Anti-sulfatide IgM antibodies in peripheral neuropathy: to test or not to test? C. Giannotta, D. Di Pietro, F. Gallia and E. Nobile-Orazio
antibodies, autoimmunity, IgM, neuropathy, sulfatide Received 20 August 2014 Accepted 28 November 2014 European Journal of Neurology 2015, 22: 879–882 doi:10.1111/ene.12658
Background and purpose: Anti-sulfatide immunoglobulin M (IgM) antibodies have been associated with diﬀerent forms of neuropathies but their diagnostic role in neuropathy remains unclear. Methods: The clinical association of increased titers of anti-sulfatide IgM antibodies in 570 patients with neuropathy and related disorders examined in our laboratory since 2004 was reviewed. Sera were tested by enzyme-linked immunosorbent assay at the initial serum dilution of 1:32 000 and titrated by serial two-fold dilution. In all positive patients IgM antibodies to myelinassociated glycoprotein (MAG) were also measured by western blot. Results: High titers of anti-sulfatide antibodies were found in 39 patients including 33 (85%) who also had anti-MAG IgM. Six patients did not have anti-MAG IgM including ﬁve in whom moderately increased anti-sulfatide titers were associated with diﬀerent forms of neuropathy. One patient with a demyelinating neuropathy and IgM monoclonal gammopathy had markedly increased anti-sulfatide titers (1:256 000). Conclusions: Increased titers of anti-sulfatide IgM antibodies are not infrequent in patients with neuropathy where they are often associated with a concomitant reactivity to MAG. A selective reactivity to sulfatide, however, is rarely found and is associated with diﬀerent forms of neuropathy limiting its usefulness in the diagnosis of neuropathy.
Introduction Increased titers of immunoglobulin M (IgM) antibodies to sulfatide have often been reported in patients with immune mediated neuropathies  even if their diagnostic role remains controversial [2,3]. These antibodies were originally associated with axonal neuropathy and IgM monoclonal gammopathy  but were subsequently associated with diﬀerent neuropathies including predominantly sensory and often painful axonal neuropathy, sensorimotor demyelinating neuropathy and small ﬁber neuropathy [5–11]. The reactivity was variably associated with IgM monoclonal gammopathy [4–11] and with a concomitant reactivity with myelinCorrespondence: E. Nobile-Orazio, Department of Medical Biotechnology and Translational Medicine, Milan University, Second Neurology, Humanitas Clinical and Research Center, Via Manzoni 56, 20089, Rozzano, Milan, Italy (tel.: +390282242209; fax: +390282242298; e-mail: [email protected]
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associated glycoprotein (MAG) [4,7–11]. These heterogeneous associations have induced some skepticism on their role in neuropathy and were not considered as a highly probable causal factor for the neuropathy in patients with IgM monoclonal gammopathy . The clinical association of anti-sulfatide IgM antibodies in our series of patients with neuropathy was reviewed to verify the possible clinical relevance of testing for these antibodies in patients with neuropathy.
Patients and methods Since 2004 the sera of over 570 patients investigated for neuropathy or related syndromes were examined in our laboratory for the presence of anti-sulfatide IgM antibodies. Only patients clinically evaluated in our institution were included so as to have access to their clinical and electrophysiological data. The diagnosis of the patients who had increased anti-sulfatide titers was
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Second Neurology, Humanitas Clinical and Research Centre, Department of Medical Biotechnology and Translational Medicine, Milan University, Rozzano, Milan, Italy
C. GIANNOTTA ET AL.
reviewed even if the desired data were not always available. In particular, since several patients had been assessed as outpatients, in some of them the conclusions but not the data of the electrophysiological studies performed elsewhere were included in the reports and so the data were not available. The Ethics Committee of Humanitas Clinical and Research Institute, Rozzano, Milan, Italy, approved this retrospective study. All sera were tested for the presence of anti-sulfatide IgM antibodies by enzyme-linked immunosorbent assay (ELISA) following our previously reported procedure . The sera were tested at the initial serum dilution of 1:32 000 and, when positive (absorbance value higher than 0.050 after subtracting the reading obtained from wells coated with bovine serum albumin), were subsequently titrated by serial two-fold dilution until negative. All positive sera were also tested, if not done before, for the presence of a serum IgM monoclonal gammopathy by serum electrophoresis and immuno ﬁxation and for anti-MAG IgM antibodies by immunoblot using human brain myelin (normal value up to 1:3200) . In patients with both reactivities the light chain speciﬁcity of each reactivity was compared by ELISA (anti-sulfatide) and immunoblot (anti-MAG) to determine whether reactivities could be related to the same antibody. The clinical and electrophysiological data of patients with anti-sulfatide and anti-MAG antibodies were compared with those of patients with only anti-MAG IgM to determine if anti-sulfatide reactivity aﬀected the clinical presentation. Statistical analysis was performed by Fisher’s exact test or Student’s t test as appropriate. All statistical analyses were performed with signiﬁcance set at the 5% level and using two-sided tests or two-sided 95% conﬁdence intervals.
Results Increased titers of anti-sulfatide IgM antibodies (1:32 000 or more) were found in 39 of the 570 patients examined (6.8%). In 33 (85%) of them high titers of anti-MAG IgM (1:6400 or more) were also found. Four of these patients had anti-sulfatide titers up to 1:64 000 whilst 19 had titers of 1:128 000 or more. Six patients (15%) only had anti-sulfatide antibodies without anti-MAG reactivity including ﬁve patients with anti-sulfatide IgM up to 1:64 000 and one with titers of 1:256 000. The clinical and electrophysiological features of patients with IgM reactivity to MAG and sulfatide did not diﬀer from those of patients with only antiMAG reactivity and were consistent with a chronic demyelinating neuropathy (Table 1). The only signiﬁ-
cant diﬀerences were a mean younger age at onset of neuropathy symptoms and reduced amplitude of the distal compound muscle action potentials of the median nerve in patients with selective anti-MAG reactivity. In 26 of the 33 patients with both reactivities, these were speciﬁc for the same light chain of IgM, which was kappa in 23 patients, lambda in two patients and both kappa and lambda in one. In the other seven patients, no light chain speciﬁcity of antisulfatide IgM was detected in three patients whilst in four this reactivity was only kappa whilst reactivity to MAG was both kappa and lambda (Table 2). Of the six patients who only had increased antisulfatide IgM titers (15% of positive patients and 1% of total patients), ﬁve had moderately increased anti-
Table 1 Comparison of clinical and electrophysiological data (mean) from patients with neuropathy and both anti-sulfatide and antiMAG IgM antibodies or anti-MAG antibodies only Clinical and electrophysiological data
Anti-MAG and anti-sulfatide antibodies
Patients with available clinical data Age of onset Sex (male/female) IgM monoclonal gammopathy Disease duration (years) Paraesthesia Sensory loss Vibratory loss Tremor Ataxic gait MRC (range 0–60) INCAT (range 0–10) Patients with available electrophysiological data Median CMAP amplitude (mV) Median distal latency (ms) Median MCV (m/s) Peroneal CMAP amplitude (mV) Peroneal distal latency (ms) Peroneal MCV (m/s) Median SAP amplitude (lV) Median SCV (m/s) Sural SAP amplitude (lV) Sural SCV (m/s)
Anti-MAG antibodies only
63.3 (21)a 25/8 20/23
54.8 6/6 9/9
0.02 ns ns
3.1 (17)a 20/22 20/23 18/22 9/23 15/22 56.7 (20)a 2.3 (22)a 17/33 (52%)
5.3 (6)a 7/9 6/9 8/9 3/8 7/9 58.3 2.7 7/12 (58%)
ns ns ns ns ns ns ns ns
8.1 32.9 0.7
11.5 31 1.5
ns ns ns
39 8 42.3
35 4 43.5
ns ns ns
MAG, myelin-associated glycoprotein; MRC, Medical Research Council; INCAT, inﬂammatory neuropathy cause and treatment; CMAP, compound muscle action potential; MCV, motor conduction velocity; SAP, sensory action potential; SCV, sensory conduction velocity. a Patients with data available in parentheses.
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ANTI-SULFATIDE AND NEUROPATHY
Table 2 Comparison of anti-MAG and anti-sulfatide IgM antibody titers and of light chain speciﬁcity of antibody reactivity Anti-MAG
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1:51 200 1:100 000 1:200 000 1:100 000 1:200 000 1:400 000 1:400 000 1:51 200 1:800 000 1:100 000 1:51 200 1:100 000 1:25 600 1:25 600 1:12 800 1:6400 1:200 000 1:6400 1:25 600 1:25 600
21 22 23 24 25 26 27 28
1:200 000 1:25 600 1:51 200 1:12 800 1:12 800 1:12 800 1:51 200 1:100 000
1:12 800 1:12 800
Kappa/ lambda Kappa Kappa Lambda Kappa Kappa Kappa Kappa Kappa Kappa Kappa Kappa Lambda Kappa Kappa Kappa Kappa Lambda Kappa Kappa Kappa/ lambda Kappa Kappa Kappa Lambda Kappa Kappa Kappa Kappa/ lambda Kappa/ lambda Kappa/ lambda Kappa Kappa/ lambda Kappa
Titer 1:1 000 000 1:256 000 1:1 000 000 1:256 000 1:128 000 1:1 000 000 1:1 000 000 1:128 000 1:128 000 1:256 000 1:128 000 1:64 000 1:500 000 1:128 000 1:32 000 1:1 000 000 1:32 000 1:1 000 000 1:32 000 1:1 000 000
1:32 000 1:32 000 1:32 000 1:32 000 1:1 000 000 1:1 000 000 1:1 000 000 1:32 000
Kappa Kappa Lambda Kappa Kappa Kappa Kappa Kappa Kappa Kappa Kappa Lambda Kappa Kappa Kappa Kappa 0 Kappa Kappa Kappa/ lambda Kappa Kappa 0 0 Kappa Kappa Kappa Kappa
1:64 000 1:1 000 000
sulfatide titers including one with minimal distal paraesthesia in one foot and normal nerve conduction studies (1:32 000), one with axonal neuropathy associated with IgG monoclonal gammopathy of undetermined signiﬁcance (MGUS) (1:32 000), one with transthyretin amyloid neuropathy (1:32 000), one with POEMS syndrome (1:32 000) and one with a subacute sensory neuronopathy without evidence of cancer (1:64 000). The only patient with markedly increased anti-sulfatide titers (1:256 000) had a chronic demyelinating neuropathy associated with IgM MGUS. At the time of our evaluation this patient had a 15-year history of neuropathy with paraesthesia, ataxia, tremor, with touch and vibratory
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loss, and distal limb weakness with a Medical Research Council sum score of 48/60 and an inﬂammatory neuropathy cause and treatment score of 7/10. Motor nerve conduction velocity was markedly reduced in the peroneal nerve (20 m/s). No additional data were available from this patient including data on response to therapy. Overall, only one of the 570 examined patients had a selective marked increase of anti-sulfatide IgM antibodies.
Discussion In this study increased titers of anti-sulfatide IgM antibodies were found in 6.8% of the patients with neuropathy or related syndromes examined in our laboratory in the last 10 years. The vast majority of positive patients (85%) also had an IgM monoclonal gammopathy and increased titers of IgM antibodies to MAG. In these patients reactivity to sulfatide was most often restricted to the same light chain of the M-protein and corresponded to that of anti-MAG antibodies suggesting that this reactivity was related to the same antibodies even if this was not conﬁrmed by absorption studies. In addition, the clinical and electrophysiological features of patients with both anti-sulfatide and anti-MAG antibodies did not diﬀer from those of patients with only anti-MAG antibodies suggesting that reactivity to sulfatide did not aﬀect the clinical phenotype compared to patients with antiMAG IgM reactivity only. Only six patients (1%) had a selective increase of anti-sulfatide antibodies. Five of them had moderately high titers that were associated with diﬀerent causes of the neuropathy whilst one had markedly increased titers that were associated with a chronic demyelinating neuropathy and IgM MGUS. Suﬃcient data were not available from this patient to establish whether this reactivity had a role in the neuropathy and particularly whether the patient improved after immune therapy. The myelin localization of sulfatide, however, may support the hypothesis that in this patient markedly increased antibodies to a myelin antigen might have contributed to the neuropathy. In any case, from this study it appears that testing for anti-sulfatide IgM only rarely provides information that may have some implications on the diagnostic assessment of patients with neuropathy, limiting its usefulness in the diagnosis of neuropathy.
Acknowledgement The study was supported by a research grant from Humanitas Research Institute. No external funding was used for the study.
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Disclosure of conflicts of interest Eduardo Nobile-Orazio serves in the Steering or Advisory Board of Baxter, Italy, CSL Behring, Italy, Kedrion, Italy, and Novartis, Switzerland. He received honoraria for lecturing from Baxter, CSL Behring and Kedrion and travel support to attend scientiﬁc meetings from Baxter and Kedrion. Francesca Gallia received travel support to attend scientiﬁc meetings from CSL Behring, Italy, and Kedrion, Italy. Claudia Giannotta and Davide di Pietro have no conﬂict of interest. All compensations and support are outside the submitted work.
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