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Verena Kraus, MD* Rajneesh Srivastava* Sudhakar Reddy Kalluri Ulrich Seidel, MD Markus Schuelke, MD Mareike Schimmel, MD Kevin Rostasy, MD Steffen Leiz, MD Stuart Hosie, MD Verena Grummel Bernhard Hemmer, MD

Potassium channel KIR4.1-specific antibodies in children with acquired demyelinating CNS disease ABSTRACT

Objective: A serum antibody against the inward rectifying potassium channel KIR4.1 (KIR4.1-IgG) was recently discovered, which is found in almost half of adult patients with multiple sclerosis. We investigated the prevalence of KIR4.1-IgG in children with acquired demyelinating disease (ADD) of the CNS. We also compared antibody responses to KIR4.1 and myelin oligodendrocyte glycoproteins (MOGs), another potential autoantigen in childhood ADDs.

Methods: We measured KIR4.1-IgG by ELISA in children with ADD (n 5 47), other neurologic disease (n 5 22), and autoimmune disease (n 5 22), and in healthy controls (HCs) (n 5 18). One hundred six samples were also measured by capture ELISA. Binding of KIR4.1-IgG human subcortical white matter was analyzed by immunofluorescence. Anti-MOG antibodies were measured using a cell-based assay. Results: KIR4.1-IgG titers were significantly higher in children with ADD compared with all control

Correspondence to Dr. Hemmer: [email protected]

groups by ELISA and capture ELISA (p , 0.0001, p , 0.0001). Overall, 27 of 47 patients with ADD (57.45%) but none of the 62 with other neurologic disease or autoimmune disease or the HCs (0%) were KIR4.1-IgG antibody positive by ELISA. Sera containing KIR4.1-IgG stained glial cells in brain tissue sections. No correlation among KIR4.1-IgG, age, or MOG-IgG was observed in the ADD group.

Conclusion: Serum antibodies to KIR4.1 are found in the majority of children with ADD but not in children with other diseases or in HCs. These findings suggest that KIR4.1 is an important target of autoantibodies in childhood ADD. Neurology® 2014;82:470–473 GLOSSARY ADD 5 acquired demyelinating disease; ADEM 5 acute disseminated encephalomyelitis; CIS 5 clinically isolated syndrome; DEM 5 demyelinating encephalomyelitis; HC 5 healthy control; IgG 5 immunoglobulin G; MDEM 5 multiphasic demyelinating encephalomyelitis; MOG 5 myelin oligodendrocyte glycoprotein; MS 5 multiple sclerosis; OD 5 optical density; OND 5 other neurologic disease.

Differences and similarities of multiple sclerosis (MS) in adults and children have been a focus of research in recent years.1,2 Clinically, MS in children is more likely to show a polysymptomatic onset, and sometimes initial symptoms include acute disseminated encephalomyelitis (ADEM) characterized by encephalopathy. Although ADEM and MS are considered autoimmune diseases of the CNS, the targets of the immune response are still largely uncertain. Several studies have demonstrated that in childhood demyelinating diseases, autoantibodies to myelin oligodendrocyte glycoprotein (MOG) are present in high titers, especially in younger children, while they are only rarely observed in adult patients with MS.3,4 Recently, an antibody to the inward rectifying potassium channel KIR 4.1 was identified in adult patients with MS.5 The aims of the current study were to address the occurrence of antibodies to KIR4.1 (KIR4.1-IgG) in children with acquired demyelinating disease (ADD) and the relation of autoantibodies to KIR4.1 and MOG. Supplemental data at www.neurology.org *These authors contributed equally to this work. From the Departments of Pediatrics (V.K.) and Neurology (R.S., S.R.K., V.G., B.H.), Klinikum rechts der Isar, Technische Universität, Munich; Department of Neuropediatrics (U.S., M.S.), Charité Universitätsmedizin, Berlin; Department of Pediatrics (M.S.), Hospital Augsburg, Germany; Department of Neuropediatrics (K.R.), University Hospital Innsbruck, Austria; Department of Pediatrics (S.L.), Hospital Dritter Orden, Munich; Department of Pediatric Surgery (S.H.), Städtisches Klinikum München GmbH, Klinikum Schwabing, Munich; and Munich Cluster for Systems Neurology (SyNergy) (B.H.), Munich, Germany. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article. 470

© 2014 American Academy of Neurology

METHODS Standard protocol approvals, registrations, and patient consents. Patients were recruited at different Departments of Pediatrics and Neuropediatrics in Germany (Technische Universität Munich, Hospital Dritter Orden Munich, Hospital Augsburg, Charité Universitätsmedizin, Berlin) and Austria (University Hospital Innsbruck) from 2009 to 2012. Controls were recruited at the Department of Pediatrics, Technische Universität Munich and the Department of Pediatric Surgery, Städtisches Klinikum München GmbH, Klinikum Schwabing, Munich. Written informed consent was obtained from the parents and adolescents aged 12 to 18 years. The ethics committees of the universities approved the study.

Patient series. In the course of a routine blood check, an additional serum tube was collected. Healthy controls (HCs) underwent venous puncture for anesthesia and operation. Fourteen controls with systemic autoimmune diseases were recruited anonymously. Fortyseven patients with ADD comprising 8 patients with clinically isolated syndrome (CIS), 3 with acute or past demyelinating encephalomyelitis (DEM) including ADEM and multiphasic DEM (MDEM), and 33 with MS were included. MS was diagnosed according to the McDonald criteria. Three patients had isolated optic neuritis (table 1; table e-1 on the Neurology® Web site at www.neurology.org). None of the patients with CIS and MS had clinical or radiologic features suggestive of neuromyelitis optica. Patients with isolated optic neuritis tested negative for aquaporin-4 antibodies by a cell-based assay.6 ELISA to determine KIR4.1-specific antibodies. We used the same methodology to measure KIR4.1-specific antibodies by ELISA as the one used in a previous study.5 HEK cells were transfected with C-terminal his-tagged KIR4.1 by reversed transfection. Cells were lysed and KIR4.1 protein isolated by cobalt bead-based affinity purification (Thermo Scientific, Waltham, MA). Successful purification of oligomeric KIR4.1 protein was validated by a gel filtration column (GE Biosciences, Pittsburgh, PA). The quality of the oligomeric protein, reflecting the posttranslational modifications of KIR4.1 protein expressed in white matter, was determined on 4% to 12% bis-tris gel (Invitrogen, Carlsbad, CA) with reducing and nonreducing conditions along with blue native gel (Invitrogen). Freshly purified protein was used in all assays because KIR4.1 protein was prone to aggregation at 4°C or in freezing/thawing cycles. Sera were diluted 1:100 and blinded to the person performing the assay. All sera and the sera to determine the cutoff were measured in the same assay. None of the samples used in the assays were thawed and refrozen. The coefficients for interassay (3.41% 6 0.86%) and intraassay (0.97% 6 0.36%) variation were within permitted limits of variance. The cutoff value was determined

Table 1

Clinical findings in all patient and control groups

Group

Mean age, y (min–max)

Sex ratio (M/F)

ADD

13.6 (6–18)

1:3.7 (10/37)

OND

9.5 (1–18)

1:1.4 (9/13)

AIa

11.6 (1–16)

1:1.7 (3/5)

HC

8.8 (3–15)

1:1 (9/9)

Abbreviations: ADD 5 acquired demyelinating disease; AI 5 autoimmune disease; HC 5 healthy control; OND 5 other neurologic disease. The mean age (range) and the sex ratio (absolute numbers) of all patient and control groups are shown. a Data of 14 anonymized patients were not available.

in each assay by the optical density (OD) obtained from the same sera of 37 HC donors (mean 1 5 SDs). Therefore, the cutoff varied slightly from assay to assay. Four KIR4.1-IgG–positive sera and KIR4.1-IgG–negative other neurologic disease (OND) sera were analyzed by immunohistochemistry for binding to human brain tissue sections as described previously.5

Capture ELISA to determine KIR4.1-specific antibodies. For sandwich ELISA, KIR4.1 oligomeric protein was also used (see above). Linear titration was performed to determine the optimal amount of monoclonal antibody coating along with sample matrix optimization with standard spiked and recovery assay. For final assay amino link (Thermo Scientific), plates were coated with 50 mL of 1.5 mg monoclonal anti-KIR4.1 antibody (Sigma) per milliliter and left overnight at 4°C with mild shaking. Plates were washed 5 times with washing buffer (0.05% Tween 20 with phosphate-buffered saline) and blocked with 200 mL of sample buffer (0.5% milk in washing buffer) at room temperature for 1 hour. Blocked plates were incubated with 50 mL of purified KIR4.1 (200 ng) in sample buffer for 2 hours at room temperature with mild shaking. After 2 hours, plates were washed 5 times with washing buffer. Washed plates were incubated with 50 mL of serum sample (1 mL serum/100 mL sample buffer) for 2 hours at room temperature. Subsequently, plates were washed with washing buffer 5 times and incubated with 50 mL of anti-human rabbit secondary antibody (Sigma) with dilution of 1:10,000 in sample buffer for 1 hour at room temperature. Plates were washed again 5 times and developed with 100 mL of stabilized TMB chromogen for 20 minutes. Subsequently, reactions were stopped with 50 mL of 1 N sulfuric acid. Control ELISA plates were incubated with purified aquaporin-4 protein to anti-KIR4.1 monoclonal antibody–coated plates and further assays were performed in the same manner. OD measurements were performed at 450 nm on a microplate reader (Tecan, Männedorf, Switzerland). All assays were run in triplicate, and the average delta OD was considered for final analysis after background subtraction from control antigen plates. The serum of 3 patients was no longer available for testing by capture ELISA (1 ADEM, 1 autoimmune disease, 1 HC).

MOG-specific antibodies. MOG-specific antibodies in sera of patients with ADD were measured using a cell-based bioassay based on a MOG-transfected glioma cell line as previously described.4 The serum of one patient with ADEM was not available for testing. Serum was added in a final dilution of 1:100. Statistical analysis. KIR4.1-IgG titers were compared among different groups using the Kruskal-Wallis and Dunn multiple comparison tests. KIR4.1-IgG titers were considered positive if they exceeded the cutoff defined by the mean and 5 SDs of 37 sera from independent OND controls that have also been used in previous assays.5 KIR4.1-IgG, age, and MOG-IgG were compared using a nonparametric Spearman correlation. A p value ,0.05 was considered significant.

RESULTS KIR4.1-specific antibodies are present in the

serum from children with ADD of the CNS. Serum samples from 47 children with ADD, 22 with OND, and 22 with autoimmune disease, and from 18 HCs were tested for KIR4.1-IgG by ELISA. Antibody reactivities were significantly higher in patients with ADD compared with HCs and patients with autoimmune disease (p , 0.0001, figure 1A). Twenty-seven patients with ADD were KIR4.1-positive (57.45%), whereas none Neurology 82

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Figure 1

Serum reactivity to KIR4.1 in children

We conclude that serum KIR4.1-IgG is found in a majority of children with ADD but not in children with other diseases or in healthy children. These antibodies bind KIR4.1 in the brain similar to KIR4.1IgG from adult patients with MS. Anti–KIR4.1-IgG titer in children with MS, CIS, ADEM, and MDEM. Serum KIR4.1-IgG was found in more

than 50% of children with MS or CIS (figure 1B). No difference was observed between children with CIS/MS and DEM (comprising ADEM and MDEM), although the number of patients was low. Two patients with isolated optic neuritis were KIR4.1-positive, and one patient was negative. Of the 8 patients with CIS, 4 developed MS during the study period from 2009 to 2012 and 3 of them had high serum KIR4.1-IgG (figure 1B). Similar results were obtained by capture ELISA (figure e-1A). Altogether, we found no major differences in KIR4.1-IgG serum titers in children with CIS, MS, or ADEM/MDEM. No correlation of anti–KIR4.1-IgG with age or anti–MOGIgG antibodies. Next, we investigated the relation

(A) Protein-based ELISA was used to detect anti-KIR4.1 serum autoantibodies. Purified recombinant KIR4.1 from HEK293 cells was covalently coupled to ELISA plates. Serum antibody binding to KIR4.1 was determined in HC and in patients with ADD of the CNS, AI, and OND. (B) Comparison of KIR4.1 antibody titers in children with different ADDs (CIS, MS), acute or multiphasic DEM, and ON. The threshold for anti-KIR4.1 antibody positivity in this assay was 1.18 OD (broken line, determined by serum KIR4.1 antibody titer measured in 37 OND sera; mean of 37 OND samples 0.4313). Groups were compared using the KruskalWallis test followed by the Dunn multiple comparison test for which p values are shown. ADD 5 acquired demyelinating disease; AI 5 autoimmune disease; CIS 5 clinically isolated syndrome; DEM 5 demyelinating encephalomyelitis; HC 5 healthy control; MS 5 multiple sclerosis; OD 5 optical density; ON 5 optic neuritis; OND 5 other neurologic disease.

of the controls had a serum titer above the cutoff. Similar results were obtained when we used a capture ELISA to determine antibodies to KIR4.1. Antibody reactivities were significantly higher in patients with ADD compared with HCs and patients with OND and autoimmune disease (p , 0.0001; figure e-1A). We also stained human brain tissue sections with serum IgG of patients with ADD and OND control sera. A specific binding to CNS cells, most likely oligodendrocytes and astrocytes, similar to the pattern seen in adult patients, was observed for the ADD but not for the OND sera (figure e-2).5 472

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between antibody responses to KIR4.1 and MOG. No correlation between KIR4.1-IgG and MOG-IgG was observed (figure e-3A). The frequency of MOG antibodies was similar in patients with and without KIR4.1-IgG (table e-2). No correlation of KIR4.1 antibodies with age was observed (figure e-3B). In summary, KIR4.1-IgG antibodies, in contrast to MOG antibodies, are not age-dependent. The lack of correlation between anti-MOG and anti-KIR4.1 antibodies suggests that the formation of these autoantibodies is a specific event in a subset of patients with ADD. DISCUSSION Recently, a serum antibody to KIR4.1 was identified in adult patients with MS. Almost half of the patients had serum KIR4.1-IgG, while the antibody was only rarely found in patients with OND.5 The antibody binds to the first extracellular domain of KIR4.1. Transfer experiments in mice have shown that KIR4.1-IgG induces loss of Kir4.1 and complement activation in vivo. KIR4.1 is an inward rectifying potassium channel expressed on oligodendrocytes and astrocytes. The channel contributes to the maintenance of the electrochemical gradient by removing potassium from the extracellular space.7 Kir4.1 knock-out mice show hypomyelination of the spinal cord and axonal degeneration, leading to severe motor deficits.8 Mutations of the KIR4.1 gene (KCNJ10) in humans cause SeSAME or EAST syndrome characterized by epilepsy, ataxia, sensorineural deafness, and tubulopathy.9 We identified serum antibodies to KIR4.1 in 57.45% of children with ADD of the CNS. KIR4.1-IgG was

predominantly found in children with MS or CIS, although 1 of 3 children with DEM also reacted to KIR4.1. The prevalence and distribution of KIR4.1IgG in children with MS/CIS is similar to the prevalence observed in adult patients. Multiple previous studies have focused on antibodies to MOG, another potential autoantigen in ADD of the CNS. Highest titers of antiMOG antibodies are found in young children with ADD and are only rarely seen in older children or even adult patients with MS.3,4,10,11 KIR4.1-IgG was not age-dependent and did not correlate with MOG antibody responses. Therefore, KIR4.1-IgG and MOG-IgG seem to characterize different subgroups of patients. MOG-IgG occurs predominantly before the age of 10 years in ADD and seems to characterize a subset of patients that are distinct from adult MS. By contrast, KIR4.1 antibodies are found in older children and adult patients with MS, suggesting similarities between these 2 groups. AUTHOR CONTRIBUTIONS Verena Kraus: design of the study, sample and data collection, analysis and interpretation of data, drafting and revising the manuscript. Rajneesh Srivastava: design of the study, data collection, analysis and interpretation of data, revising the manuscript. Sudhakar Reddy Kalluri: analysis and interpretation of data, revising the manuscript. Ulrich Seidel, Markus Schuelke, Mareike Schimmel, Kevin Rostasy, Steffan Leiz, and Stuart Hosie: sample and data collection, revising the manuscript. Verena Grummel: technical assistance, data collection, revising the manuscript. Bernhard Hemmer: design of the study, obtaining funding, interpretation of data, revising the manuscript.

STUDY FUNDING Supported by a grant from the German Ministry for Education and Research (BMBF, German Competence Network Multiple Sclerosis [KKNMS], Control-MS, 01GI0917) and a KKF grant from the Technische Universität Munich, Germany (V. Kraus).

DISCLOSURE V. Kraus was supported by a KKF grant from the Technische Universität Munich, Germany, and has received funding for travel from Merck Serono. R. Srivastava holds a patent on KIR4.1 antibody testing in MS. S.R. Kalluri, U. Seidel, M. Schuelke, and M. Schimmel report no disclosures. K. Rostasy has received speaker honoraria from Biogen Idec and Merck Serono. S. Leiz, S. Hosie, and V. Grummel report no disclosures. B. Hemmer has served on scientific advisory boards for Roche, Novartis, Bayer Schering, Merck Serono, Biogen Idec, GSK, Chugai Pharmaceuticals, Micormet, and Genzyme Corporation; serves on the international advisory board of Archives of Neurology and Experimental Neurology; has received speaker honoraria from Bayer Schering, Novartis,

Biogen Idec, Merck Serono, Roche, and Teva Pharmaceutical Industries Ltd.; and has received research support from Biogen Idec, Bayer Schering, Merck Serono, Five Prime, Metanomics, Chugai Pharmaceuticals, and Novartis. B. Hemmer holds a patent on KIR4.1 antibody testing in MS and on genetic determinant of neutralizing antibody development in interferon-b–treated patients. Go to Neurology.org for full disclosures.

Received August 21, 2012. Accepted in final form August 15, 2013. REFERENCES 1. Yeh EA, Chitnis T, Krupp L, et al. Pediatric multiple sclerosis. Nat Rev Neurol 2009;5:621–631. 2. Banwell B, Ghezzi A, Bar-Or A, Mikaeloff Y, Tardieu M. Multiple sclerosis in children: clinical diagnosis, therapeutic strategies, and future directions. Lancet Neurol 2007;6: 887–902. 3. McLaughlin KA, Chitnis T, Newcombe J, et al. Agedependent B cell autoimmunity to a myelin surface antigen in pediatric multiple sclerosis. J Immunol 2009;183: 4067–4076. 4. Brilot F, Dale RC, Selter RC, et al. Antibodies to native myelin oligodendrocyte glycoprotein in children with inflammatory demyelinating central nervous system disease. Ann Neurol 2009;66:833–842. 5. Srivastava R, Aslam M, Kalluri SR, et al. Potassium channel KIR4.1 as an immune target in multiple sclerosis. N Engl J Med 2012;367:115–123. 6. Kalluri SR, Illes Z, Srivastava R, et al. Quantification and functional characterization of antibodies to native aquaporin in neuromyelitis optica. Arch Neurol 2010;67: 1201–1208. 7. Kucheryavykh YV, Kucheryavykh LY, Nichols CG, et al. Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes. Glia 2007;55:274–281. 8. Neusch C, Rozengurt N, Jacobs RE, Lester HA, Kofuji P. Kir4.1 potassium channel subunit is crucial for oligodendrocyte development and in vivo myelination. J Neurosci 2001;21:5429–5438. 9. Bockenhauer D, Feather S, Stanescu HC, et al. Epilepsy, ataxia, sensorineural deafness, tubulopathy, and KCNJ10 mutations. N Engl J Med 2009;360:1960–1970. 10. Di Pauli F, Mader S, Rostasy K, et al. Temporal dynamics of anti-MOG antibodies in CNS demyelinating diseases. Clin Immunol 2011;138:247–254. 11. Pröbstel AK, Dornmair K, Bittner R, et al. Antibodies to MOG are transient in childhood acute disseminated encephalomyelitis. Neurology 2011;77:580–588.

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Potassium channel KIR4.1-specific antibodies in children with acquired demyelinating CNS disease Verena Kraus, Rajneesh Srivastava, Sudhakar Reddy Kalluri, et al. Neurology 2014;82;470-473 Published Online before print January 10, 2014 DOI 10.1212/WNL.0000000000000097 This information is current as of January 10, 2014 Updated Information & Services

including high resolution figures, can be found at: http://www.neurology.org/content/82/6/470.full.html

Supplementary Material

Supplementary material can be found at: http://www.neurology.org/content/suppl/2014/01/10/WNL.0000000000 000097.DC1.html

References

This article cites 11 articles, 3 of which you can access for free at: http://www.neurology.org/content/82/6/470.full.html##ref-list-1

Subspecialty Collections

This article, along with others on similar topics, appears in the following collection(s): Acute disseminated encephalomyelitis http://www.neurology.org//cgi/collection/acute_disseminated_encephal omyelitis All Demyelinating disease (CNS) http://www.neurology.org//cgi/collection/all_demyelinating_disease_cn s All Pediatric http://www.neurology.org//cgi/collection/all_pediatric Multiple sclerosis http://www.neurology.org//cgi/collection/multiple_sclerosis

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Neurology ® is the official journal of the American Academy of Neurology. Published continuously since 1951, it is now a weekly with 48 issues per year. Copyright © 2014 American Academy of Neurology. All rights reserved. Print ISSN: 0028-3878. Online ISSN: 1526-632X.

Potassium channel KIR4.1-specific antibodies in children with acquired demyelinating CNS disease.

A serum antibody against the inward rectifying potassium channel KIR4.1 (KIR4.1-IgG) was recently discovered, which is found in almost half of adult p...
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