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Childhood peripheral neuropathy with autoantibodies to myelin glycoprotein Po.

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Childhood PeriDheral
Neuropathy wiih
Autoantibodies to Myelin
Glycoprotein Po
S. Ben Jelloun-Dellagi, PhD,*t K. Dellagi, MD,*
D. Burger, PhD,$ A. Ben Younes-Chennoufi, PhD,O
F. F. Hentati, MD,f A. Steck, MD,$
and M. Ben Hamida, MDf
The serum of a 4-year-old child with severe hypertrophic peripheral neuropathy contained high-titer polyclonal antibodies, mainly of IgG class, reacting with the
30-kd Po glycoprotein of peripheral nerve. This was
demonstrated by indirect immunofluorescence, immunoblot analysis of myelin proteins, and enzyme-linked
immunosorbent assay with purified Po glycoprotein.
The antibodies did not react with myelin-associated glycoprotein, sulfated glycuronic acid-containing paragloboside (SGPGand SGLPG), or other peripheral nerve
glycolipids or gangliosides. To our knowledge this is
the first report of strong antibody activity specifically
directed against Po. This antibody may play a causative
role in the pathogenesis of the peripheral neuropathy in
this patient.
Ben Jelloun-Dellagi S, Dellagi K, Burger D,
Ben Younes-Chennoufi A, Hentati FF, Steck A,
Ben Hamida M. Childhood peripheral neuropathy
with autoantibodies to myelin glycoprotein Po.
Ann Neurol 1992:32:700-702
Serum antibodies to neural antigens have been found
in peripheral neuropathy and are thought to play a
role in the pathogenesis of the disease. Monoclonal
IgM from patients with IgM gammopathy and polyneuropathy may show antibody activity to a carbohydrate
epitope shared by several myelin-specific antigens,
namely, myelin-associated glycoprotein (MAG), Po
glycoprotein, two sulfated glycuronic acid-containing
paraglobosides referred to as SGPG and SGLPG, and
several neural cell adhesion molecules El-41. Some
patients with IgM gammopathy and peripheral neurop-
athy may express antibody activity to other peripheral
nervous system (PNS) antigens, that is, chondroitin
sulfate, various glycolipids, or Schwann cell antigens
(reviewed in IS}). Polyclonal antibodies to nerve gangliosides or to other neural antigens are also seen in
patients with peripheral neuropathy without monoclonal IgM or even, at low level, in normal healthy
persons [6-81. We describe here the occurrence of
serum polyclonal antibodies to Po glycoprotein in a
child with a severe demyelinating neuropathy. This unusual autoantibody activity could not be detected in 4 1
other patients with various peripheral nerve disorders.
Materials and Methods
Case History
A 4-year-old girl, born of unrelated parents, was admitted at
the Department of Neurology (Tunis) for severe generalized
weakness. Symptoms began 3 years earlier and worsened
progressively but the parents noticed a transient and partial
improvement between age 1 and 3 years. N o neurological
symptom was reported in the kindreds. On admission, walking was possible with steppage gait. There was generalized
weakness with hypotonia of the four limbs. All tendon reflexes were absent. N o superficial sensory loss was noted.
Deep sensation was difficult to evaluate. There was a convergent strabismus of the left eye. The mental state appeared
normal. The cerebrospinal fluid (CSF) protein level was 0.57
gmlliter. The motor conduction velocity was markedly reduced (5 m/sec in the peroneal nerves) and sensory potentials
in the legs were absent. The nerve biopsy specimen (peroneal
superficial nerve) showed severe demyelination with prominent onion-bulb formations. The diagnosis of hypertrophic
peripheral neuropathy related to chronic polyradiculoneuritis
rather than Dejerine-Sottas atrophy was proposed. The patient was lost to follow-up.
Sera were obtained from 42 patients with various PNS diseases: 10 patients with motor neuron disease, 15 patients
with Charcot-Marie-Tooth neuropathy, 9 patients with
Friedrich's ataxia, and 8 patients with the Guillain-Barre syndrome. Sera from 10 healthy persons were used as negative
controls. Sera from 2 patients with IgM gammopathy and
polyneuropathy and the HNK- 1 mouse monoclonal antibody, also reacting with MAG, were used as positive controls.
Tissues and Antigens
From the 'Laboratory of Hematology and Immunopathology, PNR
Biologie Medicale, Facult6 de M6decine de Tunis, Tunis, Tunisia;
tDepartment of Neurology, Institut National de Neurologic-La
Rabta, Tunis, Tunisia; $Department of Neurology, Centre Hopitalier Universitaire Vaudois, Lausanne, Switzerland; and §Unit6 de
Neurologie Cellulaire, Moleculaire et Clinique, INSERM, Hdpital
de la, Paris, France.
Received Mar 6, 1771, and in revised form Jun 3 and Oct 21, 1771,
and Apr 20, 1772. Accepted for publication May 3, 1772.
Address correspondence to Dr Dellagi, Laboratory of Hematology
and Immunopathology, PNR Biologie MMicale, Facult6 de MCdecine de Tunis,Rue Essafi, 1007 Tunis, Tunisia
Human brain, peripheral nerves, and other organs were obtained within 12 hours after death from a patient who had
no neurological disease. Brain and sciatic nerves were also
obtained from rat, mouse, cat, and rabbit.
Central nervous system (CNS) myelin was isolated from
human brain by sucrose density centrifugation I91 and delipidated in chloroform methanol (2 : 1 vol/vol). Homogenates
of human sciatic nerve were directly prepared in sample
buffer of polyacrylamide gel electrophoresis (1:20 vol/vol).
The extracellular derivative of MAG (dMAG) was purified
by immunoaffinity chromatography and Po glycoprotein was
electroeluted from gel slice {41.
700 Copyright 0 1992 by the American Neurological Association
Indirect ImmzlnofEuorescence
Indirect immunofluorescence on tissue sections with human
sera was performed as previously described [lo].
Western Blot Analysis
Delipidated CNS myelin and homogenates of peripheral
nerve (20-25 pg
. of protein) were examined by western blot
analysis performed according to Towbin and colleagues [1 11.
The reacting antigens were immunostained with patient or
healthy control sera diluted to 1:200, with a second step of
peroxidase-conjugated F(ab’)*goat antibodies to human IgG
or IgM using diaminobenzidine as substrate.
Enzyme-Linked lmmunosorbent Assay (ELISA)
Purified human Po (0.25 p,g/well) was bound to Covalink
NH plates (A/S Nunc, Kamstrup, Denmark) by the bifunctional linker dissuccinimidyl suberate. Then, the ELISA was
carried out as described El21 and the serum antibody titer
was calculated as that dilution which was required to obtain
an optical density of 1.0 at 4 9 0 nm.
Imm~nodetectionof Glycolipids
on Thin-Layer Chromatography
Glycolipids from human sciatic nerve and central white matter were extracted with chloroform methanol and water and
immunodetected on thin-layer chromatography plates according to Magnani and associates [13], the patient’s serum
being diluted at 1 : 100.
Results and Discussion
By indirect immunofluorescence on sections of human
sciatic nerve, the patient’s serum gave a strong and
uniform staining of all compact myelin sheaths (Fig 1).
The reactive antibodies were mostly of the IgG class,
although some reactive IgM was also observed (not
shown). The reactivity of the patient’s serum was restricted to PNS myelin, since antibody binding was not
observed in other organs such as CNS myelin, kidney,
Fig 1. Indirect immunofluorescence staining of myelin sheaths of
human sciatic nerve with the primary antibodies from the patient’s serum. (A) Cross section. (B) Longitudinal section.
Bar = 20 p n ,
Fig 2. Western blot immunostaining of proteins i n CNS myelin
extracts (lanes 1-4), PNS homogenates (lanes 3-71, purified
myelin-associated glycoprotein ( M A G ) (lanes c, e), and pariled
Po (lanes d, f ) with HNK-1 (lane I ) , human anti-MAG IgM
(lanes 2, 5), normal control immunoglobulin (lanes 3, 7), and
the patient’s immunoglobulin (lanes 4, 6), I g G (lanes c, d), and
IgM (lanes e, f ) . Lanes a and b are coomassie blue staining of
pun$ed M A G and Po, respectively, on polyactylamide gel.
Square dots = Po; round dots = MAG; asterisks = molecukzrweight markers, from top to bottom, 94, 66, 4 5 , 30, 20, and
14 kd.
liver, and muscle (not shown). O n the other hand, her
antibodies recognized sciatic nerves of rabbit, mouse,
rat, and cat to the same extent as human nerve, suggesting that the reactive antigen(s) is (are) well conserved.
The myelin antigens recognized by the patient’s serum antibodies were identified by immunostaining on
Western blot (protein antigens) and thin-layer chromatography (lipid antigens). Her serum did not react with
CNS myelin or PNS lipid extracts (not shown). As
shown in Figure 2 (lane 6), the patient’s serum strongly
recognized only a protein band of molecular weight
( M W ) 30,000, present uniquely in PNS myelin. Her
serum did not bind MAG (see Fig 2, lane 4), a protein
recognized by monoclonal IgM from patients with demyelinating polyneuropathy and gammopathy (see Fig
2, lanes 2, 5 ) and from mouse HNK-1 (see Fig 2, lane
1). These latter antibodies are known to recognize,
through the L2/HNK-l epitope, both MAG and Po,
although, with patients IgM, the signal obtained for Po
is much weaker than for the MAG 141. This explains
the faint signal observed at the Po level in Figure 2
(lane 5). The patient’s serum also reacted weakly with
a CNS and PNS protein of about 55 kD (see Fig 2,
lanes 4,6). However, we have frequently observed this
reactivity with various sera, especially when used at low
dilution, and therefore it is not significant.
The patient’s serum did not bind purified human
Brief Communication: Ben Jelloun-Dellagi et al: Autoantibodies to Po Glycoprotein
is more sensitive than Western blot immunostaining or
immunofluorescence. To our knowledge, this is the
first observation of a strong antibody response specifically directed against Po in a human demyelinating
neuropathy. Since antisera to Po have been shown to
cause demyelination of rat sciatic nerve [l5}, it seems
likely that this anti-Po activity has clinical significance
and plays a role in the pathogenesis of the neuropathy.
However, the possibility that these antibodies are induced secondarily to the demyelination process can not
be excluded.
l o g (serum dilution 1
1. Braun PE, Frail DE, Latov N. Myelin associated glycoprotein is
the antigen for a monoclonal IgM in polyneuropathy. J Neurochem 1982;39:1261-1265
2. Chou DK, Ilyas AA, EvansJE, et al. Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNKl
antibody and some IgM paraproteins in neuropathy. J Biol
Chem 1986;261:11717- 11725
3. Bollensen E, Schachner M. The peripheral myelin glycoprotein
P express the L2/HNK-1 and L3 carbohydrate structures shared
by neural adhesion molecules. Neuroscience Lett 1987;82:
4. Burger D, Simon M, Peruisseau G, Steck AJ. The epitope(s)
recognized by HNK-1 and IgM paraprotein in neuropathy
Fig 3. Enzyme-linked imrnunosorbent assay titration curves in
the present patient (RZ) and a patient with polyneuropathy
and anti-MAG gammopathy (BP). N = normal control values
standzrd deviation (n = 6).
is present on several N-linked oligosaccharide structures on
human Po and myelin associated glycoprotein. J Neurochem
5. Steck AJ, Murray N, Dellagi K, et al. Peripheral neuropathy
associated with monoclonal IgM autoantibody. Ann Neurol
MAG (see Fig 2, lanes c, e) but recognized purified
human Po and also the Po self-aggregates that are generated during the purification process 1141 (MW
56,000, 84,000, and 112,000) (see Fig 2, lanes d, f).
These data demonstrate that the patient’s antibodies
are specifically directed against Po and not against a
putative contaminating myelin protein that could be
coelectroeluted with Po during the preparation. The
antibodies were mainly of the class IgG (see Fig 2, lane
f ) although some anti-Po IgM were also observed (see
Fig 2, lane d). Since the patient’s serum did not react
with MAG or nerve gangliosides, it appeared likely
that the epitopes recognized by her antibody on the
Po protein are not the L2/HNK-1 epitopes.
Finally, the antibody titer of our patient was determined by ELISA on purified Po. As shown in Figure
3, the patient’s serum displayed a higher titer than that
of a second patient with a demyelinating polyneuropathy and anti-MAG monoclonal IgM.
The antibody activity reported here appeared unique
since it could not be detected by indirect immunofluorescence or immunoblot analysis in sera from 10 normal control subjects or from 41 patients with various
neurological diseases. These results are in disagreement with those of Quarles and colleagues 181, describing weak antibody reactivities against Po in some patients with the Guillain-Barre syndrome. However,
they screened patient’s sera by ELISA, a method that
6. Ilyas AA, Willison HJ, Quarles RH, et al. Serum antibodies to
gangliosides in Guillain-Barre syndrome. Ann Neurol 1988;
7. Cruz M, Ernerudh J, Olsson T, et al. Occurrence and isotype
of antibodies against peripheral nerve myelin in serum from
patients with peripheral neuropathy and healthy controls. J Neurol Neurosurg Psychiatry 1988;51:280-285
8. Quarles RH, Ilyas AA, Willison HJ. Antibodies to ganghosides
and myelin proteins in Guillain-Barrk syndrome. Ann Neurol
9. Norton WT, Poduslo SE. Myelination in rat brain. Method of
myelin isolation. J Neurochem 1973;21:749-757
10. Dellagi K, Dupouey P, Brouet JC, et al. Waldenstrom’s macroglobulinemia and peripheral neuropathy: a clinical and immunological study of 25 patients. Blood 1983;62:283-285
11. Towbin H , Staehlin T, Gordon J. Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Natl Acad Sci USA 1979;
12. Burger D, Perruisseau G, Steck AJ. Antibody titers in patients
with anti-MAG M-IgM gammopathy and polyneuropathy: usefulness of a simplified method for the preparation of glycolipid
antigens. J Immunol Methods 1991;140:31-36
13. Magnani JL, Nilsson B, Brockhaus M, et al. A monoclonal defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto N-Fuco pentaose 11. J Biol Chem
14. Van Den Berg LH, Sadiq SA, Thomas FP,Latov N. Characterisation of HNK-1 bearing glycoproteins in human peripheral
nerve myelin. J Neurosci Res 1990;25:295-299
15. Hughes RAC, Powel HC, Braheny SL, Brostoff S. Endoneurial
injection of antisera to myelin antigens. Muscle Nerve 1985;
702 Annals of Neurology Vol 32 No 5 November 1992
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periphery, autoantibodies, neuropathy, childhood, glycoprotein, myelin
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