated overnight different commercially available IVIG preparations (Flebogamma, [Grifols] and Octagam; Octapharma) with A␤1-40 (0.4␮gm/ml; Fig 2). Thereafter,
IVIG was analyzed for anti–A␤ antibodies using
ELISA. In both preparations, we could detect antibodies against A␤. In both IVIG preparations, we could
detect a considerable decrease of the ELISA signal after
incubation with A␤1-40 and agarose A.
To evaluate our hypothesis that these antibodies may
have an effect on A␤- peptide levels (total A␤ and A␤1Table 1. Clinical Data of the Patients Included in this Study
PNP unknown origin
PNP unknown origin
PNP unknown origin
Lambert-Eaton syndrome
None of the patients showed disturbance of the blood-brain barrier
(QALb ⬍ 8; QALb ⫽ CSFALb/SerumALb).15
MS ⫽ Multiple Sclerosis; PNP ⫽ Polyneuropathy.
Annals of Neurology
Vol 52
No 2
August 2002
Fig 1. Purification and characterization of human anti–A␤
antibody from human intravenous immunoglobulin (IVIG)
samples (Octagam; Octapharma). (A)Anti–A␤ antibody after
elution from the A␤ affinity column. We passed IVIG through
an affinity sepharose column conjugated with the A␤1-40 peptide. Anti–A␤ antibody from the purified human plasma IgG
was recovered after elution with buffer 1 (pH 2.5) followed
by buffer 2 (pH 1.5). A␤1-40 was immunoprecipitated by
affinity-purified human anti–A␤ antibody. (B) A␤ from
PDAPP mouse hippocampal homogenates was immunoprecipitated by purified human anti–A␤ antibody and detected by
monoclonal antibodies to A␤.13 A␤ ⫽ amyloid ␤; crb ⫽ cerebellum; hanti-A␤ ⫽ human purified A␤ antibody; hippo ⫽
hippocampus; IgG ⫽ immunoglobulin G (IVIG flowthrough).
42), we investigated the effects of IVIG administration
on CSF and serum A␤ levels in patients with different
neurological diseases. We detected a significant decrease in CSF levels of total A␤ and A␤1-42 after IVIG
(Table 2). Similar to the recent results in PDAPP
mice,14 a significant increase in total A␤ concentration
was observed in the serum after IVIG treatment (see
Table 2). Although there was a trend toward an increase of A␤1-42 in the serum, values did not reach statistical difference ( p ⫽ 0.06). The concentration of
Fig 2. Human anti–A␤ antibody was preabsorbed with A␤
peptide. Experiments were performed using intravenous immunoglobulin (IVIG) before and after incubation with A␤1-40.
Briefly, 100␮l of IVIG was incubated (overnight, 4°C) with
protein A–agarose or A␤1-40 at the designated concentration.
Protein A–agarose (Sigma, St. Louis, MO) was added to
IVIG (1␮l, overnight, 4°C) and removed by low-speed centrifugation. The IVIG then was analyzed for anti–A␤ antibody using enzyme-linked immunoadsorbent assay. Antibody
was recovered by incubation with eluting buffer (pH 1.5).
This experiment was repeated 3 times with similar results.
A␤ ⫽ amyloid ␤; Alp ⫽ Flebogamma, (Grifols); Oct ⫽ Octagam (Octapharma).
A␤-antibodies in the serum increased after treatment
with IVIG.
On the basis of the results by Schenk and colleagues,7
we identified specific anti–A␤ antibodies (IgG) in both
the serum and the CSF from nonimmunized humans,
which may act in an immune-mediated A␤ clearance
pathway.11 In an earlier study, human antibodies reactive with A␤ were isolated and cloned from human
B-cell lines from AD patients; however, the role of
these antibodies in AD pathogenesis remains unclear.6
In our study, we detected a significant difference in the
amount of A␤ antibodies in AD patients compared
with controls.11 These observations led us to ask
whether these anti–A␤ antibodies are detectable in
commercially available human IVIG preparations. After purification of these antibodies from IVIG, we
found that they specifically recognize A␤. Furthermore,
we investigated the effect of IVIG treatment on A␤,
A␤1-42 levels in serum and CSF. Intravenous immunoglobulin is an accepted and routinely used treatment
for several neurological and nonneurological immunemediated disorders.17 These patients are treated with
IVIG on a routine basis in our department. Although
we are aware that the patient selection for this study
has several limitations (eg, age, immune status, different diagnoses), this was the most straightforward and
feasible approach to evaluate our hypothesis.
Treatment of IVIG resulted in a significant decrease
of total A␤ and A␤1-42 in the CSF compared with
baseline. Mean A␤ antibody concentration increased in
the CSF. In contrast, the serum total A␤ and A␤ antibody concentration increased, but A␤1-42 remained
unchanged. A␤ antibody concentration increased in
the serum.
These findings suggest that A␤ peptides may pass
from the CSF to the blood and probably are metabolized locally. Recently, transporters at the blood-brain
barrier have been reported, which control the central
and peripheral exchange of smaller peptides including
Table 2. Total A␤, A␤1– 42, and A␤. Antibody Concentrations in the CSF and Serum at Baseline and after IVIG
Total A␤ (pg/ml)
A␤ antibody (ng/ml)
1–4 Days
After IVIG
7–14 Days
After IVIG
Total A␤, A␤1– 42, and A␤-antibody concentrations in the CSF and serum were assessed at baseline and 1 to 4 days (serum), 7 to 14 days
(serum), and 7 to 20 days (CSF) after IVIG (Octagam [Octapharma], 0.4 gm/kg body weight for 3 consecutive days). Values represent the
mean of triplicate determinations from a single assay.
p ⬍ 0.05.
Levels of significance ⬎0.05.
A␤ ⫽ ␤-amyloid; CSF ⫽ cerebrospinal fluid; IVIG ⫽ intravenous immunoglobulin; ns ⫽ not significant; SEM ⫽ standard error of the mean.
Dodel et al: Human Antibodies against A␤ Peptide
A␤.2,3 A similar A␤ efflux from the central to the peripheral compartment has been found in PDAPP mice
passively immunized with antibodies against A␤.14
Whether there is a specificity for shorter A␤ peptides
to cross the blood-brain barrier, as seen in the aforementioned experiments, cannot be stated at this point.
No data are available on the quantitative decrease of
A␤ concentration necessary to reduce A␤ deposition.
Therefore, one can only speculate whether the observed
reduction may have an impact on plaque formation.
Further studies, including careful dose studies in animals, are necessary. From earlier studies, however,
some information can be deduced. First, a relatively
modest A␤ clearance already reduced memory impairment in Tg2576 APP⫹PS1 mice.10 Second, an increase in A␤ concentration of approximately 1.5 times
in familial AD patients because of mutations in the
APP gene shifts the disease onset earlier by several decades. It can be assumed that already small changes in
A␤ concentrations in the CSF may have an impact on
A␤ deposition and plaque development.18
Our findings might have important implications for
the understanding of immune-mediated clearance pathways of A␤ in humans. Moreover, our data support
earlier in vitro and animal experiments that antibodies
applied outside of the blood-brain barrier may serve to
facilitate clearance of soluble peptides such as A␤ out
of the central nervous system.2,19,20 Given the relatively large volume of distribution of the peripheral as
compared with the central compartment, the addition
of anti–A␤ antibodies or other antipeptide antibodies
may considerably alter the clearance of biologically active peptides in the brain.
Finally, further studies are warranted to investigate
the role of A␤ antibodies in A␤ clearance; however,
our findings suggest that IVIG-purified A␤ antibodies
may be a potential therapeutic approach to AD.
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