to potassium channels
and glutamic acid
Abstract—A patient with thymoma-associated neuromyotonia and voltage-
gated potassium channel (Kv1.2 and Kv1.6) antibodies by immunoprecipitation
and rat brain immunolabeling was treated successfully with immunoadsorp-
tion and cyclophosphamide. Curiously, glutamic acid decarboxylase antibodies,
absent at onset, appeared later. Stiff-person syndrome was absent, but fast
blink reflex recovery suggested enhanced brainstem excitability. The range of
antibodies produced in thymoma-associated neuromyotonia is richer, and the
timing of antibody appearance more complex, than previously suspected.
C. Antozzi, MD*; C. Frassoni, PhD*; A. Vincent, FRCPath; M.C. Regondi, PhD; F. Andreetta, PhD;
P. Bernasconi, PhD; C. Ciano, MD; T. Chang, MD; F. Cornelio, MD; R. Spreafico, MD;
and R. Mantegazza, MD
Neuromyotonia (NM), a variant of peripheral nerve
hyperexcitability (PNH),1is associated with CNS in-
volvement in some patients, a condition known as
Morvan fibrillary chorea (MFC).2NM/MFC seems
linked to antibodies against voltage-gated potassium
channels (VGKCs) and may coexist with myasthenia
gravis (MG), thymoma, or lung cancer.1
Glutamic acid decarboxylase antibodies (anti-
GAD) have been reported in a patient with thymoma
and acetylcholine receptor (AChR) antibodies, but no
evidence of MG, diabetes, or stiff-person syndrome
(SPS).1Anti-GAD is also found in SPS and other
neurologic conditions.3We describe a patient with
NM/MFC whose anti-VGKC was reduced and whose
clinical condition improved by immunoadsorption,
but who subsequently developed anti-GAD. We in-
vestigated immunoreactivity of the patient’s purified
immunoglobulins (IgG) with rat brain.
A.4Two plasma volumes were treated during each session per-
formed once or twice every 2 months.
In the patient’s total IgG and CSF, anti-AChR, anti-VGKC,
and anti-GAD antibodies were assayed by radioimmunoprecipita-
tion and titin and ryanodin receptor antibodies by ELISA and
western blot. Immunoreactivity of purified IgG was investigated
on free-floating rat brain slices before (IgG-NM1) and after (IgG-
NM2) 1 year of immunoadsorption.5Monoclonal antibodies
against Kv1.2 and Kv1.6 VGKC and purified IgG from MFC (IgG-
MFC) and SPS (IgG-SPS) patients served as positive controls.
Immunoperoxidase was used for detection. Double immunofluo-
rescence combined IgG-NM1 with anti-Kv1.6, IgG-NM1 with anti-
IgG immunoadsorption used staphylococcal protein
parvalbumin (anti-PV), and IgG-NM2 with anti-PV. Confocal
images were merged with Bio-Rad Lasersharp 2000 software
(Hemel Hempstead, UK).
mood changes and dream states and had a generalized
seizure in early 2000. In August 2000, paresthesia, fascicu-
lations, hyperhydrosis, fatigability, and cramps developed.
Neoplastic markers, thyroid hormones, and serum anti-
GAD and CSF examination were negative. CT revealed
cortical thymoma. After thymomectomy, he improved tran-
siently. Anti-VGKC were detected (?400 pM; healthy con-
trols ?100 pM).
We first saw the patient in December 2001. He could
walk with walking aid, experienced dream states, and was
seizure-free on gabapentin. Thyroid antibodies became
positive, anti-VGKC remained positive (516 pM), but
AChR, titin, and ryanodine receptor antibodies remained
negative. Nerve conduction and repetitive nerve stimula-
tion were normal. EMG revealed spontaneous activity typ-
fasciculations, and multiplets). Supramaximal motor nerve
stimulation evoked compound muscle action potentials fol-
lowed by repetitive after-discharges (figure 1).
Transcranial magnetic stimulation was normal. Cortex
and spinal cord stimulation evoked biphasic motor evoked
potentials (MEPs) followed by brief repetitive discharges;
the cortical silent period was shortened or absent. Recov-
ery of the R2 component of the blink reflex was faster than
controls (see figure 1).
IgG immunoadsorption began in December 2001. The
patient improved rapidly, with immediate disappearance
of hyperhydrosis and later reduction of fasciculations. Cy-
clophosphamide (100 mg/day) was added in January 2002.
Improvement has been maintained, but in August 2002, he
became weak and gained weight. Tests revealed severe
hypothyroidism, normalized with thyroxin.
Serial EMG during treatment found no spontaneous ac-
tivity in deltoid muscles but reduced neuromyotonic dis-
charges, persistent myokymia, and multiplets in other
muscles. Amplitude and duration of after-discharges were
much reduced (see figure 1).
Anti-VGKC, assayed in IgG be-
fore and after each immunoadsorption course from Decem-
ber 2001 to January 2003, were reduced by the first course
(figure 2) and later stabilized at approximately 200 pM.
Anti-GAD were absent in February 2001; however, be-
A 41-year-old man developed
*These authors contributed equally to this work.
From the Neurology Department IV (Drs. Antozzi, Andreetta, Bernasconi,
Cornelio, and Mantegazza), Immunology and Muscular Pathology Unit,
Experimental Neurophysiology Unit (Drs. Frassoni, Regondi, and Spre-
afico), and Clinical Neurophysiology Unit (Dr. Ciano), National Neurologi-
cal Institute “Carlo Besta,” Milan, Italy; and Neurosciences Group (Drs.
Vincent and Chang), Weatherall Institute of Molecular Medicine, John Rad-
cliffe Hospital, Oxford, UK.
Supported in part by MURST (project no. 2003068749).
Received June 30, 2004. Accepted in final form December 27, 2004.
Address correspondence and reprint requests to Dr. C. Antozzi, Neurology
Department IV, Immunology and Muscular Pathology Unit, National Neu-
rologic Institute “Carlo Besta,” Via Celoria 11, 20133, Milan, Italy; e-mail:
Copyright © 2005 by AAN Enterprises, Inc.
cause immunohistochemistry suggested their presence,
they were assayed in all available samples. They were
positive from April 2002 on, when anti-VGKC were well
controlled (see figure 2). CSF was negative for anti-VGKC;
at latest assay (August 2003), anti-GAD were positive (4.6
IgG-NM1 labeled somata and
proximal dendrites of rat cortical neurons and Purkinje
cells (figure 3, A and B), as did anti-Kv1.6 (see figure 3, E
and F) and IgG-MFC (see figure 3, G and H); anti-Kv1.2
gave similar cortical staining (see figure 3C) but stained
cerebellar basket cell terminals (see figure 3D).
IgG-NM2 labeled punctate structures (interpreted as
axon terminals or fiber cross-sections) in cortex, hippocam-
pus, and cerebellum (see figure 3, I, K, and M). Scattered
interneurons were labeled in cortical layer V and hip-
pocampal CA3 region (see figure 3, I and K). Purkinje cells
and granular cell layer were also labeled (see figure 3M).
This pattern, similar to that obtained with SPS serum (see
figure 3, J, L, and N), corresponds to the GABAergic sys-
tem distribution in rat brain.
Double immunofluorescence showed that IgG-NM1 co-
localized with Kv1.6 (see figure 3O) and IgG-NM2 with
GABAergic terminals. To demonstrate differing behavior
of IgG-NM1 and IgG-NM2, we double-labeled with PV
(GABAergic marker). IgG-NM1 plus PV showed cytoplas-
mic labeling of pyramidal neurons surrounded by GABAer-
gic terminals (see figure 3P). IgG-NM2 plus PV confirmed
co-localization of extracellular staining and GABAergic
terminals (see figure 3Q).
NM plus initial CNS involvement; thus, the diagnosis
is MFC. The appearance of thyroid and GAD antibod-
ies at a time when anti-VGKC were controlled is puz-
zling. We detected anti-VGKC by two techniques:
radioimmunoprecipitation (which detects Kv1.1, 1.2
and 1.6) and immunocytochemistry, indicating that
most antibody in our patient was against Kv1.6. Corti-
cal labeling with IgG-NM1 resembled that of Kv1.2
and Kv1.6, but cerebellar labeling was similar to that
of Kv1.6, probably reflecting the variable subunit com-
position of VGKC in different brain areas.
The patient had the characteristics of
Figure 1. Neurophysiologic recordings demonstrating after-discharges before (A, C) and after 1 year (B, D) of treatment, in
ulnar nerve (A, B) and tibial nerve (C, D). Also illustrated is the rapid recovery of the R2 component of the blink reflex (E).
Figure 2. Titers of anti-VGKC (left)
and anti-GAD (right) in patient’s im-
munoglobulin during follow-up. Pre ?
before each immunoadsorption session;
Post ? after each immunoadsorption
session; VGKC ? voltage-gated potas-
sium channel; GAD ? glutamic acid
April (1 of 2) 2005
April (1 of 2) 2005
When immunohistochemistry was repeated after a Download full-text
year of immunoadsorption, IgG-NM2 showed GABA-
ergic-type labeling on cerebral, cerebellar, and hip-
pocampal neurons, and anti-GAD were confirmed by
immunoprecipitation. Anti-GAD were absent early
in the patient’s history, when CNS symptoms were
prominent, so it is unlikely they contributed to the
CNS involvement. It is also possible that anti-GAD
reduction by treatment may have prevented further
CNS involvement, although their role in promoting
CNS manifestations is unclear, particularly because
anti-GAD in rat brain slices were detected only in
serum and not CSF. The only sign of CNS involve-
ment after treatment was shortened R2 recovery,
suggesting increased brainstem excitability.6Trans-
cranial magnetic stimulation revealed reduced upper
limb silent period and absent lower limb silent peri-
od—findings that might be explained by the presence
of after-discharges following MEP.
Thymoma occurs in about 20% of NM and MFC
patients1and occasionally with SPS alone or followed
by MG.7Anti-GAD have been found in patients with
thymoma, either with or without AChR antibodies
and MG.8-10In a large series of thymomatous pa-
tients reported recently, GAD65 antibody was the
most frequently observed neuronal autoantibody,
particularly in association with neurologic disorders
other than MG.10In one patient, antibodies were
monitored over 20 years.8Anti-AChR and anti-GAD
interferon-? antibodies peaked 15 years after thy-
momectomy at a time when anti-AChR and anti-
GAD were relatively low. Findings in our patient are
similar, in that anti-GAD arose at a time when anti-
VGKC were controlled by therapy, indicating a com-
plex relationship between thymoma and antibodies
to muscle and neuronal antigens. Antibody produc-
tion possibly initiated by T cells sensitized in the
thymoma may persist in peripheral lymphoid tissue
We suggest that the finding of anti-VGKC in a
patient with PNH warrants trying immunotherapy.
IgG immunoadsorption has had long-term efficacy in
our patient, supporting a role of circulating IgG (i.e.,
anti-VGKC antibodies) in the pathogenesis of NM.
Passive transfer of purified antibodies to animal
models would be an appropriate means of investigat-
ing the role and complexity of antibody production in
these antibody-mediated disorders of the nervous
The authors thank Linda Clover for the anti-VGKC assay, Ornella
Simoncini and Cristina Cappelletti for technical assistance, Dr. R.
Liguori (Institute of Neurology, University of Bologna, Italy) for
serum from the patient with MFC, and Don Ward for help with
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Figure 3. Cerebral cortex (A, C, E, G) and cerebellum (B, D, F, H) labeled with IgG-NM1, anti-Kv1.2, anti-Kv1.6, and
IgG-MFC. Note labeling of layer V neuronal somata and apical dendrites in cortex (arrows). Purkinje cells and apical
dendrites are also labeled in cerebellum, except that anti-Kv1.2 (D) stains basket cells axon plexus (arrowheads). Scale
bar ? 60 ?m. Cerebral cortex (I, J), hippocampus (K, L), and cerebellum (M, N) labeled with IgG-NM2 (I, K, M) and SPS
serum (J, L, N). Note labeled structures (arrowheads) around neurons of layer V and hippocampal CA3 region and inter-
neurons (arrows) labeled with IgG-NM2 (I, K) and SPS (J, L). Note also labeling of Purkinje cells (arrows) by IgG-NM2
(M) and SPS (N). Scale bar ? 60 ?m. Double immunofluorescence is also shown (O, P, Q). Double-staining with IgG-
NM1 and Kv 1.6 shows co-localization on pyramidal neurons (O). Double-staining with IgG-NM1 and PV shows pyrami-
dal neurons labeled in the cytoplasm with IgG-NM1 surrounded by PV-positive (i.e., GABAergic) terminals (P). Double
staining with IgG-NM2 and PV shows co-localization of extracellular IgG-NM2 positivity and GABAergic terminals (Q).
Scale bar ? 30 ?m. IgG ? immunoglobulin; NM ? neuromyotonia; MFC ? Morvan fibrillary chorea; SPS ? stiff-person
syndrome; PV ? parvalbumin.
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