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Anovel tau mutation S320F causes a tauopathy with inclusions similar to those in Pick's disease.

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Comparison of Weakness
Progression in Inclusion
Body Myositis during
Treatment with
Methotrexate or Placebo
Umesh A. Badrising, MD,1
Marion L.C. Maat-Schieman, MD, PhD,1
Michel D. Ferrari, MD, PhD,1
Aeilko H. Zwinderman, PhD,2 Judith A.M. Wessels, MSc,3
Ferdinand C. Breedveld, MD, PhD,4
Pieter A. van Doorn, MD, PhD,5
Baziel G.M. van Engelen, MD, PhD,6
Jessica E. Hoogendijk, MD, PhD,7
Chris J. Höweler, MD, PhD,8 Aeiko E. de Jager, MD, PhD,9
Frans G.I. Jennekens, MD, PhD,10
Peter J. Koehler, MD, PhD,11
Marianne de Visser, MD, PhD,12 Alain Viddeleer, MD,1
Jan J. Verschuuren, MD, PhD,1
and Axel R. Wintzen, MD, PhD1
We investigated whether 5 to 20mg per week oral methotrexate could slow down disease progression in 44 patients with inclusion body myositis in a randomized
double-blind placebo-controlled study over 48 weeks.
Mean change of quantitative muscle strength testing sum
scores was the primary study outcome measure. Quantitative muscle strength testing sum scores declined in both
treatment groups, ⴚ0.2% for methotrexate and ⴚ3.4%
for placebo (95% confidence interval ⴝ ⴚ2.5% to
ⴙ9.1% for difference). There were also no differences in
manual muscle testing sum scores, activity scale scores
and patients’ own assessments after 48 weeks of treatment. Serum creatine kinase activity decreased significantly in the methotrexate group. We conclude that oral
methotrexate did not slow down progression of muscle
weakness but decreased serum creatine kinase activity.
Ann Neurol 2002;51:369 –372
DOI 10.1002/ana.10121
From the Departments of 1Neurology, 2Medical Statistics, 3Clinical
Pharmacology, 4Rheumatology, Leiden University Medical Center,
Leiden; 5Department of Neurology, Erasmus Medical Center Rotterdam, Rotterdam; 6Department of Neurology, University Medical
Center Nijmegen, Nijmegen; 7Department of Neurology, University Medical Center Utrecht, Utrecht; 8Department of Neurology,
University Hospital Maastricht, Maastricht; 9Department of Neurology, University Hospital Groningen, Groningen; 10Interuniversitair Steunpunt Neuromusculair Onderzoek, Baarn; 11Department of
Neurology, Atrium Medical Center, Heerlen; and 12Department of
Neurology, University of Amsterdam, Amsterdam, the Netherlands.
Received Aug 6, 2001, and in revised form Nov 16. Accepted for
publication Nov 17, 2001.
Published online Feb 27, 2002.
Address correspondence to Dr Badrising, Department of Neurology,
K5Q, Leiden University Medical Center, P.O.B. 9600, 2300 RC,
Leiden, the Netherlands. E-mail:
Inclusion body myositis (IBM) is a progressive muscle
disorder with unknown etiology. Muscle biopsy specimens show inflammation and depositions of proteins
similar to those seen in degenerative disorders,1–3 processes that do not seem to be closely related, as they do
not co-localize.4 Immunosuppressive therapies have
yielded no or only short-lasting improvement of muscle
strength.5– 8 Whether immunosuppressive treatment can
slow down disease progression has not been studied.
Oral methotrexate (MTX) is a widely used, effective,
and well-tolerated treatment in rheumatoid arthritis.9,10 The weekly regimen facilitates compliance and
MTX has low cost. In the present study, we compared
the efficacy and tolerability of MTX and placebo in
slowing down disease progression in IBM.
Patients and Methods
From April 1996 until December 2000, we conducted a nationwide, randomized, placebo-controlled, parallel-group,
double-blind trial at the Leiden University Medical Center,
after approval of the protocol by the ethics review board. All
patients gave informed consent. We included 44 patients fulfilling the diagnostic criteria11,12 for definite (n ⫽ 42) or
probable (n ⫽ 2) IBM according to a previously reported
recruitment process.12 Inclusion criteria included sufficient
residual muscle strength to evaluate changes, absence of risk
factors for MTX-induced toxicity, no use of immunosuppressive therapy for at least 6 weeks before the study, no
previous use of MTX, no use of medication interfering with
MTX pharmacokinetics or pharmacodynamics, and absence
of severe dysphagia interfering with oral medication use.
Baseline studies were carried out 2 weeks before therapy
initiation. Clinical evaluations comprised quantitative muscle
power testing (QMT) by handheld myometry assessing the
maximum voluntary contraction13 and manual muscle testing (MMT) by the five-point Medical Research Council
(MRC)14 scale. Activity limitations were evaluated according
to the Barthel Index,15 Brooke’s Grading System,16 and the
Rivermead Mobility Index.17 Laboratory studies included serum creatine kinase (CK) activity levels.
One investigator (UB) tested baseline muscle strength.
The mean scores of each of 14 muscle groups tested three
times with QMT were added to a QMT sum score. MMT
measurements resulting in a sum score were performed on 32
muscle groups. Trial medication was distributed by the hospital pharmacy. Patients were randomly assigned, using a
computer-generated schedule, to receive either MTX or an
identical-appearing placebo. The randomization schedule
used random numbers in permuted blocks of 4. The code
was concealed by the pharmacy and broken after assessment
of all patients.
A 48-week treatment period started with a dose of 5mg
per week, each 6 weeks increased by 5mg up to 20mg. To
enhance blinding, all patients were requested to decrease
their 20mg dosage by 2.5mg without explanation after routine laboratory evaluations for 3 months. After blood assessments, the dosage was restored to 20mg per week.
A blinded assessor ( JV) monitored patients with regard to
treatment schedules, 3-month routine laboratory evaluations,
© 2002 Wiley-Liss, Inc.
patient’s subjective opinion of the muscle strength and the
changes in serum CK activity levels.
To detect a difference of 100 Newtons (N) in mean
changes or a clinically important stabilization 44 patients
were required (power ⫽ 0.80; ␣ ⫽ 0.05) according to the
following assumptions: an annual decline in muscle strength
in IBM patients of 5%, a mean change in QMT sum score
of 100N over 48 weeks for placebo and zero for MTX with
a standard deviation of 100N and a dropout rate of 25%.
An intention-to-treat analysis with carry forward of last assessments in the case of missing data was performed. Statistical
tests were two-sided. The mean changes in muscle strength
sum scores were compared by mixed-model analysis of variance, with the sum score as dependent variable, randomized
treatment as factor, time as covariate and by treatment-time
interaction, and by independent-samples t-test.
Fig 1. Flowchart of assessed patients with inclusion body myositis (IBM) and progress during treatment with methotrexate
(MTX) and placebo.
including serum CK activity, and adverse events. Another
blinded assessor (UB) evaluated the QMT and MMT measurements and patients’ opinions concerning the state of
muscle weakness (scored as progression, stabilization, or improvement) at 22 and 48 weeks after treatment initiation and
activity limitations at 48 weeks. Patients who discontinued
study treatment were immediately assessed.
The primary study outcome measure was the difference in
mean change from baseline of the QMT sum scores between
the two study groups. Secondary outcome measures were the
differences in MMT sum scores, the three activity scales, the
Twenty-one patients were allotted to MTX and 23 to
placebo (Fig 1). Baseline characteristics were similar for
the two groups (Table). Significantly more patients on
MTX discontinued treatment (8 vs 1 for placebo, p ⫽
0.008, Fisher’s exact test), mostly because of adverse
events. The mean weekly dose of MTX was 14.0mg in
all treated patients and 14.6mg in those who completed the entire study.
Primary Outcome
Mean QMT sum scores declined both for MTX
(⫺0.2%) and placebo (⫺3.4%). This difference was
not significant ( p ⫽ 0.3; 95% confidence interval [CI]
⫽ ⫺2.5% to ⫹9.1% for difference). A per-protocol
analysis including only those patients who fully completed the study also showed no difference: ⫹0.9% for
Table. Characteristics of Patients at Baseline
Duration of symptoms (years)
Other autoimmune disorders
Discontinuation of immunosuppressive therapy before baseline studies
Sum score by hand-held dynamometry (N)
Sum score by MRC
Wheelchair bound
CKa (U/L)
Activity score
Barthel index (0–20)
Rivermead mobility index (0–15)
Brooke’s grading (3–22)
Mean ⫾ SD.
Normal value ⬍200U/L.
CK ⫽ creatine kinase; MRC ⫽ Medical Research Council; N ⫽ Newton.
Annals of Neurology
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(n ⫽ 21)
(n ⫽ 23)
68 (⫾8)
9 (⫾5)
2533 (⫾800)
255 (⫾34)
676 (⫾830)
69 (⫾7)
11 (⫾7)
2492 (⫾844)
247 (⫾37)
725 (⫾761)
18 (⫾2)
12 (⫾2)
6 (⫾1)
18 (⫾3)
12 (⫾3)
6 (⫾3)
Fig 2. Mean (⫾SE) changes in percent from baseline in quantitative muscle testing scores (left) and manual muscle testing scores
(right) after 22 and 48 weeks of treatment with methotrexate (MTX) (solid diamonds) or placebo (solid squares) for intention to
treat design (above) and per-protocol design (below).
MTX and ⫺2.7% for placebo ( p ⫽ 0.3; 95% CI ⫽
⫺3.3% to ⫹10.7%) (Fig 2).
Except for CK values, none of the other study parameters showed a significant difference between MTX
and placebo. MMT sum scores decreased in both
groups, ⫺0.5% for MTX and ⫺2.0% for placebo
( p ⫽ 0.2; 95% CI ⫽ ⫺1.0% to ⫹3.9% for difference). In the per-protocol analysis MMT sum score
changes were ⫺2.2% for MTX and ⫺3.8% for placebo ( p ⫽ 0.4; 95% CI ⫽ ⫺2.3% to ⫹5.4%) (see Fig
2). The scores on activity scales did not change from
baseline (see Table). Two patients had a subjective improvement in strength at 48 weeks, both from the placebo group. Twelve patients, 5 from the MTX group,
noticed no change; others felt they had deteriorated.
Serum CK values fell in both groups, but more so in
the MTX group, notably in the first treatment period:
from 676 to 274U/L for MTX and from 725 to
690U/L for placebo ( p ⫽ 0.01; 95% CI ⫽ ⫺732 to
⫺102 for difference).
Adverse Events
Four patients in the MTX group and 1 patient in the
placebo group required dose reductions because of adverse events. One patient on placebo discontinued trial
medication because of progressive muscle weakness.
Seven patients on MTX discontinued trial medication
because of nausea (n ⫽ 3), hair loss (n ⫽ 2), arthralgia
(n ⫽ 2), and progressive muscle weakness (n ⫽ 1).
We investigated whether immunosuppression with
MTX could slow down decline of muscle strength in
IBM over a near-year treatment period. Randomization
was adequate, as study groups were similar with regard
to important baseline characteristics.
We failed to find a significant difference between
MTX and placebo, possibly because of the lower than
expected decline in the placebo group (3.4% in stead
of 5%), a greater than anticipated variability in QMT
results of patients (250 Newton in stead of 100 New-
Badrising et al: Inclusion Body Myositis and Methotrexate
ton), and a greater than expected dropout rate because
of adverse events (8/21). As a result, the post hoc
power of the study turned out to be only 23%. We
had based our conservative estimation of disease progression on the only available data on the natural
course of IBM showing a decline in muscle strength of
1.4% per month (range 0.5–2.8%).18
Muscle strength testing according to both the QMT
and MMT sum scores showed a trend towards slowed
decline of muscle weakness in the MTX group. Activity scales and patients’ own assessments did not show a
difference among treatment groups. The only statistically significant finding of predefined outcome measures was a decrease in serum CK activities in the
MTX group.
Because of the low post hoc study power, as exemplified by the wide 95% confidence interval for treatment difference, we cannot completely rule out a clinically relevant effect of MTX, although this seems
unlikely. To demonstrate unequivocally such a beneficial effect, 110 patients per treatment arm would be
required over a 48-week period or 28 patients per
treatment arm over 2 years. The large patient groups
necessary for the first option would require a multinational trial. The second option is obviously unattractive. Furthermore, the relatively high incidence of adverse events make MTX a less attractive treatment
option in this disorder.
The decrease in serum CK activity levels during
treatment with MTX suggests inhibition of inflammation and is in line with other findings showing a decrease of CK activity and of signs of inflammation in
biopsies after treatment with intravenous immunoglobulin or prednisone.5,19
In conclusion, the findings of the present study do
not support the use of MTX in IBM. The clinical
course in the placebo group may provide a useful basis
for future studies in this muscle disorder.
1. Askanas V, Engel WK. Sporadic inclusion-body myositis and its
similarities to Alzheimer disease brain. Recent approaches to diagnosis and pathogenesis, and relation to aging. Scand J Rheumatol 1998;27:389 – 405.
2. Villanova M, Kawai M, Lubke U, et al. Rimmed vacuoles of
inclusion body myositis and oculopharyngeal muscular dystrophy contain amyloid precursor protein and lysosomal markers.
Brain Res 1993;603:343–347.
3. Sarkozi E, Askanas V, Engel WK. Abnormal accumulation of
prion protein mRNA in muscle fibers of patients with sporadic
inclusion-body myositis and hereditary inclusion- body myopathy. Am J Pathol 1994;145:1280 –1284.
4. Pruitt JN 2nd, Showalter CJ, Engel AG. Sporadic inclusion
body myositis: counts of different types of abnormal fibers. Ann
Neurol 1996;39:139 –143.
5. Dalakas MC, Koffman B, Fujii M, et al. A controlled study of
intravenous immunoglobulin combined with prednisone in the
treatment of IBM. Neurology 2001;56:323–327.
Annals of Neurology
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6. Dalakas MC, Sonies B, Dambrosia J, et al. Treatment of
inclusion-body myositis with IVIg: a double-blind, placebocontrolled study. Neurology 1997;48:712–716.
7. Amato AA, Barohn RJ, Jackson CE, et al. Inclusion body
myositis: treatment with intravenous immunoglobulin. Neurology 1994;44:1516 –1518.
8. Leff RL, Miller FW, Hicks J, et al. The treatment of inclusion
body myositis: a retrospective review and a randomized, prospective trial of immunosuppressive therapy. Medicine 1993;72:
9. Rau R, Schleusser B, Herborn G, Karger T. Long-term treatment of destructive rheumatoid arthritis with methotrexate.
J Rheumatol 1997;24:1881–1889.
10. Weinblatt ME, Kaplan H, Germain BF, et al. Methotrexate in
rheumatoid arthritis. A five-year prospective multicenter study.
Arthritis Rheum 1994;37:1492–1498.
11. Verschuuren JJ, Badrising UA, Wintzen AR, et al. Inclusion
body myositis. In: Emery AEH, editor. Diagnostic criteria for
neuromuscular disorders. London: Royal Society of Medicine
Press, European Neuromuscular Centre, 1997;81– 84.
12. Badrising UA, Maat-Schieman M, van Duinen SG, et al. Epidemiology of inclusion body myositis in the Netherlands: a nationwide study. Neurology 2000;55:1385–1387.
13. van der Ploeg RJO. Hand-held dynamometry. Groningen, the
Netherlands: Dijkhuizen Van Zanten bv, 1992.
14. Aids to the examination of the peripheral nervous system. 1st
ed. London: Bailliere Tindall; 1990.
15. Barthel DW, Mahoney FI. Functional evaluation; the Barthel
index. Md State Med J 1965;14:61– 65.
16. Brooke MH. A clinician’s view of neuromuscular disorders. 2nd
ed. Baltimore: Williams & Wilkins; 1986.
17. Collen FM, Wade DT, Robb GF, Bradshaw CM. The Rivermead Mobility Index: a further development of the Rivermead
Motor Assessment. Int Disabil Stud 1991;13:50 –54.
18. Lindberg C, Persson LI, Bjorkander J, Oldfors A. Inclusion
body myositis: clinical, morphological, physiological and laboratory findings in 18 cases. Acta Neurol Scand 1994;89:
19. Walter MC, Lochmuller H, Toepfer M, et al. High-dose immunoglobulin therapy in sporadic inclusion body myositis: a doubleblind, placebo-controlled study. J Neurol 2000;247:22–28.
A Novel tau Mutation,
S320F, Causes a Tauopathy
with Inclusions Similar to
Those in Pick’s Disease
constitutes the first known mutation in exon 11 of tau.
Experimentally, the S320F mutation resulted in a
markedly reduced ability of tau to promote microtubule assembly.
Sonia M. Rosso, MD,1 Esther van Herpen, MSc,2
Wout Deelen,2 Wouter Kamphorst, MD, PhD,3
Lies-Anne Severijnen,2 Rob Willemsen, PhD,2
Rivka Ravid, PhD,4 Martinus F. Niermeijer, MD, PhD,2
Dennis Dooijes, PhD,2 Michael J. Smith,5
Michel Goedert, MD, PhD,5 Peter Heutink, PhD,5
and John C. van Swieten, MD, PhD1
The proband, a travelling salesman, presented at age 38 years
with complaints of mild memory problems and spatial disorientation. Neuropsychological examination, computed tomography, and electroencephalography were normal at this
time. Nine years later, at age 47 years, memory problems and
naming difficulties had evidently worsened. Furthermore, he
had become introverted, mentally inflexible, and disinterested. Psychometric evaluation revealed fluent aphasia, wordfinding difficulties, impairment of comprehension, and abstract thinking. Extrapyramidal signs and motor neuron
disease were absent. Magnetic resonance imaging of the brain
showed moderate bilateral temporal atrophy. The patient
died at age 53 years; the proband’s mother had also died of
a similar dementing illness at age 53 years. Neither of the
mother’s parents (ages at death, 57 and 90 years) nor any of
her seven siblings were reported to have developed dementia.
Mutations in the tau gene cause familial frontotemporal
dementia and parkinsonism linked to chromosome 17. In
this article, we describe a novel missense mutation, S320F,
in the tau gene in a family with presenile dementia. To
our knowledge, it is the first mutation to be described in
exon 11 of tau. The proband died at age 53 years, after a
disease duration of 15 years, and autopsy revealed a neuropathological picture similar to Pick’s disease. Recombinant tau protein with the S320F mutation showed a
greatly reduced ability to promote microtubule assembly.
Ann Neurol 2002;51:373–376
DOI 10.1002/ana.10140
The identification of different types of mutations in
the tau gene in familial frontotemporal dementia and
parkinsonism linked to chromosome 17 (FTDP-17),
and the association of these mutations with a spectrum
of filamentous tau pathology, has established the important role of the tau gene in causing neurodegeneration.1–3 The primary effect of intronic and some coding region mutations in exon 10 is at the mRNA level,
resulting in a change in ratio of 3- to 4-repeat tau isoforms. By contrast, most missense mutations reduce
the ability of mutant tau to interact with microtubules
and other molecules, and some also stimulate the in
vitro assembly of tau into filaments.
In this article, we report a novel missense mutation
(S320F) in tau in a family with presenile dementia. It
From the Departments of 1Neurology and 2Clinical Genetics, Erasmus Medical Center, Rotterdam; 3Department of Pathology, University Hospital Vrije Universiteit, Amsterdam; 4Netherlands Brain
Bank, Amsterdam, the Netherlands; and 5Medical Research Council
Laboratory of Molecular Biology, Cambridge, United Kingdom.
Received Sep 19, 2001, and in revised form Nov 29. Accepted for
publication Nov 29, 2001.
Published online Feb 27, 2002.
Address correspondence to Dr van Swieten, Department of Neurology, University Hospital Rotterdam-Dijkzigt, Dr Molewaterplein
40, 3015 GD Rotterdam, the Netherlands.
Patient and Methods
Clinical History of the Proband
Immunohistochemistry with phosphorylation-dependent
(AT8, AT180, AT270, PHF1, MC1, and 12E8 [1:500, donated by P. Seubert, Elan Pharmaceuticals, San Francisco,
CA]) and phosphorylation-independent tau antibodies
(BR01, Tau 2) was performed, as well as with antibodies
directed against ubiquitin, ␤-amyloid, ␣-synuclein, and ␣Bcrystallin, as described previously.4
DNA Extraction and Mutational Analysis
Genomic DNA of the proband was extracted, and exons 9 to
13 of tau were amplified and sequenced as described.5 Exon
11 of tau was also sequenced from the genomic DNA of a
healthy maternal uncle of the proband (age 84 years), as well
as 50 control individuals.
Tau Extraction, Immunoblotting, and
Electron Microscopy
Sarkosyl-soluble and -insoluble tau was extracted, dephosphorylated, and analyzed as described previously,6 and incubated with BR01 tau antibody (1:2,000). The ratio of soluble 3- to 4-repeat tau was assessed using Image Master 1D
elite software (Amersham Pharmacia Biotech, United Kingdom). Dispersed filaments from the sarkosyl-insoluble fraction
were processed for electron microscopy and immunolabeled
with tau-antibodies BR01 and AT8, as described previously.6
Microtubule Assembly
Site-directed mutagenesis was used to change S320 to phenylalanine in the 3-repeat 381 and 4-repeat 412 amino acid
isoforms of human tau (numbering of 441 amino acid isoform of human tau), expressed from cDNA clones htau37
and htau46, respectively. Wild-type and mutant tau proteins
were expressed in Escherichia coli BL21(DE3), purified, and
incubated with bovine brain tubulin as described previously.7
© 2002 Wiley-Liss, Inc.
Assembly into microtubules was monitored over time by
change in turbidity at 350nm.
Sequencing of the proband’s genomic DNA showed a
C to transition in exon 11 at the second base position
of codon 320 (TCC to TTC), which results in the
substitution of serine by phenylalanine (S320F). This
change was not observed in the healthy maternal uncle
of the proband or in 100 control chromosomes.
At autopsy, the proband’s brain (weight 1,200g)
showed focal bilateral atrophy of the anterior temporal
lobes, with only very mild frontal atrophy. Severe neuronal loss and gliosis were present in the temporal cortex, cingulate gyrus, entorhinal cortex, and hippocampus. The substantia nigra was not affected. A few Pick
cells were seen in the temporal cortex. Bodian silver
staining did not show any Pick bodies or neurofibrillary tangles.
Immunohistochemical staining showed extensive tau
pathology in the form of Pick-like bodies and more
diffuse cytoplasmic staining in neurons of the frontal,
temporal, and parietal cortices; the dentate gyrus; the
amygdala; and the ventral striatum (Fig 1A–E). The
Pick-like bodies were immunoreactive with all anti-tau
antibodies tested, with the exception of antibody 12E8.
A few glial cells, probably oligodendrocytes, in affected
regions also contained tau-positive inclusions. Staining
with ␤-amyloid and ␣-synuclein was negative.
By immunoblotting, sarkosyl-insoluble tau ran as
two major bands of 60 and 64 kDa (Fig 2A). Following dephosphorylation, these bands resolved into four
bands that aligned with human tau isoforms 4R0N,
3R1N, 4R1N, and 3R2N, except in the temporal cortex, where the 3R2N band was not observed (Fig 2B).
Following dephosphorylation, soluble tau gave a pattern similar to that seen in Alzheimer’s disease (AD;
Fig 2C), with a ratio of 3- to 4-repeat tau isoforms of
0.92, compared with 1.01 in the control brain.
Electron microscopy of preparations of sarkosylinsoluble filaments showed filaments with two distinct
morphologies (Fig 1F, G). The major species (approximately 80% of filaments) was a straight filament, very
similar to the filaments seen in AD brain. The minor
species (approximately 20%) was an irregularly twisted
filament with a crossover spacing of 110 to 160nm and
a diameter of 6 to 8nm in its narrow part. Both types
of filament were decorated with BR01 and AT8 antibodies (Fig 1H).
Recombinant 3-repeat htau37 and 4-repeat htau46
with the S320F mutation showed a markedly reduced
ability to promote microtubule assembly when compared with the corresponding wild-type proteins (Fig
Fig 1. Neuropathological findings in the
proband’s brain. (A, B) Immunostaining
of the frontal cortex with the
phosphorylation-dependent anti-tau antibody AT8 shows multiple tau-positive inclusions (A), some of which resemble Pick
bodies (arrow in B), while others show a
more diffuse staining of the cytoplasm (arrowhead in B). (C) A small number of
AT8-positive glial cells are seen in the
frontal cortex. (D, E) The granule cells of
the dentate gyrus of the hippocampus contain numerous inclusions resembling Pick
bodies that are immunoreactive with AT8
(D) but not with the phosphorylationdependent anti-tau antibody 12E8 (E).
(F–H) Electron micrographs of tau filaments isolated from the proband’s brain
show unlabeled straight (F) and twisted
(G) filaments as well as a twisted filament
labeled by AT8 (H). Scale bars: 150 ␮m
(A), 100 ␮m (B–E), and 80 nm (F–H).
Annals of Neurology
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Bodian silver staining. They were immunoreactive with
all anti-tau antibodies used, with the exception of antibody 12E8, which recognizes tau phosphorylated at
S262, S356, or both. This 12E8-negative staining of
Pick bodies has also been described in sporadic PiD15 as
well as in K257T, G272V, and K369I mutations, 9,11,14
indicating that these epitopes are not substantially hyperphosphorylated in most cases with Pick-like pathology. However, the 12E8-positive staining in G389R
mutation suggests that nonphosphorylation of these sites
is not required for the formation of Pick bodies.10
Sarkosyl-insoluble tau extracted from S320F brain resolved into two major bands of 60 and 64 kDa, like the
Fig 3. Effects of the S320F mutation on the ability of threerepeat htau37 (381 amino acid isoform of human tau) and
four-repeat htau46 (412 amino acid isoform of human tau)
to promote microtubule assembly. (A) Polymerization of tubulin induced by wild-type htau37 and htau37S320F. (B) Polymerization of tubulin induced by wild-type htau46 and
htau46S320F. Microtubule assembly was monitored over time
by turbidimetry. (C) Optical densities for wild-type and mutant htau37 and htau46 at 2 minutes (expressed as percentages of wild-type htau37 and htau46 taken as 100%). Each
result is expressed as the mean ⫾ the standard error of the
mean (n ⫽ 5).
Fig 2. Sarkosyl-insoluble tau and soluble tau from the proband’s brain. (A, B) Immunoblot of sarkosyl-insoluble tau
before (A) and after (B) alkaline phosphatase treatment. (C)
Immunoblot of soluble tau after alkaline phosphatase treatment. Immunoblotting was done using the phosphorylationindependent anti-tau antibody BR01. F ⫽ frontal cortex;
T ⫽ temporal cortex; P ⫽ parietal cortex; C ⫽ cerebellum;
AD ⫽ Alzheimer’s disease, Rt ⫽ recombinant tau.
3A, B). Thus, the S320F mutation led to a 90 to 95%
reduction in the rates of microtubule assembly when
expressed as the optical density at 2 minutes (Fig 3C).
This study describes a novel mutation in exon 11 of the
tau gene in a patient with presenile dementia. S320F is
the first mutation to be described in exon 11 of tau. The
initial clinical diagnosis was AD, but neuropathological
findings closely resembled Pick’s disease (PiD). The inclusions in S320F brain were similar to those described
in sporadic PiD8 and in some other cases with tau mutations, 9 –14 except that they were undetectable with
Rosso et al: S320F Mutation in Exon 11 of the tau Gene
pattern seen in sporadic PiD8 and in K257T and
G389R mutations.10,11,13 However, following dephosphorylation, the normally abundant band corresponding
to tau isoform 3R0N was missing. Four major bands
aligning with isoforms 4R0N, 3R1N, 4R1N, and 3R2N
were observed instead. As the 60-kDa band corresponds
to the 3R0N tau isoform in AD brain,6 the presence of
a 60-kDa band in the absence of 3R0N tau in the case
described herein implies that the isoform composition of
sarkosyl-insoluble tau differed from that of AD. Previously, unexpected tau isoform patterns have been observed in the E342V mutation and in one of two families with a G389R mutation.12,13 As in the present case,
it remains to be seen whether these patterns are a direct
and general result of the tau mutations, or whether they
are limited to the individual cases within each family
studied so far. Soluble tau from S320F brain consisted
of all six isoforms, similar to that seen in AD and other
missense mutations in tau. The two distinct filament
morphologies, straight and twisted, have also been described in some cases of sporadic PiD and in some other
cases with tau mutations and a Pick-like phenotype.8,10
The S320F mutation is located within the highly conserved third microtubule-binding domain of tau. A
serine residue is found at this position in all known tau
sequences, as well as in related proteins MAP2 and
MAP4. Accordingly, recombinant tau with the S320F
mutation showed a greatly reduced ability to promote
microtubule assembly, suggesting that this may be its
primary effect. It is conceivable that this mutation has
additional effects. It is located within the core region of
the paired helical filament of AD, two residues aminoterminal of C322, which is known to be required for the
dimerization of tau.17,18 The S320F mutation removes a
potential phosphorylation site in tau. In vitro studies
have shown that microtubule-affinity regulating kinase,
protein kinase N, and cyclic adenosine monophosphate–
dependent protein kinase (in the presence of heparin)
can phosphorylate S320.19,20 It has even been suggested
that phosphorylation of this site may inhibit the assembly of tau protein into filaments.19 However, at present,
there is no evidence to suggest that S320 is phosphorylated in either normal or pathological tau in vivo.
In conclusion, the present study describes a novel
tau mutation that causes a syndrome similar to Pick’s
disease. It further underlines the relevance of tau protein dysfunction in the etiology and pathogenesis of
frontotemporal dementia in general, and Pick’s disease
in particular.
This work was supported in part by grants from the Dutch Brain
Foundation, the Internationale Stichting voor Alzheimer Onderzoek
(ISAO; JCS, PH), the Netherlands Organization for Scientific Re-
Annals of Neurology
Vol 51
No 3
March 2002
search (NWO project 940-38-005, JCS, PH), and the United Kingdom Medical Research Council (JCS, PH).
We thank Patrizia Rizzu and Wim van Noort for technical advice.
1. Poorkaj P, Bird TD, Wijsman E, et al. Tau is a candidate gene
for chromosome 17 frontotemporal dementia. Ann Neurol
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2. Hutton M, Lendon CL, Rizzu P, et al. Association of missense
and 5⬘-splice-site mutations in tau with the inherited dementia
FTDP-17. Nature 1998;393:702–705.
3. Spillantini MG, Murrell JR, Goedert M, et al. Mutation in the
tau gene in familial multiple system tauopathy with presenile
dementia. Proc Natl Acad Sci U S A 1998;95:7737–7741.
4. Rosso SM, Kamphorst W, De Graaf B, et al. Familial frontotemporal dementia with ubiquitin-positive inclusions is linked
to chromosome 17q21–22. Brain 2001;124:1948 –1957.
5. Rizzu P, van Swieten J, Joosse M, et al. High prevalence of
mutations in the microtubule-associated protein tau in a population study of frontotemporal dementia in the Netherlands.
Am J Hum Genet 1999;64:414 – 421.
6. Goedert M, Spillantini MG, Cairns NJ, Crowther RA. Tau
proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms. Neuron 1992;8:159 –168.
7. Hasegawa M, Smith MJ, Goedert M. Tau proteins with
FTDP-17 mutations have a reduced ability to promote microtubule assembly. FEBS Lett 1998;437:207–210.
8. Dickson DW. Pick’s disease: a modern approach. Brain Pathol
1998;8:339 –354.
9. Spillantini MG, Crowther RA, Kamphorst W, Heutink P, et al.
Tau pathology in two Dutch families with mutations in the
microtubule-binding region of tau. Am J Pathol 1998;153:
1359 –1363.
10. Murrell JR, Spillantini MG, Zolo P, et al. Tau gene mutation
G389R causes a tauopathy with abundant Pick body-like inclusions and axonal deposits. J Neuropathol Exp Neurol 1999;58:
11. Rizzini C, Goedert M, Hodges JR, et al. Tau gene mutation
K257T causes a tauopathy similar to Pick’s disease. J Neuropathol Exp Neurol 2000;59:990 –1001.
12. Lippa CF, Zhukareva V, Kawarai T, et al. Frontotemporal dementia with novel tau pathology and a Glu342Val tau mutation. Ann Neurol 2000;48:850 – 858.
13. Pickering-Brown S, Baker M, Yen SH, et al. Pick’s disease is
associated with mutations in the tau gene. Ann Neurol 2000;
48:859 – 867.
14. Neumann M, Schulz-Schaeffer W, Crowther RA, et al. Pick’s
disease associated with the novel tau gene mutation K369I. Ann
Neurol 2001;50:503–513.
15. Probst A, Tolnay M, Langui D, et al. Pick’s disease: hyperphosphorylated tau protein segregates to the somatoaxonal compartment. Acta Neuropathol 1996;92:588 –596.
16. Delacourte A, Robitaille Y, Sergeant N, et al. Specific pathological tau protein variants characterize Pick’s disease. J Neuropathol Exp Neurol 1996;55:159 –168.
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3- and 4-repeat tau isoforms within the PHF in Alzheimer’s
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Structure of tau protein and assembly into paired helical filaments. Biochim Biophys Acta 2000;1502:122–132.
19. Schneider A, Biernat J, von Bergen M, et al. Phosphorylation
that detaches tau protein from microtubules (Ser262, Ser214)
also protects it against aggregation into Alzheimer paired helical
filaments. Biochemistry 1999;38:3549 –3558.
20. Taniguchi T, Kawamata T, Mukai H, et al. Phosphorylation of
tau is regulated by PKN. J Biol Chem 2001;276:10025–10031.
Brain White Matter
Anatomy of Tumor Patients
Evaluated with Diffusion
Tensor Imaging
Susumu Mori, PhD,1,2 Kim Frederiksen, BS,2
Peter C. M. van Zijl, PhD,1,2 Bram Stieltjes, MD,1,2
Michael A. Kraut, MD, PhD,1
Meiyappan Solaiyappan, BS,1
and Martin G. Pomper, MD, PhD1
We applied multislice, whole-brain diffusion tensor imaging (DTI) to two patients with anaplastic astrocytoma.
Data were analyzed using DTI-based, color-coded images
and a 3-D tract reconstruction technique for the study of
altered white matter anatomy. Each tumor was near two
major white matter tracts, namely, the superior longitudinal fasciculus and the corona radiata. Those tracts were
identified using the color-coded maps, and spatial relationships with the tumors were characterized. In one patient the tumor displaced adjacent white matter tracts,
whereas in the other it infiltrated the superior longitudinal fasciclus without displacement of white matter. DTI
provides new information regarding the detailed relationship between tumor growth and nearby white matter
tracts, which may be useful for preoperative planning.
Ann Neurol 2002;51:377–380
DOI 10.1002/ana.10137
Approximately 18,000 brain tumors are diagnosed annually in the United States.1 Advancing faster than effective new brain tumor therapies are new magnetic
resonance–based techniques for characterizing these
malignancies. A significant problem in brain tumor imaging and therapy is the accurate depiction of white
matter involvement. Are there effects of tumor on
white matter pathways that are not seen on conventional T2-weighted images? That question has important implications for the accurate localization of radiation ports and for determining whether and how
extensive surgery should proceed.
Diffusion tensor imaging (DTI) is a technique that
From the 1Department of Radiology, Johns Hopkins University
School of Medicine, and the 2F. M. Kirby Research Center,
Kennedy Krieger Institute, Baltimore, MD.
Received Aug 15, 2001, and in revised form Dec 4. Accepted for
publication Dec 4, 2001.
Published online Feb 27, 2002.
Address correspondence to Dr Mori, Department of Radiology,
Johns Hopkins University School of Medicine, 217 Traylor Bldg.,
720 Rutland Ave, Baltimore, MD 21205.
can characterize the properties of water diffusion in the
brain by providing three types of information, namely,
the extent (apparent diffusion constant, or ADC) and
directionality (anisotropy) of diffusion and its predominant orientation.2 The ADC and anisotropy are believed to be related predominantly to the integrity of
axonal tracts3,4 and depict contrast that is different
from conventional T1- and T2-weighted images. Abnormalities on diffusion-weighted images due to brain
tumors have been reported,5 including an increase in
ADC within malignant tissue,6 –11 detection of changes
in white matter fiber angles,12 and ADC and anisotropy changes seen with therapy.12,13
In contrast to the aforementioned studies, in which
DTI provided a method of characterizing the tumor
itself, we focused on the capability of DTI to reveal the
effect of tumor on white matter pathways. We found
that DTI is an effective tool for delineating the effect
of tumor on nearby white matter tracts, information
that may facilitate preoperative planning.
Patients and Methods
MRI Data Acquisition
Studies were performed using a 1.5T Philips GyroscanNT
(Best, The Netherlands) system. DTI was accomplished using multislice, segmented echo-planar imaging (EPI) with
cardiac triggering (repetition time [TR] ⫽ 5 heartbeats; echo
time [TE] ⫽ 92ms) and navigator echo phase correction. A
data matrix of 128 ⫻ 95 over a field of view of 230 ⫻
173mm was obtained, acquiring 17 echoes per excitation.
Slice thickness was 3mm (coronal, 40 – 60 slices) without
gaps. Diffusion weighting was performed along 6 axes, using
a b-value of 600 s/mm2. A reference image with low diffusion weighting (b ⫽ 33s/mm2) was recorded. Measurements
were repeated 6 times to increase signal/noise. Double-echo
T2- weighted imaging (TEs of 22 and 100ms; image resolution equal to DTI) was performed for anatomic guidance.
Total examination time was 1 hour.
Diffusion tensors at each pixel were calculated using multivariate linear least square fitting2 and diagonalized. The eigenvector (v៮ 1) associated with the largest eigenvalue (␭1) was
assumed to represent the local fiber direction. Anisotropy
maps were obtained using the orientation-independent fractional anisotropy (FA).14 DTI-based color maps were created
from FA values (image intensity) and the three vector elements of v៮ 1;15,16 red indicated fibers running along the right
to left direction, green represented anterior to posterior, and
blue was superior to inferior. The average diffusion constant,
ADCav, was calculated from the trace of the diffusion tensor.
Axial and sagittal images were obtained by reslicing the 3-D
volume data. For 3-D reconstruction of tracts of interest, the
Fiber Assignment by Continuous Tracking method was employed.17–19 Tract reconstruction required 20 minutes using
an 833MHz Pentium III Workstation (Dell, Austin, TX).
Patient 1 was a male age 36 years with a right frontal anaplastic astrocytoma. Originally he presented with two partial
© 2002 Wiley-Liss, Inc.
seizures in 1 week due to a left-sided, low-grade glioma that
eventually dedifferentiated to an anaplastic astrocytoma, necessitating resection. Contralateral extension of the left frontal mass was found in follow-up imaging. Patient 2 was a
male age 45 years with a posterior left frontal anaplastic astrocytoma. He presented with aphasia, hemiparesis, and simple partial seizures involving the right face. DTI data on
healthy volunteers were obtained from our normative DTI
database (19 subjects, ages 19 – 43 years; 10 male).
Figures 1A–C show the T2-weighted image, FA map,
and ADCav map for Patient 1. The T2-bright lesion in
the left hemisphere was due to a surgical cavity. High
ADCav values (see Fig 1C) were found within the tumor, in keeping with previous reports.6 –11,13 The T2abnormal portion of the tumor (see Fig 1A) had low
FA (0.05 to 0.15; see Fig 1B). The boundary of the
tumor identified in the FA maps was not as clear as
that on the T2-weighted images, because the anisotropy
of the tumor was similar to that of gray matter.
To clarify white matter anatomy, the DTI-based
color map was compared with that of a healthy volunteer (Figs 1D–G). When the 2 subjects were compared,
several major anatomical changes become evident.
First, the corona radiata of the right hemisphere of the
patient was dislocated medially (white arrowheads).
Second, the corpus callosum genu was severely affected
in the patient. This was in contrast to the cingulum,
which was adjacent to the corpus callosum but structurally preserved.
The superior longitudinal fasciculus (slf) is a prominent white matter tract that projects through regions
occupied by the tumor. In Figure 1E, only the posterior slf can be appreciated (yellow arrowheads) in the
patient brain. Figures 1F and G depict the right slf of
the patient dislocated superiorly as it courses anteriorly.
Figure 2 shows color maps (Figs 2A–D) and T2weighted images (Fig 2E) of Patient 2. Tumor could
identified by T2 hyperintensity and low anisotropy. Although this tumor displayed similar histology to that of
Patient 1, the color maps indicate that the low anisotropy regions were not accompanied by deformation of
adjacent white matter structures. As with Patient 1, the
slf of Patient 2 lay within the path of the tumor. The
slf can be appreciated in the contralateral hemisphere
(yellow arrowheads) but not near the tumor. The slf
was apparently within the area of lowest anisotropy. To
confirm that, we reconstructed the trajectory of the slf
in the contralateral hemisphere. For the reconstruction,
the slf was identified at the slice level shown in Figure
2C, and tracking results that penetrated the identified
slf were searched. The result is shown in Figure 2E
(red), superimposed on T2-weighted images. The T2hyperintense regions correlated spatially with the brain
regions occupied by the slf.
Annals of Neurology
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March 2002
Fig 1. T2-weighted images (A), fractional an isotropy map
(B), and average diffusion constant map (C) of Patient 1.
Images in (D–E) show detailed anatomical information with
DTI-based color maps at two different axial slices (D and E),
a coronal plane (F), and a sagittal plane (G). The color maps
in the left column are from Patient 1 and the right column
from a healthy volunteer (a male age 20 years) to serve as an
anatomical guide. on the color maps, red indicates fibers running along the right to left orientation, green indicates anterior to posterior, and blue indicates inferior to superior. White
arrowheads indicate deformed corona radiata and yellow arrowheads the superior longitudinal fasciculus (slf).
A 3-D reconstruction of the corona radiata was performed for both patients and is shown in Figure 3.
The 3-D relationship of the corona radiata with the
tumor can be clearly appreciated. The corona radiata of
Patient 1 surrounds the surface of the tumor because of
mechanical compression. On the other hand, the tumor in Patient 2 did not change the trajectory of the
corona radiata, and it projected into the core of the
T2-hyperintense regions, where the tracking terminated
due to the low anisotropy.
We report the application of 3-D DTI-based white
matter anatomic studies for 2 patients with brain tumors. We found that FA alone might not provide contrast superior to that of conventional T2-weighted images to define tumors. However, the DTI-based colorcoded maps could provide unique information about
white matter architecture and its alteration due to the
tumors. This finding is in line with previous reports on
altered fiber angles due to tumor mass.12 In this study,
we also employed 3-D tract reconstruction techniques,
which greatly aided the slice-by-slice interpretation of
the color maps, especially in cases of deformed anatomy.
Both patients had anaplastic astrocytomas. However,
the effects of their tumors on the 3-D white matter
disposition were different. In Patient 1, the tumor grew
Fig 2. Color maps at three coronal levels (A–C) and a axial
level (D) of Patient 2. In all color maps, blue represents fibers
running in a superior to inferior direction, red left to right,
and green anterior to posterior. Arrowheads indicate locations
of the superior longitudinal fasciculus (slf). In (E), coordinates
of the slf in the contralateral hemisphere determined from the
3-D reconstruction technique Fiber Assignment by Continuous
Tracking are superimposed on T2-weighted images.
Fig 3. A 3-D tract reconstruction of the corona radiata in
Patients 1 (A) and 2 (B). The corona radiata (yellow) was
reconstructed using the FACT method. Regions of interest for
the tracking were identified at the posterior limb of the internal capsule. For clarity, only a part of the corona radiata that
was adjacent to the tumors (red) is shown.
discretely, compressing the corona radiata and slf,
which were dislocated medially and superiorly, respectively (see Fig 1). The corpus callosum genu was severely affected, likely due to Wallerian degeneration after surgery in the left hemisphere. Although the T2weighted image of Patient 1 (see Fig 1A) also showed
that the tumor compressed adjacent white matter, information regarding the specific white matter tracts
dislocated, and in which directions, could be obtained
only by 3-D DTI.
Unlike the tumor of Patient 1, the tumor of Patient
2 did not induce significant anatomic deformation.
That difference is best illustrated by examining the corona radiata (see Figs 2 and 3). Patient 2 (see Fig 2)
illustrates the fact that significant decreases in anisotropy may be evident even without marked anatomic
deformity. Figure 2E shows that the high T2 signal and
low FA regions spread and follow regions occupied by
the slf, consistent with the proposed theory that tumors might spread along white matter tracts.20 However, we could not conclude whether the T2 hyperintensity along the slf indicates infiltrating tumor or
Wallerian degeneration. Comprehensive, longitudinal
studies with histologic correlation are needed.
While 3-D tract reconstruction provides a new way
to evaluate white matter architecture, its limitations
should also be recognized. DTI is of limited resolution
(2 ⫻ 2 ⫻ 3mm interpolated to 1 ⫻ 1 ⫻ 3mm in the
presented work), leading to substantial partial volume
effects, including multiple populations of fibers with
Mori et al: Brain Tumor Study by Diffusion Tensor Imaging
different orientations within a pixel. Consequently, it
cannot provide information about connectivity of the
brain at the cellular level. The reconstructed tract trajectories reflect only the macroscopic configuration of
prominent fiber bundles. Another problem is the motion susceptibility of the high-resolution segmented
EPI approach, which presently limits its utility for pediatric or uncooperative patients.
In conclusion, we have demonstrated the application
of 3-D DTI to the study of white matter trajectory
using data from patients with anaplastic astrocytomas.
The DTI-based color maps and 3-D tract reconstructions provided detailed anatomic information on relationships between tumors and nearby white matter
tracts, which may be important in therapeutic planning.
This work was supported by National Institutes of Health (NIH)
(grant AG20012) and NIH/National Center for Research (NCRR)
(grant RR15241 to PVZ and SM).
1. Huncharek M, Muscat J. Treatment of recurrent high grade
astrocytoma; results of a systematic review of 1,415 patients.
Anticancer Res 1998;18:1303–1311.
2. Basser PJ, Mattiello J, LeBihan D. Estimation of the effective
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4. Henkelman R, Stanisz G, Kim J, Bronskill M. Anisotropy of
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Magn Reson Imaging 1993;5:25–31.
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the cerebral hemispheres. AJNR Am J Neuroradiol 1995;16:
9. Desprechins B, Stadnik T, Koerts G, et al. Use of diffusionweighted MR imaging in differential diagnosis between intracerebral necrotic tumors and cerebral abscesses. AJNR Am J Neuroradiol 1999;20:1252–1257.
10. Sugahara T, Korogi Y, Kochi M, et al. Usefulness of diffusionweighted MRI with echoplanar technique in the evaluation of
cellularity in gliomas. J Magn Reson Imaging 1999;9:53– 60.
11. Kim YJ, Chang KH, Song IC, et al. Brain abscess and necrotic
or cystic brain tumor: discrimination with signal intensity on
diffusion-weighted MR imaging. AJR Am J Roentgenol 1998;
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12. Wieshmann UC, Symms MR, Parker GJ, et al. Diffusion tensor imaging demonstrates deviation of fibres in normal appearing white matter adjacent to a brain tumour. J Neurol Neurosurg Psychiatry 2000;68:501–503.
13. Chenevert TL, Stegman LD, Taylor JM, et al. Diffusion magnetic resonance imaging: an early surrogate marker of therapeutic efficacy in brain tumors. J Natl Cancer Inst 2000;92:
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15. Makris N, Worth AJ, Sorensen AG, et al. Morphometry of in
vivo human white matter association pathways with diffusion
weighted magnetic resonance imaging. Ann Neurol 1997;42:
16. Pajevic S, Pierpaoli C. Color schemes to represent the orientation of anisotropic tissues from diffusion tensor data: application to white matter fiber tract mapping in the human brain.
Magn Reson Med 1999;42:526 –540.
17. Mori S, Crain BJ, Chacko VP, van Zijl PCM. Three dimensional tracking of axonal projections in the brain by magnetic
resonance imaging. Ann Neurol 1999;45:265–269.
18. Xue R, van Zijl PCM, Crain BJ, et al. In vivo threedimensional reconstruction of rat brain axonal projections by
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Neuro image 2001;14:723–735.
20. Geer CP, Grossman SA. Interstitial fluid flow along white matter tracts: a potentially important mechanism for the dissemination of primary brain tumors. J Neurooncol 1997;32:
Loss of Interhemispheric
Inhibition on the Ipsilateral
Primary Sensorimotor
Cortex in Patients with
Brachial Plexus Injury:
fMRI Study
Jen-Chuen Hsieh, MD, PhD,1,2,3,4
Henrich Cheng, MD, PhD,3 Hsiu-Min Hsieh, MS,1,2
Kwong-Kum Liao, MD,7,9 Yu-Te Wu, PhD,1,8
Tzu-Chen Yeh, MD, PhD,1,7 and Low-Tone Ho, MD1,3,4
This functional magnetic resonance imaging study verified an antagonistic pattern in which a concomitant deactivation of ipsilateral primary sensorimotor (SM1) was
coupled to the contralateral SM1 activation in healthy
controls during unimanual hand grasping. Of note, dramatic reduction of ipsilateral SM1 deactivation (loss of
antagonistic pattern) was observed during movement of
intact hands by patients with unilateral brachial plexus
injury. We propose that the disappearance of the antagonistic pattern of SM1 activities in the patients with brachial plexus injury reflects a reduction of interhemispheric inhibition, which may mirror an adaptive
mechanism to functional status.
Ann Neurol 2002;51:381–385
DOI 10.1002/ana.10149
Interhemispheric control of motor areas is important
for bimanual coordination and skill acquisition. Such
bihemispheric control of motor processes is necessary
for asynchronous bimanual movements and to inhibit
interference from the opposite hemisphere when even
simple unimanual movement is required.1 Evidence
supporting interhemispheric inhibitory mechanisms in
humans is mainly from transcranial magnetic stimula-
tion (TMS) studies.2–5 Nevertheless, TMS may activate
interneurons and pyramidal cells simultaneously, so
that the effects of TMS cannot be expected to reproduce physiological activation completely (ie, voluntary
Allison and colleagues7 used functional magnetic resonance imaging (fMRI) to investigate interhemispheric
interaction in motor control. Along with cerebral activation of primary sensorimotor cortex (SM1) contralateral to the hand movement, concomitant ipsilateral
SM1 deactivation (decreased fMRI-BOLD signal, suggesting a reduced flow and, operationally, a regional
net inhibition of neuronal activity8,9) was noted in
subjects performing finger–thumb opposition tasks.
This antagonistic pattern was interpreted as fMRI evidence of interhemispheric inhibition.7
We reason that if the ipsilateral SM1 deactivation
implicates a functional inhibition in the normal case, a
potentially central adaptive modulation in patients
with incapacitating injury to major peripheral nerves is
a reduction of deactivation in the SM1 ipsilateral to
the intact hand during unimanual movement. Operationally, there is no need to inhibit an already disabled
arm while moving the intact hand. This hypothesis was
tested on 9 patients with brachial plexus injury (BPI)
and on 9 healthy controls.
Patients and Methods
Nine healthy right-handed adults (male ⫽ 6, female ⫽ 3,
19 –24 years old) and 9 right-handed patients with brachial
plexus injury (BPI), with an injury history of 3 months to 8
years (male ⫽ 8, female ⫽ 1, right BPI ⫽ 5, left BPI ⫽ 4,
20 – 65 years old) were studied. Patients were free from other
central nervous system disease. Neurological and electrophysiological examinations of patients showed injury localizations
and extensions varying from C3 to T1, characterized by
common involvement of C6 and C7, manifested as a severe
disabling of hand-grasping. Patients complained of numbness/tingling and had various degrees of sensory deficit to
light-touch and pinprick at the involved area of innervation.
From 1Integrated Brain Research Laboratory, Department of Medical Research and Education, Taipei Veterans General Hospital; 2Institute of Neuroscience, School of Life Science, and Departments of
Anesthesiology, 4Psychiatry, 5Surgery, 6Neurology, and 7Radiology,
Faculty of Medicine, School of Medicine, and 8Institute of Radiological Sciences, National Yang-Ming University; 9Neural Regeneration Laboratory, Neurological Institute, Taipei Veterans General
Hospital, Taipei, Taiwan.
Received Aug 29, 2001, and in revised form Dec 4. Accepted for
publication Dec 4, 2001.
Published online Feb 27, 2002.
Address correspondence to Dr Hsieh, Integrated Brain Research
Unit, Department of Medical Research and Education, Taipei Veterans General Hospital, No. 201, Sect.2, Shih-Pai Rd., Taipei 112,
Taiwan. E-mail:
Consent for the study was obtained from all participants,
with a protocol approved by the institutional Ethics and Radiation Safety Committees.
The motor task consisted of simple self-paced hand grasping
at 1Hz. All subjects participated in a short training session
immediately before scanning and were under visual observation during the fMRI experiment. Each study began with a
resting phase (5 dummy ⫹ 10 scans) during which participants performed no voluntary motor activity, followed by 10
scans of unilateral hand fist, using either the right hand
(healthy controls) or the intact hand (BPI patients). Alternating blocks were repeated consecutively four times. Partic-
© 2002 Wiley-Liss, Inc.
ipants were cued with a gentle touch on the left foot before
the first scan of each block (rest, movement), to alert them
to the change of conditions.
spatially smoothed with an 8mm full width at half maximum
(FWHM) gaussian kernel, using standard SPM methods.12,13
Contrasts between movement and rest conditions
Functional Magnetic Resonance Imaging
Activation ⫽ movement ⫺ rest;
Images were acquired using a 3.0T Bruker MedSpec S300
system (Bruker, Karlsrube, Germany) with a quadrature
head. Subjects’ heads were immobilized with a vacuum-beam
pad in the scanner. Subjects’ eyes were closed during the experiment. Functional data were acquired with a T2*weighted gradient-echo EPI using BOLD contrast (TR/TE/␪
⫽ 1,700ms/50ms/90°, the slice thickness ⫽ 4mm, interslice
interval ⫽ 1mm, field of view (FOV) ⫽ 250mm, 64 ⫻ 64
⫻ 16 matrix). For each slice, 85 images were acquired. Brain
signals in the bilateral SM1 were specifically observed. The
anatomical images were acquired using a high-resolution T1weighted, 3D gradient-echo pulse sequence (MDEFT: Modified Driven Equilibrium Fourier Transform; TR/TE/TI ⫽
88.1ms/4.12ms/650ms, 128 ⫻ 128 ⫻ 128 matrix, FOV ⫽
Image Analysis
Data were analyzed with statistical parametric mapping (SPM99 software from the Wellcome Department of Cognitive Neurology, London), running under Matlab 6.0 (Mathworks, Sherbon, MA) on a Sun
workstation. Images of right BPI patients (using left hands
for the experiment) were flipped10,11 for a homogeneous motor representation. The first five images were discarded from
analysis, to eliminate nonequilibrium effects of magnetization. Scans were realigned, normalized, time-corrected, and
deactivation ⫽ rest ⫺ movement
were examined by voxel-specific t-tests {SPM(t)} across all
participants of the same group. t-scores were subsequently
transformed to the unit normal Z-distribution to create a
statistical parametric map {SPM(z)} for each contrast. Regionally specific differences that survived an uncorrected
threshold of p ⬍ 0.001 (Z ⫽ 3.09, with cluster size ⫽ 5
pixels) were considered statistically significant. Maxima were
localized on the normalized structural images and labeled using the nomenclature of Talairach. Spatial extent (significant
voxel numbers) within SM1 (areas 2, 3, and 4) was calculated using Talairach Daemon (Research Imaging Center,
University of Texas).
BETWEEN-GROUP COMPARISONS ON IPSILATERAL SM1 ACTIVITIES. The random effect model was exploited to quan-
tify the ipsilateral SM1 activity difference between groups,
using a balanced-design approach.14 In brief, first-order contrast images of the paired conditions (deactivation ⫽ rest minus movement) from each subject were processed with a
second-order contrast comparison (between groups), to delineate interaction of groups and conditions. This method
accommodated randomness in differential responses by comparing mean activation with variability in activation from
Table 1. Brain Activity Changes in the SM1 during Unimanual Hand-Grasping*
Normal Control
BPI Patient
Contralateral (Lt) hemisphere
56 ⫺34 ⫺36
⫺40 ⫺24
⫺38 ⫺30
⫺48 ⫺20
⫺38 ⫺26
⫺26 ⫺12
⫺58 ⫺26
⫺54 ⫺26
Ipsilateral (Rt) hemisphere
6.01 2545
40 ⫺18
38 ⫺26
56 ⫺10
*Threshold at p ⬍ 0.001, cluster size ⫽ 5; Inf denotes Zmax ⬎ 8.0. Depicts significant cluster peaks within the (de)activated volume(s) in SM1.
Stereotactic coordinates of peak activation are expressed in millimeters and refer to mediolateral position (x) relative to midline (positive ⫽
right), anteroposterior position (y) relative to the anterior commissure (positive ⫽ anterior), and superior-inferior position (z) relative to the
commissural line (positive ⫽ superior). Voxels indicate the overall spatial extent of SM1 clusters.
Annals of Neurology
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March 2002
subject to subject, hence group to group. The analysis
yielded information on the existence of quantitative differences between BPI patients and the healthy controls in terms
of movement-related brain activity changes in SM1, as indexed to the resting baseline. Differences were considered
significant at uncorrected p ⬍ 0.05 (Z ⬎ 1.75; cluster size ⫽
5). Spatial extent was calculated.
Table 1 and Figure a,b present the grouped results and
maps of controls and BPI patients, respectively, using
within-group analysis of SM1 activity. In healthy controls, task-related activations were found preponderantly in the contralateral (left) SM1 and slightly/discretely in the ipsilateral (right) SM1. Prominent
deactivation was observed in the ipsilateral SM1 of the
control group (see Fig a). However, the BPI patients
lost ipsilateral SM1 deactivation in the presence of similar contralateral SM1 activation (see Fig b). Table 2
and Figure c show results and statistical maps for
between-group comparisons of the ipsilateral SM1 activities. Significant group-condition interaction was detected. The data showed significantly less deactivation
in the ipsilateral SM1 in BPI patients compared with
healthy controls.
Concomitant ipsilateral SM1 deactivation (decreased
BOLD response) associated with the contralateral SM1
activation was verified in our healthy controls (see Figure a and Table 1). Our findings are congruent to previous fMRI reports, using more demanding finger–
thumb opposition tasks,7,15 and are corroborated by a
combined positron-emission tomography–TMS study
in which a negative covariance between the right cerebral blood flow (rCBF) of contralateral primary motor
(decreased rCBF) and the TMS-stimulated ipsilateral
primary motor cortex (increased rCBF) was observed.16
The findings from brain imaging studies are further substantiated by electroencephalographic data. Pfurtscheller
and colleagues17 reported that imaging a unilateral hand
movement induces an amplitude attenuation of eventrelated desynchronization in the contralateral SM1,
while triggering in parallel a significant amplitude enhancement or event-related synchronization (ERS) in
the ipsilateral SM1. The authors interpret the ERS as
deactivation or active inhibition of ipsilateral sensorimotor structures for optimal performance of the lateralized
These converging findings lend strong support to the
theory that interhemispheric inhibition, manifesting an
antagonistic pattern of brain activities, is a critical part
of the bihemispheric control mechanism for voluntary
movement. In this study, the ipsilateral SM1 deactivation (in the context of decreased BOLD, decreased
rCBF, and ERS) is interpreted as an expression of ei-
Fig. Maps of brain activity changes in the primary sensorimotor cortices during unimanual movement. (a) Healthy control:
activation is seen in the contralateral SM1 with a concomitant deactivation in the ipsilateral SM1 (circled in red). (b)
BPI patients: contralateral SM1 activation was similar to that
of the healthy controls while the ipsilateral SM1 deactivation
was lost. Slight but nonsignificant deactivation was seen. (c)
Interaction between groups and conditions: BPI patients manifested significantly less ipsilateral SM1 deactivation (right
hemisphere) than healthy controls. Images were displayed at
y ⫽ ⫺24 and x ⫽ 36 (see annotations in Table 1), respectively, for coronal and sagittal visualizations. In a and b,
warm colors (red-yellow) denote activation while the cold colors (blue-cyan) indicate deactivation. Cold colors in c indicate
significant interaction (less deactivation in the BPI patients
than the healthy controls). Color bars to the right of the images denote the values of Zmax.
ther a callosal mechanism of interhemispheric inhibition7 (cf Reddy et al15) or a net inhibition from the
orthodromic and transsynaptic input exerted from the
Hsieh et al: Loss of Ipsilateral SM1 Inhibition in Brachial Plexus Injury
Table 2. Between-Group Comparisons of Ipsilateral SM1 Activities*
Rest-Movement (deactivation)
Patient ⬍ Normal
Normal ⬍ Patient
*Threshold at p ⬍ 0.05, cluster size ⫽ 5; see Table 1 footnote. Depicts significant cluster peaks within the ipsilateral SM1. Voxels indicate the
overall spatial extent of SM1 clusters.
stimulated hemisphere to the other,16 and, in part,
modulated by thalamocortical systems.17
The present study provides insightful access to the
functional implication of ipsilateral SM1 deactivation
through its comparative approach. A dramatic reduction
of ipsilateral SM1 deactivation (loss of antagonistic pattern) was confirmed in unilateral BPI patients during
movement of their intact hands (see Tables 1, 2, Figure
b,c). Differences of SM1 activities between the two
groups cannot be ascribed to the performance difference18 or sensory deficit,10,11 as all the participants were
trained to conduct, using their intact hands, the simple
hand-grasping at 1Hz without forceful exertion.
Reorganization in brain motor systems after lesions in
the peripheral, as well as in the central nervous system, is
well documented.19,20 The mechanisms underlying the
disappearance of the antagonistic pattern of SM1 activities in BPI patients remains to be determined. One
plausible explanation is a reduction of interhemispheric
inhibition, which could be one facet of overall plasticity
of BPI, mirroring the functional status of the patients in
an operational context. There is essentially no need for
the brain to “inhibit” unlikely potential interference
from an already disabled limb. A longitudinal study on a
larger group of patients to correlate changes with either
the severity of functional impairment or the degree of
restitution of function after therapeutic intervention will
be heuristic, to explore the origin of these changes and
to disclose the unexplored mechanisms of functional
plasticity, by penetrating both sides of signal changes observed in functional brain imaging studies.
This work was supported by grants from the National Science
Council (882314B075081, 892314B075054, 892314B075159,
J-CH), Taipei-Veterans General Hospital (89362, 89402, 89400,
91361, J-CH), Ministry of Education (89BFA221401, J-CH,
Y-TW, T-CY), and NHRI-GT-EX 89B921C of Taiwan (HC).
Special thanks to Mr Chi-Cher Chou for MRI technical assistance.
Annals of Neurology
Vol 51
No 3
March 2002
1. Geffen GM, Jones DL, Geffen LB. Interhemispheric control of
manual motor activity. Behav Brain Res 1994;64:131–140.
2. Ferbert A, Priori A, Rothwell JC, et al. Interhemispheric inhibition of the human motor cortex. J Physiol 1992;453:525–546.
3. Liepert J, Tegenthoff M, Malin JP. Changes of inhibitory interneurons during transcallosal stimulations. J Neural Transm
4. Meyer BU, Roricht S, Woiciechowsky C. Topography of fibers
in the human corpus callosum mediating interhemispheric inhibition between the motor cortices. Ann Neurol 1998;43:
360 –369.
5. Netz J, Ziemann U, Homberg V. Hemispheric asymmetry of
transcallosal inhibition in man. Exp Brain Res 1995;104:
6. Reddy H, Lassonde M, Bemasconi N, et al. An fMRI study of
the lateralization of motor cortex activation in acallosal patients.
NeuroReport 2000;11:2409 –2413.
7. Allison JD, Meador KJ, Loring DW, et al. Functional MRI
cerebral activation and deactivation during finger movement.
Neurology 2000;54:135–142.
8. Ingvar M, Hsieh J-C. The image of pain. In: Wall PD, Melzack
R, eds. Textbook of pain. 4th ed. Edinburgh: Churchill Livingstone, 1999:215–234.
9. Hsieh J-C, Stone-Elander S, Ingvar M. Anticipatory coping of
pain expressed in the human anterior cingulate cortex: a positron
emission tomography study. Neurosci Lett 1999;262:61– 64.
10. Hsieh J-C, Belfrage M, Stone-Elander S, et al. Central representation of chronic ongoing neuropathic pain studied by
positron emission tomography. Pain 1995;63:225–236.
11. Hsieh J-C, Meyerson BA, Ingvar M. PET study on central processing of pain in trigeminal neuropathy. Eur J Pain 1999;3:
51– 65.
12. Ashburner J, Friston KJ. Nonlinear spatial normalization using
basis functions. Hum Brain Mapp 1999;7:254 –266.
13. Friston KJ, Buechel C, Fink GR, et al. Psychophysiological and
modulatory interactions in neuroimaging. Neuroimage 1997;6:
218 –229.
14. Friston KJ, Holmes AP, Price CJ, et al. Multisubject fMRI studies and conjunction analyses. Neuroimage 1999;10:385–396.
15. Reddy H, Matthews PM, Lassonde M. Functional MRI cerebral activation and deactivation during finger movement. Neurology 2000;55:1244.
16. Fox P, Ingham R, George MS, et al. Imaging human intracerebral connectivity by PET during TMS. NeuroReport 1997;
17. Pfurtscheller G, Neuper C, Flotzinger D, Pregenzer M. EEGbased discrimination between imagination of right and left
hand movement. Electroencephalogr Clin Neurophysiol 1997;
103:642– 651.
18. Rao SM, Bandettini PA, Binder JR, et al. Relationship between
finger movement rate and functional magnetic resonance signal
change in human primary motor cortex. J Cerebr Blood Flow
Metab 1996;16:1250 –1254.
19. Cohen LG, Ziemann U, Chen R. Mechanisms, functional relevance and modulation of plasticity in the human central nervous system. Electroencephalogr Clin Neurophysiol Suppl
1999;51:174 –182.
20. Boroojerdi B, Ziemann U, Chen R, et al. Mechanisms underlying human motor system plasticity. Muscle Nerve 2001;24:
602– 613.
Hereditary Motor and
Sensory Neuropathy with
Minifascicle Formation in a
Patient with 46XY Pure
Gonadal Dysgenesis: A New
Clinical Entity
Kazuma Sugie, MD, PhD,
Naonobu Futamura, MD, PhD,1
Akio Suzumura, MD, PhD,1 Genshu Tate, MD, PhD,3
and Fujio Umehara, MD, PhD4
This case report is of a patient with 46XY pure gonadal
dysgenesis, who presented with chronic progressive motor and sensory polyneuropathy. The sural nerve biopsy
exhibited minifascicle formations accompanied by a
marked decrease in myelinated fibers. This is the first report of polyneuropathy with minifascicle formations in
46XY pure gonadal dysgenesis. Because a similar polyneuropathy was recently reported in a case with 46XY
partial gonadal dysgenesis, it is possible that these cases
represent a new type of hereditary motor and sensory
neuropathy associated with gonadal dysgenesis.
Ann Neurol 2002;51:385–388
DOI 10.1002/ana.10150
From the 1Department of Neurology, Nara Medical University, Nara;
Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo; 3Department of Surgical Pathology, Showa University Fujigaoka Hospital, Kanagawa; and 4Third Department of Internal Medicine,
Kagoshima University School of Medicine, Kagoshima, Japan.
Received May 30, 2001, and in revised form Oct 30. Accepted for
publication Dec 5, 2001.
Published online Feb 27, 2002.
Address correspondence to Dr Sugie, Department of Ultrastructural
Research, National Institute of Neuroscience, National Center of
Neurology and Psychiatry, Tokyo. E-mail:
46XY gonadal dysgenesis (GD) is a very rare disorder
of sexual differentiation, classified as (1) 46XY pure
GD, characterized by bilateral streak gonads1; and (2)
46XY partial GD, characterized by one streak gonad
and a testis. Both are associated with an abnormality of
testis differentiation in phenotypic females.
A patient with polyneuropathy with minifascicle formation in 46XY partial GD was recently reported by
Umehara and colleagues.2 Because the case presented a
mutation of the human desert hedgehog (DHH) gene,
these investigators suspected that the DHH gene might
play a critical role in both male gonadal differentiation
and perineurial formation. This case report is of a patient with 46XY pure GD with hereditary motor and
sensory neuropathy (HMSN), with minifascicle formation. The pathological findings in our case were almost
identical to those in Umehara’s case, and both cases
have consanguinity and GD. Minifascicle formation of
the sural nerve is very rare, and only observed in regenerating nerves after transection.3 Therefore, we considered the possibility that these 2 patients represent a
new category of HMSN.
Case Report
This 47-year-old patient was born by normal delivery and
raised as a female. Her parents were first cousins; her paternal grandfather and her maternal great-grandfather were
brothers. There was no abnormality in her developmental
milestones, although she was aware of primary amenorhea
from puberty. At age 39, she noticed distal numbness and
weakness in all four extremities, which gradually progressed
to include distal dominant severe loss of superficial and deep
sensation, as well as moderate weakness. She had not noticed
bladder dysfunction or severe constipation.
On her first examinations at age 47, her height was
156cm, and her weight 60kg. Her intellect was normal. No
abnormality of the cranial nerves was observed. Tendon reflexes were not elicitable. A Babinski sign was absent. Peripheral nerves were not palpable. Sex characteristics included
poorly developed breasts and sparse pubic hair. Gynecological examination revealed normal female external genita, a
blinded vagina, and an immature uterus. Laboratory examination showed a very high level of LH (29.7mIU/ml; normal: male, 1.8 –5.2; female, 1.8 –7.6), follicle-stimulating
hormone (109mIU/ml; normal: male, 2.9 – 8.2; female, 5.2–
14.4). Her testosterone level was very low (0.04mIU/ml; normal 2.7–10.7) in the serum; estradiol was also low (⬍10
pg/ml; normal: male, 15– 60; female, 25–100). Pelvic magnetic resonance imaging showed bilateral streak gonads. Mature ovary and testis were undetectable. Motor nerve conduction velocity was 37.2m/sec in the median and 32.3m/sec in
the tibial; it was not evoked in the peroneal nerve. A nerve
conduction block was not observed. Sensory nerve action potentials were not evoked in all examined nerves including the
sural nerve. Electromyograms showed neurogenic changes
and some denervation potentials in the four extremities. No
abnormalities were found on the electroencephalogram. The
coefficient variation of the R–R intervals on electrocardiogram was normal.
© 2002 Wiley-Liss, Inc.
Fig 1. (a) Light microscopic findings of sural nerve specimens
stained with toluidine blue. Sural nerve contains many small
fascicles. (b) Many fascicles have smaller fascicles inside them.
(c) Electron microscopic findings of sural nerve specimens.
Minifascicles contain several myelinated and unmyelinated
fibers. (d) Unmyelinated fibers appear normal. (e) Higher
magnification of c. Note that perineurial cell (filled star) is
surrounded by basal lamina (small arrow). In contrast, the
adjacent cell (open star) is partially surrounded by basal lamina (large arrow), however, its process lacks the basal lamina
(arrowhead). Bars ⫽ 100 ␮m in a, 25 ␮m in b, 12 ␮m in
c, 4 ␮m in d, 3 ␮m in e.
Sural Nerve Biopsy
A nerve biopsy was performed on the left sural nerve between the external malleolus and the Achilles tendon. A portion of the specimen was fixed in buffered 3% isotonic glutaraldehyde at pH 7.4, and then postfixed in osmium
tetroxide. One fragment of the specimen was embedded in
epoxy resin. The other fragment was macerated in 66%
glycelin for 48 hours before dissection in pure glycerin for
teased nerve analysis. Semithin sections of epon-embedded
specimens were stained with toluidine blue. The diameter
distribution of myelinated and unmyelinated fibers was de-
Annals of Neurology
Vol 51
No 3
March 2002
termined using a Luzex size analyzer. For the electron microscopic examination, ultrathin sections were stained with uranyl acetate and lead citrate. Another portion of the specimen
was fixed in 10% neutral formalin and embedded in paraffin
for immunohistochemistry; 5␮m-thick paraffin-embedded
sections were deparaffinized and rehydrated in a graded alcohol series. They were reacted with two primary antibodies,
monoclonal antihuman epithelial membrane antigen antibody (1:100) (EMA; DAKO, Kyoto, Japan) and rabbit polyclonal anticow S-100 protein antibody (1:200) (DAKO,
Kyoto, Japan), and then stained using the avidin-biotin-
peroxidase complex method (Vector Laboratories, Burlingame, CA) as described.2
The most distinct finding of the sural nerve was the minifascicle formations (Fig 1a and b). The whole nerve contained about 60 fascicles. The fascicle diameter ranged within
20 to 70 ␮m. The densities of both large and small myelinated nerve fibers were markedly decreased (Fig 2). Fibers
with relatively thinner myelin compared with their axon diameter, which suggest remyelination, were frequently observed. Onion bulb formations or myelin ovoid structures
were not noted. Electron microscopic examination showed
that each minifascicle contained some axon–Schwann cell
units, separated by several layers of flattened cell processes.
The perineurium of minifascicles were composed of a few
layers of flattened cell processes (see Fig 1c and d). In the
perineurium, the outer layer of the cells had a basal lamina,
which is one of the ultrastructural characteristics of perineurial cells (see Fig 1e). In contrast, the inner layer of the cell
bodies were surrounded by basal lamina; however, the process of the cells was not covered with basal lamina. Thus,
these cells had an intermediate character between fibroblast
and perineurial cell. The fiber density of unmyelinated fibers
of the patient was 80,206/mm2, which did not vary from
normal controls (40,000 –90,000/mm2) (see Fig 1d). Teased
fiber analysis demonstrated that about 40% of myelinated
fibers displayed segmental demyelination and remyelination
(Fig 3). Some myelinated fibers with short internodes were
also found. There was no axonal degeneration. On immunohistchemistry, perineurial cells in this case showed similar
staining patterns (eg, positive for EMA but negative for
S-100 protein) to those of normal controls (data not shown).
Gene Analysis
Chromosomal analysis, carried out by informed consent,
showed 46XY. Gene analysis showed neither duplication nor
deletion in the peripheral myelin protein 22 (PMP22 gene).
No mutation was found in the sex-determining region Y
(SRY) gene. We investigated the structure of the human
DHH gene. This gene contains three exons that encode a
Fig 2. Myelinated fiber diameter-frequency histogram of sural
nerve. The densities of both large and small myelinated nerve
fibers markedly decreased in our patient. The normal control,
with sural nerve taken from the same location as our patient,
was age-matched with our patient.
Fig 3. Teased nerve analysis showing segmental remyelination.
Bar ⫽ 25 ␮m.
polypeptide of 396 amino acids mapped to chromosome
12q12–q13.1.4 We examined exons 1 to 3 of the DHH gene
by directly sequencing genomic DNA samples from the patient, using the method previously reported.5,6 As a result,
again, no mutation was found in the three exons of the
DHH gene.
On the basis of the clinical findings, we diagnosed the
patient as having polyneuropathy. She denied having
any similar diseases in her family. However, because
her parents were first cousins and consanguinity was
also noted in the older generation, it is possible that
her symptoms were genetic. This patient most probably represents a new subtype of HMSN.
Our patient had 46XY pure GD associated with abnormality of testis differentiation in a phenotypic female. 46XY pure GD is a very rare condition characterized by an immature uterus, a blinded vagina,
bilateral streak gonads, and no ovary. Some genes related to sexual differentiation, such as the SRY gene,
have been discovered in place of testis differentiation
factor (TDF).7,8 It is now considered that testis differentiation is obstructed by abnormality of the SRY gene
in 46XY GD.9 Since no mutations of the SRY gene
were found in our case, abnormal testis differentiation
of our case might be due to another novel gene.
Another characteristic of this case was the minifascicle formation in the sural nerve. As far as we were
aware, this finding was very rare and was shown in
only the case reported by Umehara and colleagues. Although minifascicles were reportedly observed in nerve
grafts,3 traumatic changes,10 leprosy,11 and tumors,12
these disorders were excluded by clinical and appropriate laboratory examinations in our patient. Their case
exhibited clinical sensory polyneuropathy and subclinical motor neuropathy, confirmed by abnormal nerve
conduction study. Similar minifascicle formation was
noted in the sural nerve, and the chromosomal analysis
showed 46XY partial GD. The case shares many common histopathological and gynecological findings with
ours. The parents of the 2 patients were first cousins,
suggesting the existence of recessive inheritance. It is
possible that the 2 cases represent a new clinical subtype of HMSN.
Because it was suspected that minifascicle formation
Sugie et al: HMSN with Minifascicles in 46XY GD
was caused by an abnormality of the formation of the
perineurium, some genes related to the cascade of sexual
differentiation are possibly associated with the formation
of the perineurium as well. Umehara and colleagues5
found a mutation in the DHH gene in their case and
suggested that DHH may be associated with both male
gonadal differentiation and perineurial formation. In
contrast, there were no mutations in the DHH gene of
our patient, which may imply that an unknown causative gene, other than the DHH gene, also plays a critical role in both sexual and perineurial differentiation.
Because very similar polyneuropathy accompanied with
minifascicle formation was observed in two different
types of GD, it is possible that the common factors
might be involved in the two types of GD and neural
differentiation. Further investigation of similar cases, either pathologically or genetically, will be necessary.
This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (FU), the Naito
Foundation (FU), and Brain Science Foundation (FU).
We thank Dr Donald H. Silberberg, Department of Neurology,
University of Pennsylvania School of Medicine, Philadelphia, for
critical reading of the manuscript.
1. Vilain E, Jaubert F, Fellous M, McElreavey K. Pathology of
46,XY pure gonadal dysgenesis: absence of testis differentiation
associated with mutations in the testis-determining factor. Differentiation 1993;52:151–159.
2. Umehara F, Yamaguchi N, Kodama D, et al. Polyneuropathy
with minifascicle formation in a patient with 46XY mixed gonadal dysgenesis. Acta Neuropathol (Berl) 1999;98:309 –312.
3. Ahmed AM, Weller RO. The blood–nerve barrier and reconstitution of the perineurium following nerve grafting. Neuropathol Appl Neurobiol 1979 ;5:469 – 483.
4. Tate G, Endo Y, Mitsuya T. Assignment of desert hedgehog to
human chromosome bands 12q12–q13.1 by in situ hybridization. Cytogenet Cell Genet 2000;88:93–94.
5. Umehara F, Tate G, Itoh K, et al. A novel mutation of desert
hedgehog in a patient with 46,XY partial gonadal dysgenesis
accompanied by minifascicular neuropathy. Am J Hum Genet
6. Tate G, Kishimoto K, Mitsuya T. Expression of sonic hedgehog
and its receptor patched/smoothened in human cancer cell lines
and embryonic organs. J Biochem Mol Biol Biophys 2000;4:
7. Berta P, Hawkins JR, Sinclair AH, et al. Genetic evidence
equating SRY and the testis-determining factor. Nature 1990;
348:448 –50.
8. Tajima T, Nakae J, Shinohara N, Fujieda K. A novel mutation
localized in the 3⬘ non-HMG box region of the SRY gene in
46,XY gonadal dysgenesis. Hum Mol Genet 1994;3:
9. Behzadian MA, Tho SP, McDonough PG. The presence of the
testicular determining sequence, SRY, in 46,XY females with
gonadal dysgenesis (Swyer syndrome). Am J Obstet Gynecol
10. Jurecka W, Ammerer HP, Lassmann H. Regeneration of a
transected peripheral nerve. An autoradiographic and electron
microscopic study. Acta Neuropathol (Berl) 1975;32:299 –312.
Annals of Neurology
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March 2002
11. Vallat JM, Leboutet MJ, Henry P. Endoneurial proliferation of
perineurial cells in leprosy. Acta Neuropathol (Berl) 1991;81:
336 –338.
12. Mitsumoto H, Wilbourn AJ, Goren H. Perineurioma as the
cause of localized hypertrophic neuropathy. Muscle Nerve
1980;3:403– 412.
Septo-optic Dysplasia
Associated with a New
Mitochondrial cytochrome b
Markus Schuelke, MD,1 Heiko Krude, MD,2
Barbara Finckh, PhD,3 Ertan Mayatepek, MD,4
Antoon Janssen,7 Michael Schmelz, MD,5
Friedrich Trefz, MD,6 Frans Trijbels, PhD,7
and Jan Smeitink, MD, PhD7
We report on a 25-year-old patient with isolated mitochondrial complex III deficiency and a new heteroplasmic mutation (T14849C) in the cytochrome b gene. He
suffered from septo-optic dysplasia, retinitis pigmentosa,
exercise intolerance, hypertrophic cardiomyopathy, and
rhabdomyolysis. A HESX1 mutation was excluded as a
cause of his septo-optic dysplasia. Low ␣-tocopherol concentrations in his muscles and an elevated urinary leukotriene E4 excretion indicate increased production of reactive oxygen species.
Ann Neurol 2002;51:388 –392
DOI 10.1002/ana.10151
Respiratory chain complex III, a multi-protein structure of eleven structural subunits, is located at the
inner mitochondrial membrane and catalyzes the
electron transfer from ubiquinol to cytochrome c.
Cytochrome b is its only mitochondrial DNA
From the Departments 1Neuropediatrics, 2Pediatric Endocrinology,
of Charité University Hospital, Berlin; 3Children’s Hospital, University of Hamburg; 4Division of Metabolic and Endocrine Diseases, University Children’s Hospital, Heidelberg; 5Department of
Internal Medicine, Kreiskrankenhaus Bad Urach, Germany; 6Diagnostic Center for Metabolic Diseases, Reutlingen, Germany; 7Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, University Medical Center, Nijmegen, The Netherlands.
Received Jul 25, 2001, and in revised form Nov 15. Accepted for
publication Dec 11, 2001.
Published online Feb 27, 2002.
Address correspondence to Dr Schuelke, Charité University Hospital, Department of Neuropediatrics, Augustenburger Platz 1,
D-13353 Berlin, Germany. E-mail:
(mtDNA)-encoded subunit. Isolated complex IIIdeficiency is rare and mutations have only been
described in one assembly gene (BCS1L) and in the
cytochrome b gene.1–3 All cytochrome b mutations published so far are heteroplasmic and seem to have arisen
sporadically, as neither affected parents nor siblings are
known. The phenotypes of patients with isolated complex III deficiency are heterogeneous. The age of onset
ranges from early childhood until adulthood.2,4 Patients with cytochrome b mutations suffer mostly from
exercise intolerance, hypertrophic cardiomyopathy or a
Parkinson-like movement disorder. We present a patient with a new heteroplasmic mtDNA mutation in
the cytochrome b gene. In addition to the usual clinical
findings, he suffered from septo-optic dysplasia and
retinitis pigmentosa.
Case Report
The index patient is the second child of healthy nonconsanguineous parents (see Fig 2A). His elder sister is healthy.
Symptoms started at the age of 7 months with muscle hypotonia and increased daytime sleepiness. Language and motor development were retarded. He started walking unsupported at the age of 30 months. He attended a school for the
mentally disabled. At the age of 20 years, he suffered from a
gastroenteritis, which resulted in severe lactic acidosis of
31mmol/L (normal, ⬍1.7) and rhabdomyolysis. At 24 years
of age, his length was 18cm below, and his head circumference was 3cm below the third centile. He showed marked
dysdiadochokinesia and gait ataxia. He suffered from exercise
intolerance after 3 minutes at 50watt bicycle ergometry. The
electrocardiogram showed features of Wolff-Parkinson-White
syndrome; echocardiography showed left ventricular hypertrophy without cardiac outlet obstruction. The echo density
of both kidneys was increased, and the serum creatinine was
repeatedly ⬎110␮mol/L (normal, 57– 88).
Ophthalmologic examination indicated a peripheral retinitis pigmentosa with narrowing of retinal blood vessels, a
small pale optic disc, an impaired vision of 60% and bilateral
peripheral visual field defects. Amplitudes in the electroretinogram were reduced. Cranial magnetic resonance imaging
(cMRI) showed features of a septo-optic dysplasia and cerebellar hypoplasia (Fig 1).
Biochemical Measurements
After informed consent according to the Declaration of Helsinki, skin and muscle biopsies were performed in the index
patient. Complex I, II, III, and IV activities were measured
spectrophotometrically in the 600g supernatant of the muscle
homogenate (Table).5 Citrate synthase was used as the mitochondrial reference enzyme. Estimation of the oxidation rates
and of ATP/phosphocreatine production was performed as
previously described.4
Approximately 107 cultured fibroblasts were homogenized.
Deficiencies of cytochrome c oxidase and citrate synthase
were excluded in the homogenate first. Then we measured
complex I, II⫹III, III, and IV activities. This had to be done
in a mitochondrial enriched fraction, because oxidative phosphorylation activity is comparatively low in fibroblasts, even
Fig 1. Cranial magnetic resonance image (cMRI) of the patient, showing typical features of septo-optic dysplasia: absence
of the septum pellucidum; hypoplasia of the corpus callosum,
the hypothalamus and the pituitary gland. Additionally, the
cerebellar vermis is absent and the foraminae of Luschka are
in normal individuals. The process of enrichment involves
several centrifugation steps, and citrate synthase tends to leak
from the mitochondrial matrix. Therefore, the membrane
bound cytochrome c oxidase is used as the reference. Urinary
leukotriene E4 concentrations, the total radical-trapping antioxidant parameter of plasma (TRAP) as well as the concentrations of ␣-tocopherol and coenzyme Q10 in a 1:40 muscle
homogenate (w/v) were estimated as previously described.6,7
All measurements were repeated twice.
DNA Studies
DNA was prepared from muscle tissue and from fibroblasts
by standard procedures. The whole cytochrome b gene was
polymerase chain reaction-amplified with the oligonucleotides nt14695–14717 (forward) and nt15946 –15926 (reverse). All numbers refer to GenBank sequence NC_001807.
The 1,252bp fragment was sequenced bidirectionally (BigDye, Applied Biosystems) with the nested primers: nt14695–
14717, nt14925–14944, nt15381–15400, nt15600 –15621,
nt15806 –15825, forward and nt15946 –15926, nt15710 –
15691, nt15499 –15480, nt15254 –15232, nt15024 –15003,
reverse. The results were checked in the family and in 100
healthy controls by restriction analysis: A 186bp fragment
was generated with the primer pair nt14817–14836 (forward) and nt15002–14817 (reverse). In the presence of the
T14849C-mutation the 186bp fragment was cut by HaeIII
into 155- and 31bp fragments. These were separated on a
3.5% agarose gel, stained with ethidium bromide and analyzed densitometrically. Signal intensities were normalized to
© 2002 Wiley-Liss, Inc.
Table. Results of Biochemical Measurements in Patient’s Fresh Skeletal Muscle and in Cultured Fibroblasts
Oxidation rates in fresh muscle
[1-14C] pyruvate ⫹ malate
[1-14C] pyruvate carnitine
[U-14C] malate ⫹ pyruvate ⫹ malonate
[U-14C] malate ⫹ acylcarnitine ⫹ malonate
[U-14C] malate ⫹ acylcarnitine ⫹ arsenite
[1, 4-14C] succinate ⫹ acylcarnitine
Control Range
ATP ⫹ phosphocreatine production rate in fresh muscle
From pyruvate
Activity in fresh muscle
NADH:Q1 oxidoreductase
Succinate dehydrogenase
Decylubiquinol: cytochrome c oxidoreductase
Cytochrome c oxidase
Citrate synthase
Activity in cultured fibroblasts
NADH:Q1 oxidoreductase
Succinate:cytochrome c oxidoreductase
Decylubiquinol: cytochrome c oxidoreductase
Cytochrome c oxidase
Values of complex III activities were based on three (muscle) and two (fibroblasts) separate measurements.
nmol/hr/mU citrate synthase.
mU/U citrate synthase.
mU/mg protein.
mU/U cytochrome c oxidase.
fragment length. The most common mtDNA mutations
A3243G, T3271C, G3460A, T4160C, A4317G, A8344G,
T8356C, T8993C/G, G11778A, G14459A, T14484C, and
large deletions were excluded in the patient as previously described.8 The four exons of the HESX1 gene were PCRamplified and sequenced bidirectionally with the intronic
primers: (Ex1F) 5⬘-GCA GAG GCC AGA GCT GTT G-3⬘,
We found an isolated complex III-deficiency in muscle
and in cultured fibroblasts. The oxidation rates of several
substrates and the ATP and phosphocreatine production
rate from pyruvate were normal despite complex III deficiency (see Table). Histologically, the muscle fiber diameters were slightly reduced. No ragged-red fibers
could be detected. The morphology of the mitochondria
was normal on electron microscopy. Sequence analysis of
the mitochondrial cytochrome b gene showed two known
homoplasmic polymorphisms (C14770T, A15326G)
and a new T14849C transition. It was heteroplasmic in
different tissues and was absent in other family members
Annals of Neurology
Vol 51
No 3
March 2002
and in 100 normal controls (Fig 2A). Endocrinological
testing showed growth hormone deficiency (5.79ng/ml
after maximum stimulation, normal ⬎10) and latent
central hypothyroidism (T4 5.47␮g/dl, normal 6.2–14;
thyroid-stimulating hormone 4.06␮E/ml after maximum stimulation). Cortisol, testosteron levels, serum
electrolytes, and osmolarity were normal.
Because reactive oxygen species (ROS) might be involved in the pathogenesis of muscle weakness and
other neurological manifestations,9 we measured the lipophilic antioxidants in muscle and plasma. The
TRAP was slightly reduced (737␮mol/L: normal 800 –
1200). ␣-Tocopherol concentrations were reduced in
muscle (105nmol/g muscle protein: normal 150-318)
but normal in plasma. The ubiquinone levels in muscle
and plasma were normal. The urinary excretion of leukotriene E4, an in vivo marker for lipid peroxidation,10
was elevated in two independent samples (71.3 and
78.5nmol/mol creatinine: normal 35.2 ⫾ 8.9).
Complex III deficiency caused by mutations in the cytochrome b gene is characterized by exercise intolerance,
episodic myoglobinuria, hypertrophic cardiomyopathy,
Wolff-Parkinson-White syndrome and cerebellar atro-
Fig 2. (A) Pedigree of the family and restriction analysis for
quantification of wild-type (wt) and T14849C mutant (mt)
mtDNA in peripheral blood leukocytes (L), muscle (M), and
fibroblasts (F). The results of the densitometric measurements
are given in percentage below the bands. (B) Alignment of the
polypeptide sequences of different species in the vicinity of the
Ser35Pro-mutation. The serine at this position is highly conserved. (C) The 3D structure of cytochrome b (1BCC.pdb) as
solved by X-ray crystallography. The Ser35 position is marked
by an arrow. It abolishes the hydrogen bond (dotted line)
between the hydroxylic group of the serine and the ubiquinone
at (Qi)-binding site.
phy.2,3 In addition to these findings, our patient suffered from retinitis pigmentosa and septo-optic dysplasia. These features have not been described in other
patients with complex III-deficiency.
We found a new T14849C mutation in the mtDNA
that fulfills the “canonic criteria” for pathogenesis.2 It
causes an exchange of a highly conserved serine to proline at position 35 (see Fig 2B). As can be derived from
the crystal structure of complex III, the serine35 is essential for binding of ubiquinone at the Qi site of cytochrome b (see Fig 2C) and also constitutes the binding
site of antimycin A.11 The importance of serine35 for
antimycin A binding is demonstrated by a strain of
Leishmania tarentolae, in which a Ser35Ile-mutation
confers antibiotic resistance to antimycin A.12 Exclusion
of the mutation in 100 healthy individuals of the same
ethnic background excludes a common polymorphism.
The degree of heteroplasmy varies between tissues. The
highest degree of 69% corresponds to the clinically most
affected muscle tissue. However, it remains unexplained
that there is no difference in residual complex III activity
between muscle and cultured fibroblasts (both ⬃70%)
despite a considerable difference in heteroplasmy (69 vs
12%). The same applies to the normal oxidation and
ATP production rates despite complex III deficiency.
However, our patient’s complex III deficiency is mild
and does not necessarily influence the mitochondrial oxidative capacity in vitro, which has been measured under
optimal conditions. Under in vivo conditions, the complex III deficiency might well be associated with decreased ATP production.8
Septo-optic dysplasia (de Morsier syndrome, MIM
182230) is characterized by hypoplasia of the optic
nerves, agenesis of the septum pellucidum, and hypothalamic dysfunction. The most frequent endocrine defect is growth hormone deficiency.13 Our patient fulfills all these defining criteria. Most cases occur
spontaneously and the genetic basis is unknown. Recently a missense mutation in the homeobox gene
HESX1 was detected in one familial case,14 but we excluded a HESX1 mutation in our patient. The coincidence of septo-optic dysplasia with a mitochondrial
mutation prompted us to seek another pathogenetic explanation, which might link both findings. Some mitochondrial mutations have been shown to increase
production of reactive oxygen species (ROS). Rana and
colleagues9 found increased ROS-levels in cybrids carrying a 4 bp deletion in the cytochrome b gene. Their
patient had atrophy of both cerebral and cerebellar
hemispheres.15 Geromel and colleagues16 demonstrated
massive superoxide induced apoptosis in fibroblasts carrying the mitochondrial T8993G (NARP) mutation.
In 1 of these patients, agenesis of the cerebellar vermis
was described.17 Other mitochondriopathies (PDHc
and COX deficiencies) have been reported to occur together with brain malformations such as optic atrophy,
Schuelke et al: Septo-optic Dysplasia
agenesis of the cerebellar vermis, and hypoplasia of the
corpus callosum.18,19 All these findings indicate that
ROS might play a role in pathogenic events that result
in brain malformation. We found indirect evidence for
increased ROS production in our patient. The concentration of ␣-tocopherol, a free radical quenching
molecule, was reduced in the muscle tissue. In addition, urinary excretion of leukotriene E4, an indirect in
vivo marker for lipid peroxidation6 was elevated twice
the normal range. Our patient also had a retinitis pigmentosa, which is known to be associated with ROSrelated diseases.20
Therefore, the association of complex III deficiency
and septo-optic dysplasia seems to be more than a coincidental finding. However, from the present data, we
cannot determine with certainty whether complex III
deficiency itself, or the increased ROS production secondary to it, might be the cause for the neurodevelopmental disorder in our patient.
We gratefully acknowledge the financial support of the Deutsche
Forschungsgemeinschaft (SFB 577 TP B4), and the parents’ selfhelp group “Helft dem muskelkranken Kind” in Hamburg is gratefully acknowledged.
We thank our patient and his family for participation in this study,
Dr Christine Gerstenfeld and Dr Christoph Hübner for critical review of the manuscript, Dr Eckart Apfelstedt-Sylla and Dr Constantin Kriegbaum for performing the ophthalmological assessment.
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Annals of Neurology
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March 2002
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