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Detection and characterization of MuSK antibodies in seronegative myasthenia gravis.

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Detection and
Characterization of MuSK
Antibodies in Seronegative
Myasthenia Gravis
John McConville, MRCP, DPhil,1
Maria Elena Farrugia, MRCP,1 David Beeson, PhD,1
Uday Kishore, PhD,1 Richard Metcalfe, FRCP,2
John Newsom-Davis, FRS,1 and
Angela Vincent, FRCPath1
Antibodies to rat muscle specific kinase, MuSK, have recently been identified in some generalized “seronegative”
myasthenia gravis (SNMG) patients, who are often females with marked bulbar symptoms. Using immunoprecipitation of 125I-labelled-human MuSK, 27 of 66 (41%)
seronegative patients were positive, but 18 ocular SNMG
patients, 105 AChR antibody positive MG patients, and
108 controls were negative. The antibodies are of high
affinity (Kds around 100 pM) with titers between 1 and
200 nM. They bind to the extracellular Ig-like domains
of soluble or native MuSK. Surprisingly they are predominantly in the IgG4 subclass. MuSK-antibody associated
MG may be differnet in etiological and pathological
Ann Neurol 2004;55:580 –584
Eighty-five percent of myasthenia gravis (MG) patients have autoantibodies to the muscle acetylcholine
receptor (AChR).1 Patients without AChR antibodies
(seronegative MG [SNMG]) may have more frequent
bulbar involvement and less thymic pathology and often are resistant to conventional immunosuppression.2–5 Recently, IgG autoantibodies to the musclespecific kinase, MuSK, were identified in 70% of
patients with generalized SNMG,6 and antibodies to
a 110kDa protein, identified as MuSK, were found to
From the 1Weatherall Institute of Molecular Medicine and Department of Clinical Neurology, Oxford; and 2Department of Clinical
Neurology, Southern General Hospital, Glasgow, Scotland, United
Received Sep 25, 2003, and in revised form Jan 13, 2004. Accepted
for publication Jan 13, 2004.
Current address for Dr McConville: Department of Neurology,
Royal Victoria Hospitals Trust, Grosvenor Road, Belfast BT12,
Northern Ireland, United Kindgom.
Published online Mar 22, 2004, in Wiley InterScience
( DOI: 10.1002/ana.20061
Address correspondence to Dr Vincent, Neurosciences Group,
Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom.
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
be associated with oculobulbar symptoms.7 Here, we
describe new assays for detecting MuSK antibodies,
demonstrate their high disease specificity, and investigate their epitope specificity, affinity, and IgG subclass.
MuSK, or its polypeptide fragments, or soluble agrin as a
control, were added to the serum before addition of 125IMuSK.
MuSK Antibody IgG Subclasses
Subjects and Methods
Patients and Controls
Generalized SNMG patients (n ⫽ 66) all had attended
the Oxford clinic between 1987 and 2002 with welldefined MG (decrement on repetitive stimulation or jitter
on single fiber electromyography and/or a clear response to
edrophonium) and repeated negative AChR antibody titers.
Sex, age, and clinical features at first clinic presentation
were obtained from their records. Ocular SNMG patients
(n ⫽ 18) were diagnosed in Oxford, or by R.M. in Glasgow. AChR-Ab–positive sera were taken either from the
Oxford clinic or the routine AChR-Ab assay service. Control sera were from 38 volunteers, 30 community controls,
and 40 patients with other immune-mediated neurological
Serum (0.5␮l) was added to 5fmol 125I-human MuSK
and incubated overnight with 10␮l anti–human IgG1,
5␮l anti–human IgG2, 5␮l anti–human IgG3, 2.5␮l
anti–human IgG4 (monoclonal antibodies from Binding
Site, Birmingham, UK), or no antibody. The complexes
were precipitated with sheep anti–mouse IgG (Binding Site;
preabsorbed with 10% human control serum), with normal
mouse serum added to aid precipitation, and precipitated
I MuSK washed and counted as above. The enzymelinked immunosorbent assay (ELISA) for anti–MuSK IgG
subclasses6 used biotinylated monoclonal mouse anti–human IgG1 (1:100; Calbiochem, San Diego, CA); anti–human IgG2 (1:500; Sigma, St. Louis, MO); anti–human
IgG3 (1:1,500; Sigma); or anti–human IgG4 (1:1,000;
Calbiochem), as second layers, and detected their binding
with streptavidin-horseradish peroxidase (Sigma) at 1 to
Cloning of MuSK Ectodomains
Human MuSK was cloned by reverse transcription polymerase chain reaction from poly(A)⫹ RNA isolated from
the TE671 human rhabdomyosarcoma cell line.8 Expression constructs were generated in pSecTag (Invitrogen, La
Jolla, CA), with coding sequences inserted between an Ig␬
signal sequence at the N terminus and a His6 tag at the C
terminus. MuSK1-4 encoded the entire extracellular domains (nucleotides 107–1526, numbering as in Valenzuela
and colleagues9), MuSK 1-2 contained nucleotides 107 to
715, and MuSK 3-4 contained nucleotides 711 to 1526.
Human embryonic kidney (HEK) 293 cells were transiently
transfected with the constructs. Recombinant proteins were
harvested from serum-free culture supernatants (UltraCHO; Cambrex, Berkshire, UK) and purified using nickel
affinity chromatography (Probond; Invitrogen). Human
muscle agrin was used as a control antigen.6
MuSK Antibody Radioimmunoprecipitation Assay
and Epitope Mapping
Ten micrograms of purified human MuSK was labeled with
I, as described for other proteins,10 to a specific activity of
5 to 10 ⫻ 103 cpm/fmol. Five microliters of human plasma
or serum was added to 5fmol of MuSK and incubated for 1
hour at room temperature or overnight at 4°C in 0.02M
phosphate buffer/0.1% Triton X-100/5% fetal calf serum,
and IgG–MuSK complexes were precipitated with 50␮l
sheep anti–human IgG (Binding Site, Birmingham, UK).
The precipitates were centrifuged, washed, and counted for
I. All positive sera were titrated and the results were expressed as nanomoles of 125I-MuSK precipitated per liter of
serum. To determine the affinities, we incubated a limiting
amount of each serum overnight with different concentrations of 125I-MuSK; Kds were determined using GraphPad
Prism (GraphPad Software, San Diego, CA.). For epitope
mapping, culture supernatants containing 10-fold excess of
MuSK Antibodies Binding to Native MuSK on
MuSK–Green Fluorescent Protein Transfected
Human Embryonic Kidney 293 Cells
The sequence encoding enhanced green fluorescent protein
(EGFP; derived from EGFP-N1; Clontech, Palo Alto, CA),
was ligated into the naturally occurring restriction site EcoRI
at nucleotide position 2607 in the MuSK cDNA sequence,
so that the expressed product contained all but the
C-terminal 27 amino acids of MuSK followed by EGFP.
HEK 293 cells were transiently transfected using PEI.11
Trypsin-suspended cells (approximately 5 ⫻ 106) were incubated for 40 minutes in serum (1:40 dilution in phosphatebuffered saline/5% fetal calf serum). After washing, cells were
incubated with anti–human IgG PE (Sigma) at 1 to 30 and
fluorocytometry was conducted using a Becton Dickinson
(San Jose, CA) FACScan 2.
Antibodies to Human MuSK
Overall, radioimmunoprecipitation of 125I-MuSK extracellular domains detected IgG antibodies in 27/66
SNMG patients (eg, Fig 1A). MuSK-Abs were not
found in 18 patients with ocular SNMG (Ocular).
Low levels of 125I precipitation were found in a few
MG or control samples (see Fig 1A), but in these
cases, in contrast with the clear MuSK-Ab–positive
samples (Fig 2), 125I precipitation was nonspecific because it was not blocked by an excess of unlabeled
MuSK (data not shown). Titers ranged from 0.3 to
200nM (median, 20nM). All samples previously positive with the rat MuSK ELISA6 were strongly positive by immunoprecipitation of 125I-human MuSK
McConville et al: MuSK Antibody Characterization
Fig 1. Radioimmunoprecipitation assay for MuSK IgG antibodies. (A) Scatterplot of counts precipitated (cpm) by 5␮l serum or
plasma. The line is drawn at the mean ⫹ 3 SDs of the healthy control values; the neurological controls gave a similar range. The
few sera with low positive values were shown to bind nonspecifically. Antibody affinities (inset) ranged between 55 and 108pM.
(B) Correlation between binding of IgG antibodies to human MuSK-GFP expressed by human embryonic kidney (HEK) cells, measured by FACS, and the immunoprecipitation results. Examples of the FACS analysis, for one MuSK antibody–positive serum and
one control serum, are shown in the inset. SNMG ⫽ seronegative myasthenia gravis; AChR ⫽ acetylcholine receptor; GFP ⫽ green
fluorescent protein.
(r2⫽0.88; p ⬍ 0.0001 for accurate titers). All four
sera tested showed high-affinity binding with Kd values of less than 1nM (eg, see Fig 1A inset).
Patients with MuSK antibodies were usually female
(M:F, 4:23) and presented between 5 and 56 years
(median, 24 years), and 11 of 27 had prominent bulbar symptoms, whereas MuSK-Ab–negative patients
were often male (M:F, 14:25; p ⫽ 0.03, Fisher’s exact
test) and presented between 1 and 78 years (median,
37), and only 6 of 39 ( p ⫽ 0.03) had prominent bulbar symptoms.
Antibodies Binding to Native MuSK
We used the fluorescence activated cell sorter to detect
human IgG binding to the surface of HEK 293 cells
expressing human MuSK tagged with EGFP (MuSKGFP, see Fig 1B, inset). Clearly positive results were
obtained with each of the MuSK-Ab–positive sera, the
Annals of Neurology
Vol 55
No 4
April 2004
results correlating with the precipitation assay (see Fig
1B). None of the healthy control sera, or five SNMG
sera negative for binding to MuSK by radioimmunoprecipitation, bound to the MuSK-GFP–expressing
Epitopes in the Extracellular Domains of Human
Preincubation of sera with control protein (agrin) did
not alter precipitation. Preincubation with MuSK1-2
(see Fig. 2A) reduced the precipitation of all nine sera
tested (for examples, see Fig 2B), whereas MuSK 3-4
reduced precipitation of only five of nine sera. As expected, addition of both MuSK 1-2 and MuSK 3-4, or
of MuSK 1-4, reduced precipitation to control levels in
all cases (see Fig 2B).
Fig 2. (A) Diagramatic representation of MuSK and its domains that were expressed as soluble proteins. (B) Binding of
MuSK antibodies to 125I-MuSK in the presence of unlabeled
MuSK domains. Precipitation was substantially reduced in all
sera tested by incubation with forms that contained MuSK1-4,
whereas MuSK3-4 reduced precipitation in only some sera.
Precipitation in the presence of agrin, a control recombinant
protein, was similar to that by serum alone.
We found that MuSK antibodies bind to extracellular IgG-like domains of MuSK, both when expressed
in HEK 293 cells, or in solution. MuSK has a major
role during development of the neuromuscular junction (see Liyanage and colleagues13 and Hopf and
Hoch14), and MuSK antibodies reduce agrin-induced
clustering of AChRs in mouse C2C12 myotubes in
vitro,6 but it is not yet clear how they affect the neuromuscular junction in vivo. Although pathogenic effects were observed after transfer of SNMG plasmas to
mice,4,15,16 and most of the plasmas tested are now
known to be MuSK-Ab positive (A. Vincent, unpublished observations), there was little effect on total
AChR numbers.15,16 The results are complicated by
the existence of a non-IgG plasma factor in SNMG
that reduces AChR function17; although distinct, this
factor is present in many MuSK-Ab–positive SNMG
patients.17 IgG MuSK antibodies do not appear to affect AChR function directly.17
One hypothetical pathogenic mechanism would be
that MuSK antibodies activate complement which induces lysis of the AChR-containing postsynaptic
membrane. However, in contrast with AChR-Ab
MG, where complement-fixing IgG1 and IgG3 subclasses predominate,18,19 MuSK antibodies were
mainly IgG4 which is strong complement activators.
Together with the relative lack of thymic pathology,2,3 and the tendency to marked bulbar12 and neck
MuSK Antibody IgG Subclasses
Surprisingly, MuSK antibodies were predominantly
IgG4, with some IgG2 (Fig 3A). To confirm these
findings, we used a different set of mouse monoclonal
subclass–specific antibodies in an ELISA assay (see Fig
3B) with very similar results.
We confirmed, using two novel assays, that a proportion of patients with generalized MG without AChR
antibodies have increased levels of MuSK antibodies
and these antibodies are not present in patients with
AChR antibody–positive MG, in patients with purely
ocular SNMB, or in controls. The proportion of
MuSK antibody–positive patients was lower than our
previous report on a smaller number of SNMG cases,6 probably because the first study inadvertently included more patients with severe or refractory disease,
who we now know are more likely to be MuSK antibody positive.12 The simplicity, high specificity, and
sensitivity of the radioimmunoprecipitation assay
should make it useful in the routine diagnosis of this
form of MG.
Fig 3. IgG subclasses of MuSK antibodies. (A) IgG subclasses
measured by immunoprecipitation. (B) Results confirmed by
ELISA (below). In both cases, the MuSK antibodies were
largely in the IgG4 and IgG2 subclasses. MuSK-Ab Patients
1and 2 were tested in both assays.
McConville et al: MuSK Antibody Characterization
or respiratory weakness,20 these findings imply differences between MuSK-Ab MG and AChR-Ab MG in
cause and pathological mechanisms. Because IgG2 antibodies frequently are directed at carbohydrate antigens on bacteria, we tried to inhibit the antibodies
with appropriate sugars, but without success (L. Jacobson, U. Kishore, and A. Vincent, unpublished observations). Moreover, the MuSK antibodies bind
with high affinity (similar to that of AChR antibodies18) to the native MuSK molecule. Thus, our current evidence favors affinity maturation of an immune response directed against MuSK itself, rather
than cross-reactivity with a bacterial glycoprotein or
Note Added in Proof
Using sheep polyclonal antisera to IgG subclasses, we
have recently found up to 30% of the MuSK antibody
to be in the IgG1 subclass. However, IgG4 predominates in all sera examined so far.
J.M. was supported by a Clinical Training Fellowship from The
Wellcome Trust.
We are very grateful to Dr W. Hoch for his advice and the rat
MuSK constructs, and the ongoing support of the Myasthenia Gravis Association, the Muscular Dystrophy Campaign, and the French
Association against myopathies.
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antibodies, detection, seronegative, myasthenia, characterization, musk, gravis
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