Lower Motor Neuron Syndromes Defined by Patterns of Weakness, Nerve Conduction Abnormahties, and High Titers of Antiglycolipid Antibodies A. Pestronk, MD," V. Chaudhry, MD,? E. L. Feldman, MD, PhD,$ J. W. Griffin, MD,S D. R. Cornblath, MD,t E. H. Denys, MD,§ M. Glasberg, MD," R. W. Kuncl, MD, PhD,S R. K. Olney, MD,n and W. C. Yee, MD#

We studied 74 patients with progressive, asymmetrical lower motor neuron syndromes. Clinical features of these patients, including age, sex, disease duration, patterns of weakness, and reflex changes, were evaluated by review of records. In each patient the clinical features were compared to the type of nerve conduction abnormalities and to the specificities of high-titer serum antiglycolipid antibodies. Antibody specificities were determined by an enzyme-linked immunosorbent assay using purified glycolipids and carbohydrates as substrates. Our results show that high titers of antibodies to glycolipids are common in sera of patients with lower motor neuron syndromes. Selective patterns of reactivity indicate that specific carbohydrate epitopes on the glycolipids are the targets of the high-titer antibodies in individual patients with lower motor neuron syndromes. Several distinct lower motor neuron syndromes can be identified based on clinical, physiological, and antiglycolipid antibody characteristics. These syndromes include multifocal motor neuropathy with evidence of multifocal conduction block on motor, but not sensory, axons and frequent (84%) high titers of anti-GM1 ganglioside antibodies; a lower motor neuron syndrome with predominantly distal weakness early in the disease course, no conduction block, and a high incidence (64%) of anti-GM1 antibodies; and a lower motor neuron syndrome with predominant early weakness in proximal muscles and serum antibodies to asialoGMl that do not cross-react with GM1 ganglioside. Pestronk A, Chaudhry V, Feldman EL, Griffin JW, Cornblath DR, Denys EH, Glasberg M, Kuncl RW, Olney RK, Yee WC. Lower motor neuron syndromes defined by patterns of weakness, nerve conduction abnormalities, and high titers of antiglycolipid antibodies. Ann Neurol 1990;27:316-326

There is increasing evidence that lower motor neuron (MN) syndromes are often associated with high titers of antibodies to GM1 and other glycolipids. This association was initially reported in cases of motor neuron disease in which an IgM paraprotein reacted with GM1 and GDlb gangliosides [1,21. More recently, we found polyclonal anti-GM ganglioside antibodies in the serum of patients with a treatable lower M N syndrome 131. High titers of IgM anti-GM1 ganglioside antibodies occurred in 2 patients with a progressive, multifocal motor neuropathy that improved after treatment with cyclophosphamide. Lower titers of anti-GM1 antibodies are common in amyotrophic lateral sclerosis (ALS) 14-61, especially in patients with prominent lower MN changes {4}.

The anti-GM1 antibodies in patients with lower MN syndromes are generally thought to react with the carbohydrate moiety on gangliosides 171. Although they are often polyclonal, the antibodies appear to be remarkably selective, reacting with only a h t e d epitope on the carbohydrate moiety of GM1. To further characterize the specificities of antiglycolipid antibodies in lower M N syndromes, we studied sera from 74 such patients. Antibody specificities were then compared to clinical and electrophysiological data in the same patients. Our results show that antiglycolipid antibodies with several different fine specificities occur in high titer in patients with lower MN syndromes. Sera from individual patients may contain antibodies with one or several of these specificities. Patterns of antiglycolipid

From the Departments of Neurology, "Washington University School of Medicine, St Louis, MO, tJohns Hopkins School of Medicine, Baltimore, MD, KJniversity of Michigan Medical Center, Ann Arbor, MI, §Pacific Medical Center, Sari Francisco, CA, "Henry Ford Hospital, Detroit, MI, 'University of California School of Medicine, San Francisco, CA, and #University of Manitoba School of Medicine, Winnipeg, Manitoba, Canada.

Received Apr 10, 1989, and in revised form JuI 13 and Aug 24. Accepted for publication Aug 25, 1989. Address correspondence to D~ Pestronk, Dept of Neurology, Box 8111, Washington university School of Medicine, 660 s. Euc]id Ave, St Louis, MO 63110,

316 Copyright 0 1990 by the American Neurological Association

activity correlate with the types of clinical and physiological abnormalities in patients with lower MN syndromes.

Methods In October 1987, we began studying the sera of patients with motor system disorders for the presence of antiglycolipid antibodies. For each serum sample we also recorded clinical and epidemiological information and the results of electrophysiological testing. Before serum testing patients were classified as having classic ALS or a purely lower M N syndrome. Of more than 500 patients evaluated in this manner, 74 were identified as having nonfamilial, asymmetrical lower M N syndromes that had been progressive for more than 1 year. All patients had weakness and wasting, with electrophysiological evidence of denervation in one or more extremities that was not attributable to focal nerve or root lesions. Patients with bulbar dysfunction or upper M N signs (e.g., spasticity, 3+/4 or brisker deep tendon reflexes in more than one muscle group or in a single weak muscle group, or Babinski signs) were excluded. No patient had sensory signs or electrophysiological evidence of a peripheral neuropathy involving sensory fibers. For each patient with a lower M N syndrome we compared the clinical pattern of disease, the electrophysiological findings, and the titers and fine specificities of serum antiglycolipid antibodies. All clinical and physiological evaluation was carried out independently of serum testing. The clinical features evaluated included patient age and sex, length of disease, area of involvement at disease onset (proximal vs distal, arms vs legs), and reflexes. We were able to obtain data to characterize more than 80% of our patients in each clinical subcategory. Nerve conduction findings were evaluated for the presence of conduction block, other evidence of myelin changes on motor fibers such as diffuse or focal slowing of nerve conduction velocities, and axonal loss. Twentyfive of the 74 patients with lower MN syndromes had multifocal motor conduction block 13, 81.

washing ( x 8) was performed with 1% BSA without detergent. All sera were tested in duplicate. Serum was examined by adding 100 p1 of dilutions (1:50-1 : 100,000 in PBS with 1% BSA) to wells for 5 hours (overnight for the GGNBSA). The binding of immunoglobulin to glycolipids or sugars was measured using overnight exposure (2 hours for the GGN-BSA) to specific goat anti-human IgM or IgG linked to horseradish peroxidase (Cappel, Durham, NC) in PBS with 1% BSA (working dilution, 1:20,000). Color was developed by adding 100 p1 substrate buffer (0.1 M citrate buffer, p H 4.5, with 0.004% H202 and 0.1% O-phenylenediamine) for 20 to 30 minutes until a standard positive control reached an optical density (OD) of 1.0. Optical density was then read for the test and simultaneously performed control sera at 450 nM. The average OD of normal control sera was subtracted from the OD of test sera at each dilution. For virtually all sera the most linear changes in OD occurred at serial dilutions that produced OD readings between 0.040 and 0.220 above control. However, most positive sera gave readings above this range (>0.300) at a standard dilution of 1: 100. We therefore derived an antibody titer to obtain a more accurate estimate of the comparative levels of reactivity in different sera. All sera were diluted through the range where they gave OD readings of 0.040 to 0.220 above control. These readings were then extrapolated to the value that would be expected at a standard dilution of 1: 100 and multiplied by 1,000. For example, in one test for IgM versus GM1 in serum from Patient 2, dilutions of 1:1,600, 1:3,200, and 1:6,400 gave OD readings in the linear range of 0.201, 0.122, and 0.66, respectively. Using our formula, (0.201 x 16)

+ (0.122 3

x 32)

+ (0.066 x

64)

l,ooo

we calculated a titer for IgM versus GMI of 3,781. We compared the primary OD data at a dilution of 1: 100 with calculated titers of antiglycolipid antibodies. Our results showed that high titers (>800) of serum antiglycolipid antibodies were not well or reliably expressed by the OD value Antibody Assays of the test sera at a 1:100 dilution. Based on these findings Serum was assayed for antibodies to gangliosides, glycolipantibody levels are reported in this paper in terms of titers ids, and carbohydrates using enzyme-linked immunosorbent rather than OD. Serum antibody titers to Galpl-3GalNAc assay (ELISA) methodology [4, 51. Substrates were ob(GGN) were determined by subtracting titers of serum antitained from commercial sources: GMI, GDl,, GTlb, GM3 body to BSA from those to GGN-BSA. In general, a serum gangliosides, asialo-GM1 (GAl), and bovine serum albumin antibody with a high titer of X was detectable (>3 SD over (BSA) from Sigma (St Louis, MO); GM2 from Calbiochem control) in our assays up to a dilution of at least UX. (San Diego, CA); GDlb from Bio-Carb (Lund, Sweden); and Based on initial results of testing sera from more than 300 the glycoconjugate Galp 1-3GalNAc~-0-2-(2-carbomethoxy- patients {4], we designated titers of IgM anti-GM1 ganglioethyl-thio) ethyl-BSA (GGN-BSA) from Pierce (Rockford, side antibodies greater than 350 units as high positive values. IL). Substrates were attached to wells of microtiter plates by High titers were more than 12 standard deviations above the 2 methods. For gangliosides, 400 ng in 50 p1 of methanol average of a panel of 10 normal control serum samples and was added to wells and evaporated to dryness. For GGNover 8 times greater than the titer of 40 units that we desigBSA and BSA, 500 ng in 100 p1 of 0.01 M phosphatenated as positive in our previous study of patients with ALS buffered saline (PBS), p H 7.2, with 0.15 M NaCl was added [4]. In normal control patients none of 41 serum samples had IgM anti-GM1 titers greater than 350. In disease control to wells and incubated overnight at 4°C. Any remaining patients without lower M N syndromes, 11% (19 of 175) had binding sites were blocked with 100 p1 of 5% normal goat anti-GM1 titers greater than 350; nearly all of these had serum in 1% BSA in PBS overnight at 4°C. Plates of protein disorders generally considered to be autoimmune in origin glycoconjugates but not of glycolipids were then washed 10 [4}. High values for other antibodies included IgG versus times with 1% BSA and 0.05% Tween-20 in PBS. GM1 2 500 units, IgM versus GA1 2 600, IgG versus GA1 Subsequent steps were performed at 4°C. Between steps,

Pestronk et al: Lower Motor Neuron Syndromes 317

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We conducted blocking experiments to obtain information about the cross-reactivity of anti-GM1 sera with other gangliosides. To test for cross-reactivity, antibodies were blocked with GM1 or GAI before measurement in ELISA assays. Sera were first diluted to between 1:100 and 1:2,000. Varying amounts of GM1 ganglioside (up to 16 pg000 p1) or GAl (up to 20 &lo0 p1) were added to the serum and incubated overnight at 4°C. Residual IgM binding activity to different gangliosides was then determined by ELISA and compared to titers measured without GMI or GA1 blockade.

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not studied further. No relationship was found between the presence of high-titer antiglycolipid antibodies and levels of total IgM or IgG except in patients with evidence of monoclonal IgM serum proteins in whom total IgM was sometimes elevated.

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F i g 1 . l g M antibody titers versus G M l and GAI in patients with lower motor neuron ( M N ) syndromes or chronic injammatory hmyelinating polyneuropathy (ClDP) and in n o m l subjects. Those with M N I1 and M N 111 had no conduction block but had D > P and P > D weakness, respectively. Data from 3 lower M N syndrome patients (Patients 28, 35, and 49) who could not be subclassifiedare not included. These patients had IgM antibody titers vwsus GMI of 30, 831, and 799 and antibody titers versus GAI of 187, 1,520, and 1,093, respectively. Note that high titers ofantibodies versus GMI and GAI are common in lower M N syndromepatients and rare i n ClDP and normal subjects. Anti-GMI antibodies are common only in multifocal motor neuropathy ( M M N ) and M N I1 patients. AntiGAI antibodies occur with approximately equal frequeny in all 3 lower M N syndromes. (D = distal; P = proximal.)

500, IgM versus GGN 2 850, and IgG versus G G N 2 500. In each instance none of 22 normal control subjects and less than 10% of 89 control subjects with myasthenia gravis (54 patients), chronic inflammatory demyelinating polyneuropathy (CIDP, 24 patients), or systemic lupus (11 patients) had antibody titers above the threshold high value. Figure 1 illustrates data from normal control subjects and control subjects with CIDP for IgM versus GM1 and GAI. In preliminary studies, IgA class antibodies to the tested glycolipids were not detected in a series of patients with lower MN syndromes and control subjects. Thus, they were 2

318 Annals of Neurology Vol 27 N o 3 March 1990

Results During the evaluation of a series of patients with slowly progressive lower MN syndromes, it appeared that the distribution of weakness in most patients, especially in the early stages of disease, could be divided into 2 broad syndromes. In some patients weakness remained largely confined to distal (D) muscles for a long period of time (months to years). In others proximal (P) muscles were predominantly affected at early stages. In this study we compared these clinical patterns of weakness with the results of nerve conduction and antibody testing.

Nerve Conduction Testing In our series 25 patients were found to have evidence of selective motor conduction block in one or more nerves at locations other than common sites of entrapment (Table 1). Thirty-two percent (8 of 25) were female. Sixty-eight percent (17 of 25) were age 45 or younger at the onset of disease. Of the patients with conduction block who had selective involvement, all had more weakness in distal muscles than in proximal muscles (D > P). The hands were predominantly involved in 21 and the feet and ankles in 2. Reflex loss outside the area of clinical weakness occurred at some time during the disease course in 59% of patients with conduction block. The reflex loss was generalized in some patients and occurred only at the ankles in others. There were 49 patients without motor conduction block. Forty-two had evidence only of axonal loss. Seven (Patients 3, 11, 14, 28, 19, 30, 52) had slowing of some motor conduction velocities indicative of motor axon demyelination. Forty-six of the patients without motor conduction block could be classified according to their clinical pattern of weakness. Twenty-eight had a D > P pattern of weakness (Table 2). Among these patients 36% (10 of 28) were female.

Table 1 . h e r Motor Neuron Syndrome Patients with Conduction Block Serum Antibodiesqb

Clinical Syndrome Length of Anti-GM1 Antibody Disease (yr) Reflexes Cross-Reactivityd

Patient

Age and SexC Weakness

1 2 4 5 6 9 13

51, M 43, F 30, M 24, M 63, F 33, M 32, M

D>P,H D>P,H D>P,H D>P,H D > P, H Arms D>P,F

3 2 3 4 10

15 16 17 19 24 26 31 32 33 39

75, M 48, M 69, M 32, M 62, M 48, F 58, M 34, M 63, M 29, F

D > P, H D>P,F D>P,H D>P,H D>P,H D>P,H D>P,H D>P,H D>P,H Arms

20 8 2

41 46 51 53 54

40, M 30, M 61, M 30, F 47, M

Arms, legs Arms D > P, H Arms, legs D>P,H

10 20 2 3

56 73 74

52, F 51, F 46, F

D > P, H D>P,H D > P,H

10 2 15

GM1 only GM1 only GM1 only

3

6 3 6 2

1,535 3,880 1,490 (I&) 0 458 356 1,570

GAi GM1 only

3,972 1,841 1 14,250' 1,248 (I&) 582 1,400 34,7 80' 275 467 1,345

GM1 only GMl only GM1 only GAi GAl

969 506 (I&) 13,693 707 (I&) 302 (I&)

GM2 GM1 only, GAL GMl only GMI only GA1 GM2 GM1 only

5

Anti-GM1 Titer

GM1 only GM1 only

Other Antiglycolipid Antibodies' (total titer) GM2 (2,980) GAi (9,450)

GA1 (1,350) GM2 (676)

GGN (718) GA1 (1,201) GA1 (13,125) -g

GM2 (1,657) GGN (I@) (1,590) GGN (2,035) GA1 (20,325) GA1 (1,6.20) GGN (1,184)

67 1 389 146

"Antibodies to GMI, GAI, and GM2 were IgM unless otherwise noted. GGN antibodies were IgG unless noted. Numbers in parentheses indicate antibody titer. Note that 84% of patients have high titers of and-GM, antibodies. bAntibody titers: sera with a hgh titer of X units were generally significantlyabove background at a dilution of X. Values 2 350 units for IgM versus GM1 and 2 500-850 (see Methods section) for other antibodies were considered high positive. All antibody titers 2 350 units are listed. 'At time of serum testing. d G A ~= anti-GM1 antibody cross-reactive with GA1, Galpl-3GalNAc (GGN) and, in some cases, with GDlb; GM2 = anti-GM1 antibody cross-reactive with GM2; GM1 only = anti-GM1 antibody not cross-reactive with GA1, GM2. or GGN. eGAl = anti-GAl antibody not cross-reactive with GM,; GM2 = anti-GM2 antibody not cross-reactive with GM,; G G N = anti-Galpl3GalNAc antibody not cross-reactive with GM, or GA1. 'Monoclonal IgM paraprotein detected by immunofutation. =No titer of IgG or IgM antibodies to GMI, GA,, GM2, or GGN 2 350.

D = distal; P = proximal; H = hands; F = feet; area of weakness.

+

= present outside the area of weakness; - = absent at one or more locations outside the

Fifty-four percent (14 of 26) were age 45 or younger at the onset of weakness. The hands were predominantly involved in 17 and the legs in 11. Reflex loss outside the area of clinical weakness occurred in 48% (12 of 25). Eighteen patients had a P > D pattern of weakness (Table 3). None had conduction block. Eleven percent ( 2 of 18) were female. Twenty-eight percent (5 of 18) were age 45 or younger at disease onset. The arms were predominantly affected in 15 patients and

the legs in 3. Reflex loss occurred in only 19% ( 3 of

16). Antiganglioside Antih'y Testing

(See Fig 1.) IgM autoantibodies directed against epitopes containing the GGN moiety have been reported to be associated with lower MN syndromes [l-61. We used an ELISA assay to screen the sera from our 74 patients

ANTIBODY TITERS AND PREVALENCE.

Pestronk et d: Lower Motor Neuron Syndromes 319

Table 2. Lower Motor Neuron Syndrome Patients with D > P Weakness and No Conduction Block Serum Antibodies" Clinical Syndrome Patient

Age and Sexb Weakness

3 10 14 20 22 27 29

37, M 31, M 29, M 66, F 50, M 36, F 40, M

A A L

30 34 37 38 40 42 43 45 47 50 52 55 57 58 63 65 66 68 70 71 72

54, M 25, M 72, F M 70, M 32, M 68, F 45, M

L A A A A

F 64, F 66, F 67, M 54, F 44, M 68, M

76, F 33, M 25, M 28, M 61, F 58, M

L L A A

L L A L L A A L A L A A A A A L

Length of Disease (yr) Reflexes 5 3 4 7 5 5

Anti-GM1 Antibody Cross-Reactivity

2,875' 0 0 37 1 680 (I&) 429 0

GAI

GM1 only GM1 only GM1 only

9 2

6,742 68 1 648 688 89,400' 0 3,010 455 368 7 2,600' 739 701 480 0 65 5 0 0 0 376 0

GAI GA I GMI only GMI only GM1 only

10

5 5 10 2 2 1

GA 1 GMI only GM2 GA I GM1 only GM1 only GA1

1 7 15 6 3 5 2 8 5 2 2 25

Other Antiglycolipid Antibodies Anti-GM1 Titerb (total titer)

GAI

GM1 only

GAI (4,885) -d

GAI (450) GA1 (1,46 1) G G N (1,144) GA1 (1,830) GGN (621) GAI (433)

GA1 (639)

-d GM2 (2,375)

GA1 (10,225) GA1 (801) GM2 (382)

-d -d GGN(1gM) (737)

0

"Antibody titers are abbreviated as in Table 1. Note that a majority of patients have anti-GM1 antibodies. bAt time of serum testing. 'Monoclonal IgM paraprotein detected by immunofixation. dNo titer of IgG or IgM antibodies to GM,, GA,, GM2, or GGN t 350.

D = distal; P = proximal; A = arms; L = legs; area of weakness; GGN = Galpl-3GalNAc.

+

= present outside the area of weakness;

with lower MN syndromes for antibody reactivity to three compounds that contained this epitope (Fig 2), including GM 1 ganglioside, asialo-GM1 ganglioside, and the G G N disaccharide linked to BSA. We found that 77% (57 of 74) of sera from these patients had high titers of antibodies that reacted with one or more of these three compounds. Sera from 38 patients (51%) had high titers (2350) of IgM antibody reactivity to GM1 ganglioside (range, 356-114,250 units; mean, 9,646; median, 799; see Fig 1). Sera from 27 patients (36%) had high titers (2600) of IgM antibody reactivity to GA1 (range, 629-83,100; mean, 6,293; 320 Annals of Neurology Vol 27

N o 3 March 1990

- =

absent at one or more locations outside the

median, 1,461; see Fig 1). The prevalence of high-titer IgM antibodies to GM1 and to GAI was significantly greater ( p < 0.0005) in patients with lower MN syndromes than in normal subjects (see Fig 1);in a control group with CIDP, an immune-mediated neuropathy and a seemingly closely related disorder (see Fig 1)19, 101; or in a group of 65 patients with other nonneural immune disorders (data not shown) C41. Some lower MN syndrome sera without IgM reactivity to GM1, GA1, or G G N had high titers of IgG directed against these compounds. Nine sera, without high IgM anti-GM1 reactivity, had high titers of IgG

Table 3. Lower Motor Neuron Syndrome Patients with P > D Weakness and No Conduction Block

Clinical Syndrome Patient

Age and Sexb

Weakness

Length of Disease (yr)

7 8 11 12 18 21 23 25 36 44 48 59 60 61 62 64 67 69

56, M 53, M 74, M 58, M 60, M 46, M 56, M 66, M 61, M 45, M 44, M 37, M 44, M 50, M 63, F 53, M 68, M 54, F

A L L A A A A A A A A A A A A A A L

2 5 20 6 3 8 7 2 8 2 7 13 2 3 1 4 1 3

Serum Antibodies" Reflexes

Reactive Pattern

Titer 650 1,002 876 1,479 2,115; 1,041 629

483; 540 1,106 770 382 893 1,228 972 473; 392

+

"Antibody titers are abbreviated as in Table 1. GM1 + GA1 = anti-GM, antibody cross-reactive with GA,. Note that few patients have high titers of anti-GM1 antibodies. However, 50% have high titers of antibodies to GA, or the Galpl-3GalNAc (GGN) disaccharide. bAt time of serum testing. 'No titer of IgG or IgM antibodies to GM,, GA1, GM2, or G G N 2 350. D = distal; P = proximal; A = arms; L = legs; + = present outside the area of weakness; - = absent at one or more locations outside the area of weakness.

anti-GM1 antibodies (range, 506-1,490; mean, 934). Five sera without high IgM anti-GAl or any anti-GM1 activity had high titers of IgG anti-GA1 antibodies (range, 508-1,106; mean, 857). Finally, in 3 sera the only high-titer antibodies detected were IgG versus GGN (titers = 540, 621, and 972). Monoclonal antibodies were detected in 7 lower M N syndrome sera. In all 6 in which the paraprotein was IgM (lambda in 5 and kappa in l), high titers of anti-GM1 antibodies were detected (see Tables 1 and 2). One serum sample with an IgG kappa paraprotein (Patient 7 1 ) had no detectable antibody reactivity to GM1, GA1, or G G N . We next determined antibody reactivity in lower M N syndrome sera to gangliosides with different degrees of structural similarity to GMI and GA1, including GM2, GM3, GDI,, GDlb, and GTlb gangliosides. We found that lower M N syndrome sera with high levels of antiganglioside activity most often had antibodies with reactivity to only 1 or 2 gangliosides (Tables 1-4). Four common limited patterns were seen. The most frequent was selective reactivity to GM1 alone (see Tables 1-3). Another common pattern was reactivity to GM1 and GAI (see

Structure

ound

Gal/31-3GalNAcfil-4Ga~1-4Glc~1-1'Ceramide 3

GM1

I

Neu5Aca2 GAL

Galfi1-3GalNAcfil-4Gal/31-4Glcfil-1'Ceramide

GM2

GalNAcfi1-4Gal.,91-4Glc~l-1'Ceramide 3

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Neu5Aca2 ~~

Fig 2. Common ant,igenic targets in lower M N syndrome patientJ. Strur+,,eor," ,f, G M I ganglioside, asialo-GMl ganglioside l G M 2 ganglioside. ( G A I Aan6 Ll_r

GLYCOLIPID REACTIVITIES.

Tables 1-3 and example 1 in Table 4). Some of these latter antibodies also reacted with GDlb and GGN, but reactivity to the other glycolipids studied was generally low or absent. Other sera had antibodies that bound only to GM1 and GM2 (see Tables 1-3 and example 2 in Table 4) or to GA1 alone (see Tables 1-3). Serum cross-reactivity was often broader than for individual antibodies. A common pattern was binding to GMI, GM2, and GA1 (Table 4). Pestronk et al: Lower Motor Neuron Syndromes 321

Table 4. Blocking Experimentsa

Titer

Example 1 Example 2 Example 3

Anti-GM1 3,880 Reduction GMi 97% Reduction GAi 93% Anti-GAl 9,450 Reduction by GMi 70% Reduction bv, GAi 96% Anti-GMz 0 Reduction by GM1 Reduction by GAT

1,535 99% 2% 0

1,345 100% 15% 535 0%

2,980 50% 4%

78% 1,657 2% 7%

'Comparison of the ability of GM1 ganglioside (16 pgl100 p1) and GAI glycolipid (20 pgl100 pl) to block serum antibodies versus GM, ganglioside, versus asialo-GM, ganglioside (GA,), and versus GM2 ganglioside. In example 1 GM1 blocked almost all anti-GM, activity and a similar amount of GA1 activity. GA, also blocked both GM, and GA1 activity. In example 2 GM1 blocked both GM, and GMZ antibody activity. GA, had no effect on either. In example 3 GMI blockade eliminated a large proportion of anti-GM, reactivity but resulted in no change in anti-GA, activity. GA1 blockade reduced anti-GA, activity without significant effects on anti-GM1 or GM2.

GM1 BLOCKING EXPERIMENTS. We have previously found that the antiganglioside antibodies in patients with lower MN syndromes are generally polyclonal [5]. A finding that polyclonal IgM in a serum binds to several gangliosides could reflect several different underlying properties of the antibodies. Individual antibodies could cross-react generally with all of the gangliosides. Alternatively, the antibodies reacting with each ganglioside could be different. A combination of these possibilities might also occur. We tested the cross-reactivities of the antiganglioside antibodies in our patients with lower M N syndromes by comparing the relative ability of pure GM1 or GA1 to block antibody activity to GM1, GA1, and GM2. Data from illustrative examples are given in Table 4. In some sera with antibodies to GM1 and another ganglioside, there was cross-reactivity of individual antibodies to both. In example 1 (Patient 2 ) serum IgM reacted with GM1 and GA1. There was also reactivity to GDlb and G G N but not to the other gangliosides tested. After blocking with GMl ganglioside or with GA1, the titers of anti-GM1 and anti-GAl antibodies were reduced by similar magnitudes. This result suggests that most of the anti-GM1 antibodies crossreacted with GAl. However, the titer of anti-GM1 antibodies was lower than the anti-GA1 titer. Also, some of the anti-GAl antibodies were not blocked by GMI. Thus, some of the anti-GAl antibodies probably did not cross-react with GMl. Example 2 (Patient 1) provides data from a serum with IgM anti-GM1 cross-reactivity against GM2. Blocking with an excess of GM1 eliminated anti-GM1 activity and reduced anti-GM2 titers by a similar num-

322 Annals of Neurology Vol 27

ber of units. There was also excess anti-GM2 activity that was not blocked by GM1. In example 3 (Patient 39) there was serum antibodv reactivity Lainst GM1, GM2, and GA1. Howeve;, there was no apparent cross-reactivity of anti-GM1 antibodies with either GM2 or GA1. Blocking with GMl reduced titers of anti-GM1 activity but produced no change in anti-GM2 and anti-GAl antibody titers. Blocking with GA1 reduced titers against GA1 but not against GM1 or GM2.

No 3 March 1990

COMPARATIVE ANTIBODY TITERS. We conducted further tests for evidence of IgM reactivity to the GGN epitope in the sera of patients with lower MN syndromes by comparing titers of antibodies to GM1 and GA1 with those to GGN. We found a significant correlation (Y = 0.83, p < 0.001) between the titer of IgM against GGN and the lower of the two titers to GM1 or GAl. This suggests that, in many patients with high titers of antibody to both GM1 and GA1, G G N is a major part of the reactive epitope. However, in a few patients with high titers of antibodies to both GMl and GA1, little reactivity to GGN was found. These sera were also generally distinguished by their low titers of antibodies to GDlb and low levels of blocking of antiGAl titers by GM1 and of anti-GM1 titers by GA1 (e.g., example 3, Table 4). This pattern of results suggests the occurrence of two different IgM antiglycolipid antibodies in the serum of such patients, one reacting to GM1 and the other to GA1.

Antibody Reactivity Versus Clinical and Physiological Patterns Most (21 of 25, 84%) patients with lower M N syndromes and physiological evidence of conduction block had high titers of serum anti-GM1 ganglioside antibody titers (see Table 1 and Fig 1). Seventeen of the 21 positive sera had high titers of IgM class anti-GM1 antibodies. In 4 others the high anti-GM1 titers were only of the IgG class. A majority of the lower M N syndrome patients with the highest titers of anti-GM1 antibodies (e.g., 12 of the 17 with titers > 1,200 units) had motor conduction block. The median value of the high-titer sera was 1,345 units. Three patterns of anti-GM1 specificity were found: versus GM1 alone, versus GM1 and GA1, and versus GMl and GM2. The most selective anti-GM1 antibody, which did not react against any of the other gangliosides tested, occurred most frequently (14 of 2 1 high-titer sera). Several patients' sera also had evidence of antibodies that reacted with GAl alone; however, in all but 2 cases (Patients 5 and 54), high titers of anti-GM1 antibodies were also present. PATIENTS WITH CONDUCTION BLOCK.

D

>

P WEAKNESS AND NO CONDUCTION BLOCK.

High titers of anti-GM 1 ganglioside antibodies were

also common (18 of 28,64%) in lower MN syndrome patients without conduction block who had a D > P pattern of weakness (see Table 2 and Fig 1). In 17 of the 18 high-titer anti-GM1 sera, antibodies of the IgM class were seen. The remaining 5 patients with very high IgM anti-GM1 titers (>1,200 units) were in this group. One patient had high titers of IgG, but not IgM, anti-GM1. The median value of the high-titer anti-GM1 sera was 688 units. The anti-GM1 antibody with the most selective reactivity (against GM1 alone) was found in 10 high-titer sera in the D > P lower M N syndrome group. Anti-GM1 antibodies that also reacted with GA1 were found in another 7 sera. In 2 other patients, high titers of other antibodies without anti-GM1 activity were detected. These reacted selectively with GA1 in one and the G G N disaccharide in the other. P > D WEAKNESS AND N O CONDUCTION BLOCK. In lower MN syndrome patients with a P > D pattern of weakness, high titers of antibodies to GMl were much less frequent that in the previous 2 groups ( p < 0.001; see Table 3 and Fig 1). Only 11% (2 of 18) had high titers of IgM anti-GM1 antibodies. One other patient had anti-GM1 antibodies of only the IgG class. Antibodies to nonsialated carbohydrate epitopes were more common. Ten patients (56%) had a high titer of anti-GAl antibody reactivity, 6 of IgM class and 4 with only IgG versus GA1. The frequency of high-titer IgM anti-GAl antibodies (6 of 18, 33%) in the P > D lower MN syndrome group was significantly higher than that found in control subjects with CIDP ( p < 0.002) or in normal control subjects ( p < 0.001; see Fig 1). Seven patients (39%) had a selective overall pattern of high antibody (IgM or IgG) reactivity that was directed against GA1 but not GM1. This selective pattern of reactivity was significantly more common in the P > D lower M N syndrome group than in the previous 2 groups (6%; p < 0.001). Two lower MN syndrome patients with a P > D pattern of weakness,

without high titers of IgM or IgG reactivity to any of the glycolipids tested, had high levels of IgG against the GGN disaccharide. Discussion Our results show that antibodies to GM1, to similar glycolipids, and to carbohydrate epitopes on GMl and GA1 are common in sera of patients with lower MN syndromes (see Fig 1). Individual sera generally showed limited and selective patterns of reactivity, indicating that specific carbohydrate epitopes are the targets of the antibodies in lower M N syndrome patients. Some features of the antibody specificity correlate with aspects of the clinical syndrome in lower MN syndrome patients.

Clinical Cowekztions The clinical features of our patients with lower MN syndromes most closely resemble those described by many authors since Duchenne as the progressive spinal muscular atrophy form of motor neuron disease in adults Ell-151. However, it appears that acquired lower M N syndromes can be subdivided into two or more categories based on clinical, physiological, and immunological criteria. The most distinct of these are seen in patients with multifocal motor neuropathy t37. We previously described 2 such patients with a threepart syndrome of progressive lower MN dysfunction, conduction block selectively on motor axons, and high titers of serum IgM antibodies to GM1 ganghoside. Descriptions of several other similar patients with lower MN syndromes have only included data that documented motor conduction block [lb, 171 or antiGM1 antibodies {I, 27. In the present series of lower MN syndrome patients, 25 had a syndrome of asymmetrical motor weakness with physiological evidence of motor conduction block in addition to axonal loss (Tables 1 and 5). Eighty-four percent (2 1

MULTIFOCAL MOTOR NEUROPATHY.

Tabk 5 . h e r Motor Neuron Syndromesa Variable

Multifocal Motor Neuropathy

Distal Lower MN Syndrome

Proximal Lower MN Syndrome

Pattern of weakness Area of weakness at onset (arms :legs) Age of onset Males :females Nerve conduction High-titer antibody reactivity

D > P 6.7 : 1 68% 5 45 yr 2.1 : 1 AL, CB GMI (84%)

D>P 1.5:l 54% 5 45 yr 1.8: 1 AL, DM (2 1 %) GMi (64%)

P > D 5:l 28% 5 45 yr 8 :1 AL, DM (6%) GAI and G G N (50%)

"Patients with multifocal motor neuropathy were selected for the presence of conduction block. Those with distal or proximal lower MN syndromes had no conduction block but had D > P or P > D weakness, respectively.

D = distal; P = proximal; AL = axon loss; CB = conduction block; DM = features compatible with motor demyelination, e.g., slow nerve conduction velocities or prolonged distal or F-wave latencies; GM, = GM1 ganglioside; GA1 = asialo-GM, ganghoside; GGN = G@l3GalNAc disaccharide.

Pestronk et al: Lower Motor Neuron Syndromes 323

of 25) had high titers of anti-GM1 antibodies. Most patients with multifocal motor neuropathy in this series had a syndrome of weakness that began at a relatively young age ( D weakness (M. Glasberg, unpublished observations). Other such patients may be found using improved methods for detecting proximal motor conduction block. Our initial experience with treatment of patients with multifocal motor neuropathy suggests that they often have a characteristic response to immunosuppressive medications. Seven have been treated with high-dose corticosteroid therapy. Most had an exacerbation of weakness shortly after initiation of the steroid treatment. Only 1 showed sustained improvement. In contrast, at least 5 patients who have been treated with cyclophosphamide have regained motor function when their titers of anti-GM1 antibodies were reduced by 75% or more. Thus, identification of the multifocal motor neuropathy syndrome is important because many such patients can improve after immunosuppressive therapy. However, because of the potential serious side effects of cyclophosphamide, treatment should probably be reserved for those patients with severe, disabling, or clearly progressive weakness in addition to the typical physiological and immunological features of the syndrome.

> D LOWER MN SYNDROME. Another subgroup of 18 lower MN syndrome patients in this series differed from patients with multifocal motor neuropathy based on clinical, nerve conduction, and immunological criteria (see Tables 3 and 5, Fig 1). Clinically, the disease was especially common in men and onset was often relatively late. On examination, the weakness was predominant in proximal muscles and often remained confined mainly to 1 or 2 extremities over periods of at least 3 to 5 years. Conduction block in the P > D lower MN syndrome was unusual. Serum testing showed evidence of high titers of antiglycolipid antibodies in most lower MN syndrome patients with the P > D pattern of weakness. However, the most common pattern of high-titer serum antibody reactivity was to glycolipids or sugars that did not contain sialic acid. Based on these characteristics, patients with a predominantly proximal asymmetrical lower M N disorder, physiological evidence of axonopathy without conduction block, and antibodies to glycolipid or carbohydrate epitopes have a clinically recognizable syndrome (see Table 5). The frequency of antiganglioside or carbohydrate antibodies suggests that this syndrome, like multifocal motor neuropathy, may be asP

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sociated with autoimmune mechanisms. There is, however, no information about the response of this syndrome to treatment, and the use of immunosuppressive therapy for such patients should be considered experimental. D > P LOWER M N SYNDROME WITHOUT CONDUCTION BLOCK. The remaining patients in OLE series

formed a group that was intermediate between the first two (see Tables 2 and 5, Fig 1). However, based on clinical and immunological criteria, the lower MN syndrome patients with a D > P pattern of weakness without conduction block seem most similar to those with the full syndrome of multifocal motor neuropathy. Both groups had weakness in distal muscles that was greater than that in proximal muscles. In addition, high titers of anti-GM ganglioside antibodies were common in these groups (18 164%) of 28 patients with the D > P lower M N syndrome). Further testing and follow-up study will be required to decide whether the absence of conduction block in individual patients indicates a different disease syndrome or merely an inability to detect the physiological changes at the time of testing. In any case the finding of a high titer of antiGM1 antibodies (especially 2 6 0 0 units) in a patient in this group suggests that careful follow-up physiological testing to look for motor conduction block is indicated to identify those with the potentially treatable syndrome, multifocal motor neuropathy. In this study we have not considered patients with classic ALS with prominent or predominant lower M N signs. We have previously shown that anti-GM1 antibodies are common in the sera of such patients [4, 5). However, high titers of anti-GM1 antibodies (2350 units) are present in only a minority (10%). Further studies are needed to determine whether high-titer patterns of other serum antibodies are associated with the different clinical forms of classic ALS.

OTHER MN SYNDROMES.

Antibody Specificities in M N Patients The results of this and earlier studies 17, 181 suggest that targets of the antibodies in sera from lower MN syndrome patients include carbohydrate moieties (see Fig 2). Antibodies to the carbohydrate moieties of gangliosides can occur as a secondary response to nervous system damage [19) or after immunization with tissue extracts (including neural tissue) or with gangliosides, such as GM1. Antibodies generated in this manner tend to react with a wide spectrum of glycolipid antigens [20, 21). For example, immunization of rats with GMI produces high titers of anti-GM1 antibodies that cross-react with GM2, GM3, GDl,, and GA1, as well as with GM1 (V. Chaudhry, unpublished observations). In contrast to the antibodies generated by immuni-

zation with GM1 or neural tissue, antibodies in individual lower M N syndrome patients tend to react with gangliosides very selectively. The selectivity probably results from the binding of antibodies only to particular epitopes on the carbohydrate moieties of gangliosides. The specificities are illustrated by 3 findings: ( 1 ) The most common anti-GM1 antibodies in patients with multifocal motor neuropathy and in D > P lower M N syndrome patients did not cross-react with any of the other glycolipids tested. This remarkably specific pattern of polyclonal antibody reactivity has previously been described only in studies of monoclonal antiGM1 antibodies (22, 23). ( 2 ) Anti-GAl antibodies often did not cross-react with glycolipids that contained sialic acid. ( 3 ) Antibodies were identified in sera from lower MN syndrome patients that reacted against the G G N moiety only when it was not part of a glycolipid. Such results suggest that the specificity, as well as the titers, of antibody responses in patients may have diagnostic utility in identifying lower MN syndromes. It is not known whether the lipid structure in GM1 can influence antibody binding. However, analysis of the ganglioside blocking and comparative binding experiments suggests that targets of antibodies of lower MN syndrome patients often include 2 to 4 sugar epitopes. The carbohydrate GGN, the terminal 2 sugars on GM1 ganglioside, is commonly part of the epitope for a serum antibody in most of the lower MN syndrome patients in this series. In some cases the antiGM1 antibodies appear to recognize most of the terminal carbohydrate structure on GM1 (see Fig 2). This antibody specificity is the most common pattern of serum reactivity found in patients with multifocal motor neuropathy (see Table 1). A similar pattern of reactivity has been observed in human and experimental monoclonal antibodies against GM1 123). In other patients G G N may serve as the entire epitope (see Fig 2), either as part of the GM1 molecule or as an isolated carbohydrate moiety C7, 241. G G N occurs naturally in compounds other than gangliosides. It is known as the “T antigen” 1251, forming a major part of the carbohydrate moieties on glycophorin A, an erythrocyte glycoprotein that is a determinant of the M and N blood groups 126, 271. G G N is also present on immunoglobulins 128) and on neural glycoproteins 129, 30). It is known to be immunogenic 131, 32) but is normally hidden from immune attack by a sialic acid attached to each sugar [26, 331. It is not known whether such self-antigens could play a role in the generation of anti-GM1 antibodies in lower MN syndrome patients. The relationship between antiglycolipid activity and damage to motor axons also remains uncertain. We conclude that measurement of antiglycolipid antibodies may be clinically useful in defining and treating clinical subgroups of lower MN syndromes.

Quantitation of antiganglioside antibodies provides a useful guide to the treatment of one subgroup of lower MN syndromes, multifocal motor neuropathy [ 3). Measurement of anti-GM 1 ganglioside antibodies and their specificities also provides a diagnostic clue to the presence of this treatable disorder. Another lower MN syndrome, with a P > D pattern of weakness, is generally associated with a different pattern of antibodies than is found in multifocal motor neuropathy. Further study of the general and fine specificities of the antibodies in lower MN syndromes and their crossreaction with other glycolipids and glycoproteins may provide clues to the events that stimulate these antibodies and to the pathogenesis of lower MN syndromes.

Supported by grants 1 RO1 NS26616,l RO1 AG07438, and lPOl NS22849 from the National Institutes of Health, the Muscular Dystrophy Association, the Amyotrophic Lateral Sclerosis Association, and the Jay Slotkin Fund for Neuromuscular Research. We are grateful to Christine F. Salemi for assistance in the preparation of the manuscript and to Brad Winslow and Robert N. Adams for technical assistance.

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