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Vaibhav Singh MScE Danieumllle van Pelt MDMarcel P Stoop PhDChristoph Stingl DI (FH)Immy A Ketelslegers
MD PhDRinze F Neuteboom
MD PhDCoriene E Catsman-
Berrevoets MD PhDTheo M Luider PhDRogier Q Hintzen MD
PhD
Correspondence toDr Hintzenrhintzenerasmusmcnl
Supplemental dataat Neurologyorgnn
Gray matterndashrelated proteins areassociated with childhood-onset multiplesclerosis
ABSTRACT
Objective To identify CSF biomarkers for multiple sclerosis (MS) in children with an initialacquired CNS demyelinating syndrome (ADS)
Methods CSF was collected from a cohort of 39 children with initial ADS 18 of whom were diag-nosed with MS and 21 of whom had a monophasic disease course Proteomic analysis of trypsi-nized CSF (20 mL) was performed by nano-liquid chromatography Orbitrap mass spectrometryUnivariate statistical analysis was used to identify differentially abundant proteins betweenchildhood-onset MS and monophasic ADS
Results A total of 2260 peptides corresponding to 318 proteins were identified in the total setof samples Of these 2260 peptides 88 were identified as being most distinctive between MSand ADS Fifty-three peptides corresponding to 14 proteins had higher abundance in childrenwith MS compared to children with monophasic ADS Twelve of these 14 proteins were linkedto neuronal functions and structures such as synapses axons and CNS proteases (eg neuro-fascin carboxypeptidase E brevican core protein and contactin-2) The other 2 were functionallyrelated to immune function The 35 peptides identified with decreased abundance in children withMS corresponded to 7 proteins Six of them were linked to innate immune function (eg hapto-globin haptoglobin-related protein C4b-binding protein alpha chain and monocyte differentia-tion antigen CD14) and 1 was linked to cellular adhesion (protein diaphanous homolog 1)
Conclusion At first onset of ADS CSF of children diagnosed with MS showed increased abun-dance of CNS gray matterndashrelated proteins whereas CSF of children with a monophasic diseasecourse showed increased abundance of innate immunityndashrelated proteins Neurol Neuroimmunol
Neuroinflamm 20152e155 doi 101212NXI0000000000000155
GLOSSARYADS 5 acquired demyelinating syndrome ADEM 5 acute disseminated encephalomyelitis CIS 5 clinically isolated syn-drome LC-MS 5 nano-liquid chromatography Orbitrap mass spectrometry MS 5 multiple sclerosis NMO 5 neuromyelitisoptica ON 5 optic neuritis RRMS 5 relapsing remitting MS TM 5 transverse myelitis
A few percent of all patients with multiple sclerosis (MS) experience their first event in childhood1
In children such a first event can present with a spectrum of clinical features of acquired demy-elinating syndrome (ADS) including optic neuritis (ON) transverse myelitis (TM) acute dis-seminated encephalomyelitis (ADEM) neuromyelitis optica (NMO) and other clinicallymonofocal or polyfocal symptoms2 In most children with ADS the disease course remainsmonophasic However approximately 21ndash32 of these children will subsequently be diag-nosed with MS34 Current MS diagnosis is based on a combination of clinical features CSFfindings andMRI criteria for dissemination in time and space135 These factors are insufficient topredict the disease course at first event A biomarker that helps to differentiate between childrenwith monophasic ADS and those subsequently diagnosed with MS is needed Moreover identi-fication of CSF proteins that are associated with childhood-onset MS can provide further insightinto the disease pathophysiology So far 1 study has compared the CSF of children with MS and
From the MS Centre ErasMS Department of Neurology Erasmus MC Rotterdam the Netherlands
Funding information and disclosures are provided at the end of the article Go to Neurologyorgnn for full disclosure forms The Article ProcessingCharge was paid by the authors
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40(CC BY-NC-ND) which permits downloading and sharing the work provided it is properly cited The work cannot be changed in any way or usedcommercially
Neurologyorgnn copy 2015 American Academy of Neurology 1
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
monophasic ADS and this study suggested dis-turbed axoglial biology during early MSevents6 In the present study we investigatedCSF a body fluid that reflects ongoing CNSpathology7 in a fully unbiased manner Sampleswere analyzed by high-resolution sensitivenano-liquid chromatography Orbitrap massspectrometry (LC-MS)8 Our aim was to findCSF protein markers expressed during a firstevent of CNS demyelination that can help todistinguish children with monophasic ADS(n 5 21) from children with MS (n 5 18)
METHODS Patients Children younger than 18 years old
who presented with a first acquired demyelinating event were
identified by the Dutch Study Group for Pediatric MS which in-
cludes 13 major pediatric neurology centers in the Netherlands
as described earlier9 Children were diagnosed with MS if they
had a second demyelinating attack of the CNS andor MRI evi-
dence of a new lesion at least 1 month after onset1 Clinical fea-
tures and physical examination defined initial clinical phenotypes
of the children This study included 41 children with ADS 22 of
whom had a monophasic disease course and 19 of whom were
diagnosed with MS The CSF samples were collected at first
clinical presentation Children were symptomatic at the time of
sampling Children were not on immunomodulatory treatment at
the time of sampling CSF samples were collected processed and
stored following previously described protocols810
Standard protocol approvals registrations and patientconsents The study was approved by the Clinical Research Eth-ics Board of Erasmus University Written informed consent was
obtained from all patients andor their families
Sample preparation and LC-MS measurements CSF sam-
ples (20 mL) were digested using an in-solution trypsin digestion
protocol as previously described11 Prepared samples were
analyzed by LC-MSMS using an Ultimate 3000 nano RSLC
system (Thermo Fischer Scientific Germering Germany)
online coupled to a hybrid linear ion trapOrbitrap mass
spectrometer (LTQ Orbitrap XL Thermo Fisher Scientific
Bremen Germany) using a data-dependent acquisition method
LC-MS data were analyzed using the Progenesis LC-MS software
package (version 36 Nonlinear Dynamics Ltd Newcastle-upon-
Tyne United Kingdom) Detailed LC-MS measurements and
protein identification and quantification information are
available in the e-Methods at Neurologyorgnn
Statistical analysis The Wilcoxon test (unpaired 2-tailed) was
used to analyze the differences in the abundance of peptides
between children with MS and those with monophasic ADS
p values 001 were considered statistically significant Relative
quantitative differences of peptide abundances between samples
of children with MS and samples of children with monophasic
ADS were calculated as log2 ratio between median abundances of
both groups A set of significantly distinct peptides and proteins
was determined by applying the following stringent criteria (1)
peptides that had at least 15-fold difference in expression at a
p value 001 (2) protein identified by at least 2 peptides and
(3) at least 40 differentially abundant peptides (p 001) per
protein whereby peptides of a given protein were required to
have representation in the same direction (increased or
decreased abundance in MS) In the above dataset we also
excluded those proteins that had only 1 peptide differentially
abundant out of a total 2 To verify these findings and to
determine the statistical background level we performed a
permutation analysis on the entire dataset (2260 peptides)
between samples of children with MS (n 5 18) and samples of
children with monophasic ADS (n 5 21) included with the
abovementioned criteria Through this process we determined
the statistical background level on a set of significantly distinct
peptides The random permutation test was repeated 1000 times
on the dataset with randomized sample group assignment and
the resulting thresholds were saved We used the R software
package for all calculations and graphics (R version 302)12
For other calculations we used SPSS 150 (SPSS Chicago IL)
and Microsoft Excel 2010
RESULTS Patient characteristics We analyzed CSFsamples of 18 children with MS and 21 with mono-phasic ADS One patient with MS and 1 patient withmonophasic ADS were excluded because 200required alignment vectors (weak alignment) werefound during Progenesis LC-MS analysis Of the 18children diagnosed with MS 5 had ON 2 had TM 3had clinical monofocal symptoms and 8 had clinicalpolyfocal symptoms as their presenting symptoms atonset Of the 21 children with monophasic ADS 2had ON 2 had TM 8 had clinical polyfocalsymptoms and 9 had ADEM as their presentingsymptoms at onset No significant differences wereobserved between the 2 groups in terms of sex CSFlevels of total protein and albumin albumin CSFserum quotient CSF leukocyte count and CSFIgG concentration The mean age at onset ofchildren diagnosed with MS (1417 6 15 years)was found to be significantly higher than that ofchildren with monophasic ADS (689 6 49 years)reflecting the epidemiology of these phenotypes Inaddition elevated CSF IgG index and positiveoligoclonal bands were more frequent in childrenwith MS (p 001) Patient characteristics areshown in table 1
Identification of proteins that discriminate MS from
monophasic ADSWe detected 50119 peptide precur-sors from Progenesis label-free analysis LC-MSexperiment from all trypsin-digested protein in CSFsamples A Mascot database search in the humansubset of the Uniprot database resulted into 2260unique peptides that corresponded to 318 proteins(table e-1) The total protein concentrations ofdigested peptide samples quantified (integrated UVarea at 214 nm) during LC-MS measurements didnot show any significant difference (p 5 054)between CSF samples of children with MS andthose of children with monophasic ADS To checktechnical variability we measured 12 reference samples(pooled CSF samples from all patients) at regularintervals Here too the total protein concentrations ofdigested peptide samples quantified (integrated UV
2 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
area at 214 nm) during LC-MS measurements did notshow any difference between reference group 1 (n5 6)and reference group 2 (n 5 6) (reference group 138294 6 15328 reference group 2 42934 6
66190 p 5 03) In addition a number of MSMSfragmentation spectra did not show any significantdifference between samples of children with MS andthose of children with monophasic ADS In particularthe measured MSMS fragmentation spectra for MSand monophasic ADS samples were 16796 6 1795and 16971 6 1153 (p 5 072) respectivelyMoreover the database identified MSMS spectra forMS and monophasic ADS samples as 1607 6 280and 1626 6 256 (p 5 083) respectively
Comparing the abundance of identified peptides(n 5 2260) from CSF samples of children withMS and children with monophasic ADS using thestringent criteria described in the statistical analysiswe found a total of 88 differentially abundant pep-tides (tables e-2 and e-3) Of these 88 peptides 53were significantly increased (table 2 and table e-2) and35 significantly decreased in CSF samples of childrenwith MS (table 2 and table e-3) compared with sam-ples of children with monophasic ADS Peptides withincreased abundance (n 5 53) in the MS group cor-responded to 14 proteins and peptides with decreasedabundance (n 5 35) corresponded to 7 proteins Aninventory of these 21 proteins is given in table 2Moreover fold expression difference between theMS and monophasic ADS groups and statistical signif-icance are plotted simultaneously for our entire dataset(n5 2260 peptides) as a volcano plot (figure 1) Thispermutation analysis resulted in 20 6 41 (median 9)
false-positive peptide markers which indicated thatour observations are not due to chance alone becausemore than 90 of the permutations yielded 0ndash4 sig-nificant hits (ie in 900) Only 4 times out of 1000were more than 88 hits with low p value detected (falsediscovery rate 04) Contrasting this to mere back-ground chance 88 peptides (true hits) were identifiedfrom the actual dataset Therefore comparison of per-mutated data with the real data indicated that theoccurrence of differentially abundant peptides relatedto MS or monophasic ADS was highly significant (p0001) The outcome of the permutation test is shownas a histogram (figure e-1)
Identified proteins with increased abundance(n 5 14) in MS (table 2 and table e-2) were the fol-lowing amyloid-like protein 2 (22 2 significant pep-tides for a total of 2 peptides) neurofascin (33)carboxypeptidase E (23) neuronal growth regulator1 (23) contactin-2 (49) amyloid beta A4 (611)brevican core protein (57) disintegrin and metallopro-teinase domain-containing protein 22 (22) tyrosine-protein phosphatase non-receptor type substrate1 (34) dickkopf-related protein 3 (611) neuronalcell adhesion molecule (918) Ig kappa chain V-IIIregion POM (22) Ig gamma-1 chain C region(511) and kallikrein-6 (47) Proteins identifiedwith decreased abundance (n 5 7) in MS (table 2and table e-3) were the following apolipoproteinB-100 (717) C4b-binding protein alpha chain(24) haptoglobin (1829) haptoglobin-related pro-tein (24) leucine-rich alpha-2-glycoprotein (23)monocyte differentiation antigen CD14 (33) andprotein diaphanous homolog 1 (22) The function of
Table 1 Clinical characteristics of children with multiple sclerosis and monophasic acquired demyelinating syndrome
Monophasic ADS (n 5 21) MS (n 5 18)p Valuea
monophasicADS vs MSMean 6 SD Median Range Mean 6 SD Median Range
Age at onset y 689 6 491 (n 5 21) 581 114ndash1711 1417 6 150 (n 5 18) 1429 1114ndash1621 001
Sex females 714 NA NA 611 NA NA NS
Protein gL 036 6 021 (n 5 21) 03 018ndash115 033 6 014 (n 5 17) 032 019ndash075 NS
Albumin gL 023 6 020 (n 5 11) 016 011ndash079 018 6 007 (n 5 14) 017 008ndash036 NS
Leukocytes 3106 3712 6 3491 (n 5 20) 285 1ndash118 2098 6 2568 (n 5 17) 13 10ndash87 NS
IgG gL 005 6 007 (n 5 11) 003 001ndash027 005 6 003 (n 5 13) 005 003ndash012 NS
IgG index 061 6 010 (n 5 12) 058 05ndash079 136 6 072 (n 5 17) 1 067ndash3 001
Elevated IgGindexb n ()
2 (167) NA NA 16 (941) NA NA 001
Positive OCB n () 2 (125) (n 5 16) NA NA 14 (875) (n 5 16) NA NA 001
Relapsing disease n () 0 (0) NA NA 18 (100) NA NA 001
Abbreviations ADS 5 acquired demyelinating syndrome MS 5 multiple sclerosis NA 5 not applicable NS 5 not significant OCB 5 oligoclonal bandsThis table shows clinical characteristics and routine CSF findings of children with MS (at disease onset) and children with monophasic ADS Data arepresented as the mean 6 SD or median and range In case of missing data the number of patients with available data is indicated in parenthesesaCalculated by Mann-Whitney test and p 001 was considered significantb IgG index considered elevated at 068 or higher4
Neurology Neuroimmunology amp Neuroinflammation 3
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these 14 proteins and overlap with previous stud-ies613 are summarized in table 3 Among the 14 pro-teins with increased abundance in MS 12 wereassociated with CNS structure and functions (86)especially the gray matter (table 3) whereas only 17of the total identified proteins were related to CNSstructure and functions The 7 proteins with decreasedabundance in MS (relative to the monophasic ADSgroup) were components of the innate immune systemand inflammation (table 4)
For each peptide that passed (53 increased and 35decreased in MS) all stringent criteria (described inthe statistical analysis) the associated protein data-base search identification details p value fold expres-sion difference total number of peptides per proteinnumber of peptides identified below 001 medianabundance and frequencyoccurrence of identifica-tions by MSMS spectra are shown in tables e-2and e-3 Proteins identified in the current study didnot exhibit any myelin-related proteins (eg myelin
Table 2 Identification of proteins differentially abundant in CSF samples of children with MS (n 5 18) andsamples of children with monophasic ADS (n 5 21)
Trend in MSa DescriptionSigntotalb
Fold changemean (minndashmax)c p Valued mean (minndashmax)
Increased abundancein MS (n 5 14)
Amyloid-like protein 2 22 38 (2ndash55) 0006 (0003ndash0008)
Neurofascin 33 25 (21ndash3) 0002 (0001ndash0002)
Carboxypeptidase E 23 24 (21ndash26) 00004 (00002ndash00005)
Neuronal growth regulator 1 23 23 (21ndash25) 00008 (00008ndash00009)
Contactin-2 49 23 (15ndash32) 0002 (00005ndash0003)
Amyloid beta A4 protein 611 22 (18ndash26) 0002 (000003ndash0008)
Brevican core protein 57 21 (17ndash26) 0002 (00001ndash0005)
Disintegrin and metalloproteinasedomain-containing protein 22
22 19 (17ndash21) 00004 (00001ndash00006)
Tyrosine-protein phosphatasenon-receptor type substrate 1
34 19 (15ndash21) 0003 (0001ndash0006)
Dickkopf-related protein 3 611 18 (16ndash2) 0002 (000004ndash0006)
Neuronal cell adhesion molecule 918 18 (16ndash21) 0004 (0001ndash0009)
Ig kappa chain V-III region POM 22 18 (17ndash18) 0002 (00009ndash0003)
Ig gamma-1 chain C region 511 17 (15ndash18) 00016 (00002ndash0005)
Kallikrein-6 47 17 (15ndash19) 0001 (00006ndash0004)
Decreased abundancein MS (n 5 7)
Apolipoprotein B-100 717 661 (35ndash3930) 00026 (000002ndash001)
C4b-binding protein alpha chain 24 81 (29ndash134) 0008 (0007ndash0008)
Haptoglobin 1829 37 (2ndash121) 0002 (000004ndash001)
Haptoglobin-related protein 24 33 (31ndash35) 00002 (000001ndash00005)
Leucine-rich alpha-2-glycoprotein 23 26 (23ndash3) 0008 (0007ndash0009)
Monocyte differentiation antigen CD14 33 19 (18ndash2) 0006 (0004ndash0009)
Protein diaphanous homolog 1 22 18 (17ndash19) 0005 (0001ndash0009)
Abbreviations ADS 5 acquired demyelinating syndrome MS 5 multiple sclerosisThe table shows 21 proteins of which 14 were identified with increased abundance in CSF samples of children with MSand 7 were identified with decreased abundance in CSF samples of children with MS The table includes the direction ofdifference (trend) in MS name of the protein (description) fold expression difference and p value All proteins given in thetable were identified with at least 2 unique peptides differentially abundant peptides (p 001) had at least 15-folddifference in expression (median) between groups and for the same protein 40 of identified peptides were differentiallyabundant with the expression in the same direction (ie either higher or lower in MS) of the same protein Details of eachpeptide of the indicated proteins are listed in tables e-2 and e-3a Protein abundance is either significantly increased or decreased in children with MS compared to children withmonophasic ADSbNumber of differentially identified peptides p 001total number of identified peptides for the same proteinc Fold expression difference calculated based on median abundance from 18 patients with MS and 21 patients with ADSShown is the average of fold difference for all peptides of the same protein Minimum and maximum range for the same isindicateddCalculated by Wilcoxon test Given in the table is the mean p value and range for all differentially abundant peptides of thesame protein
4 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
oligodendrocyte glycoprotein and myelin basicprotein)
We performed a correlation analysis to examinethe influence of age at onset on the abundance ofthe 88 candidate peptides in children with MS and
monophasic ADS We found a mean coefficient ofdetermination (6SD) of 004 6 006 for MS and005 6 005 for monophasic ADS Thus no signif-icant correlation was found between age and peptideabundance for children with MS and monophasicADS for all 88 peptides
DISCUSSION In the current study we used a LC-MS proteomic approach to search for differences inCSF proteome between children with MS andchildren with monophasic ADS A benefit of thisOrbitrap technique is the ability to identifyrelatively vast amounts of different peptides andassess their abundances in a small sample volumeWe observed a striking difference between the 2groups (children with monophasic ADS vs MS)using stringent statistical criteria We searched forthe known functions of the 88 peptides correspondingto 21 distinctive proteins (14 increased and 7decreased in abundance in MS) using biologicaldatabases (wwwgeneontologyorg and wwwnextprotorg) and literature Recently 2 research groups613
demonstrated the identification of axoglial and graymatter proteins using mass spectrometry in CSF ofpatients with MS Similar to our study Dhaunchaket al6 compared CSF of 8 children with MS with CSFof 11 children with monophasic ADS The overlap ofsome proteins in the MS group is noticeable (egcarboxypeptidase E) despite clear differences insample handling such as depletion of abundantproteins with possible carrier function for otherproteins and exclusion of proteins with less than 5 kDaweight (table 3)
Our results show overlap with molecules identi-fied in the CSF of adults with acute-onset MS bySchutzer et al13 who performed mass spectrometryanalysis in CSF of patients with clinically isolatedsyndrome (CIS) vs those with established relapsingremitting MS (RRMS) and controls (table 3) Theyfound proteins that distinguished patients with CISfrom both patients with established RRMS and con-trols For example they found a significant increase inkallikrein-6 and dickkopf-related protein 3 at firstclinical onset compared to patients with establishedRRMS and controls (table 3) They also found a sig-nificant increase in contactin-2 (neuronal membraneprotein) in patients with a first attack of MS relativeto those with established RRMS (table 3)13
Our findings illustrate that the neurodegenerativearm of MS neuropathology is already active at the ear-liest stage of clinical disease also in children Consis-tent with the findings of others and our own previousstudies614ndash16 we again observed a striking lack ofmyelin proteins in these clear-cut cases of acute demy-elination This may not directly imply absence ofsuch free proteins in this type of pathology but rather
Figure 1 Volcano plot
Peptides (n 5 2260) showing distribution of fold change and statistical significance In thisplot each point represents a peptide and shows the ratio between CSF samples of childrenwith multiple sclerosis (MS) (n5 18) and samples of children with monophasic acquire demy-elinating syndromes (ADS) (n5 21) plotted against the level of statistical significance Y-axisshows p values obtained (plotted at log10) from a Wilcoxon test performed between abun-dances of peptides X-axis shows the ratio of the median between MS and monophasic ADSsamples (plotted on log2) (A) Above the dashed horizontal line red points (n 5 53 peptides)were found with increased abundance (right side of the vertical line) and green points (n5 35peptides) with decreased abundance (left side of the vertical line) in the MS group (comparedto the monophasic ADS group) (B) Peptides shown in gray below the dashed horizontal linedid not pass the stringent statistical criteria for identification of a candidate peptide
Neurology Neuroimmunology amp Neuroinflammation 5
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
it may reflect the specific physicochemical propertiesof the hydrophobic myelin components and perhapsdifferent pathways of elimination from the CSF (egvia draining macrophages)17 In any case we must becautious in using the dominant presence of CNS graymatter over white matter proteins as proof that neuro-degeneration is a primary event in MS and wouldprecede demyelination The presence of CNS graymatter may simply represent damage by inflamma-tion and the molecules identified may lead to a better
understanding of this presumed inflammation-induced neurodegeneration It should be stressed thatnot all differentially abundant proteins were overrep-resented in MS some were underrepresented point-ing at more complex mechanisms such as aperturbation in the physiology of the axoglial appara-tus (table 2 and tables e-2 and e-3)6 A confoundingfactor in our study could be the fact that the groupswere not matched according to age because of theoccurrence of monophasic disease at younger ages
Table 3 Function of proteins identified with increased abundance in CSF samples of children with MS compared with samples of children withmonophasic ADS
Protein (accession no) Functions and expressions Dhaunchak et al6 Schutzer et al13
Amyloid-like protein 2 (Q06481) Amyloid precursor protein family memoryprocesses concentrated in synapses18 regulatesneuronal stem cell differentiation during corticaldevelopment
3 variant
Neurofascin (O94856) Synapse formation located at CNS paranodaldomain and expressed by oligodendrocytes20
target for autoantibody-mediated axonal injury inMS21
NS 3
Contactin-2 (Q02246) Axon connection majorly expressed on CNSjuxtaparanodal domain1920
NS
Amyloid beta A4 protein (P05067) Component of amyloid plaques (Alzheimer brains)35
neuronal adhesion3 3
Brevican core protein (Q96GW7) Major proteoglycan in perisynaptic extracellularmatrix of brain inhibits neurite outgrowth fromcerebellar granule neurons23
NS Decreased expression in first-attack CIS-MS relative toestablished RRMS and controls
Carboxypeptidase E (P16870) Found in brain and throughout neuroendocrinesystem involved in synthesis of mostneuropeptides36
Same trend (4 sign21 total p 5 0004)
3
Neuronal growth regulator1 (Q7Z3B1)
Promotes outgrowth and is expressed on reactiveastrocytes after entorhinal cortex lesion (in mice)37
NS 3
Tyrosine-protein phosphatase non-receptor type substrate 1 (P78324)
Supports adhesion of cerebellar neurons neuriteoutgrowth and glial cell attachment28
3 3
Neuronal cell adhesion molecule(Q92823)
Localized at the node of Ranvier and in unmyelinatedaxons paranodal region of CNS axoglial contact19
NS
Disintegrin and metalloproteinasedomain-containing protein 22(Q9P0K1)
Expressed in the juxtaparanodal complex19 Not same trend (2 sign10 total p 5 001 002)
3
Dickkopf-related protein 3 (Q9UBP4) Expressed in the brain and spinal cord synapseformation13
3
Kallikrein-6 (Q92876) Elevated in active MS26 regulates early CNSdemyelination in a viral (mouse) model of MS26
3
Ig gamma-1 chain C region (P01857) Elevated in adult MS and CIS compared tononinflammatory neurologic diseases38
3 3
Ig kappa chain V-III region POM(P01624)
Elevated in adult MS compared to noninflammatoryand inflammatory neurologic diseases38
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis NS 5 not significant RRMS 5
relapsing remitting MS3 5 protein was not identified variant 5 variant of same protein was found with increased expression in first-attack CIS-MS group compared toestablished RRMS and controls 5 increased expression in first-attack CIS-MS group compared to established RRMS and controls 5 protein wasfound with increased expression in first-attack CIS-MS relative to RRMS but with decreased expression in first-attack CIS-MS vs controls NS 5 proteinwas detected in pediatric CSF samples but no statistical difference in their abundance was reported between MS and monophasic ADS group comparisonsame trend (trend is the direction of difference in MS) 5 when abundance of the protein was compared between 2 group (MS vs monophasic ADS)expression of the identified protein showed overlap with our study not same trend 5 when abundance of protein was compared expression of theidentified protein did not show overlap with our studyNodes paranodes juxtaparanodes (begins at the innermost axoglia junction of paranode) and internodes are structural parts of a myelinated axonSummary of the function of differentially abundant increased protein markers in MS and their overlap with previous studies Most of the proteins wereassigned as either neuronal or immune-related molecules based on previous reports and database searches The first column shows the name of the proteinand Uniprot accession number The second column shows the function of the proteins The third column shows comparison with a previous study onchildren with MS (Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourth column showsoverlap with the work of Schutzer et al who used CIS-MS (n 5 9 age 18ndash42 years) and established MS and controls (n 5 6 age 31ndash54 years)
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
We doubt that this influenced the results howeverbecause we did not see an age effect on the abundanceof the 21 identified proteins
Of the 14 proteins with increased abundance inMS (tables 2 and 3) 2 were associated with the amy-loid beta A4 protein family Amyloid-like protein 2 ismainly concentrated at neuronal synapses18 and has arole in memory processes whereas amyloid beta A4protein is associated with neurite growth neuronaladhesion and axonogenesis (table 3) Six proteins(contactin-219 neurofascin20 neuronal growth regu-lator 1 brevican core protein6 neuronal cell adhesionmolecule19 and disintegrin and metalloproteinasedomain-containing protein 22)19 are located at theparanodal and juxtaparanodal region of the CNS ofmyelinated axons (table 3) Contactin-2 is an axonalglycoprotein at the juxtaparanodal domain of myeli-nated axons13 Neurofascin plays a role in the assem-bly of nodes of Ranvier in the CNS21 Two isoformsof neurofascin have been shown to interact withcontactin-associated protein and contactin-1 to forma paranodal junction that attaches the myelin loop tothe axon and helps to separate voltage-gated sodiumchannels at nodal and potassium channels at juxtapar-anodal regions20 Disruption of neurofascin localiza-tion reveals early changes preceding demyelinationand remyelination in MS22 Of interest contactin-2and neurofascin have previously been reported asautoimmune targets in MS20 Neuronal growth
regulator 1 is located at the paranodal region of theCNS and plays a role in axoglia contact at the node ofRanvier Identification of contactin-2 and neurofas-cin in the CSF of children with MS is consistent witha previous study6 however they did not find a sig-nificant difference in the CSF of children with MSand monophasic ADS (tables 2 and 3) Brevican coreprotein is known as a CNS-specific proteoglycan19 atthe surface of neuroglial sheaths where it is enrichedin perisynaptic extracellular matrix23
Among the 14 proteins with increased abun-dance in MS the proteasespeptidases protein car-boxypeptidase E plays a role in the synthesis ofmost neuropeptides24 Disintegrin and metalloprotei-nase domain-containing protein 22 is highly expressedin the brain and localized at the juxtaparanode19 Thismolecule is presumed to be a major neuronal recep-tor25 Another brain-related protease is kallikrein-6which is a secreted serine protease13 It regulates earlyCNS demyelination in a viral model (expression in thebrain and spinal cord of mice) of MS26 In additionelevated levels of kallikrein-6 in CSF have been re-ported in adults with MS compared to neurologic con-trols (table 3)27
Among the other proteins with increased abun-dance in MS tyrosine-protein phosphatase non-receptor type substrate 1 is implicated in neuriteoutgrowth and glia cell attachment and has beenshown to support adhesions of cerebellar neurons28
Table 4 Function of proteins identified with decreased abundance in CSF samples of children with MScompared to samples of children with monophasic ADS
Protein (accession no) FunctionsDhaunchaket al6
Schutzeret al13
Apolipoprotein B-100 (P04114) Innate immune-related not produced inCNS39
3 3
C4b-binding protein alpha chain (P04003) Innate immune defense involved incomplement activity31
3 3
Haptoglobin (P00738) Innate immune defense29 3 3
Haptoglobin-related protein (P00739) Innate immune defense40 3 3
Leucine-rich alpha-2-glycoprotein (P02750) Induced by inflammation and involved incell adhesion high expression in the deepcerebral cortex25 novel marker ofgranulocytic differentiation29
3 3
Monocyte differentiation antigen CD14(P08571)
Main modulator of innate immune system32 3 3
Protein diaphanous homolog 1 (O60610) Coordinates cellular dynamics byregulating microfilament and microtubulefunction role in cellndashmatrix adhesionsvariant related to innate immune function34
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis3 5 Protein was not identifiedSummary of the function of differentially abundant decreased protein markers in MS and their overlap with previousstudies Most of the proteins were assigned as either neuronal or immune-related molecules based on previous reports anddatabase searches The first column shows the name of the protein and Uniprot accession number The second columnshows the function of the proteins The third column shows comparison with a previous study on children with MS(Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourthcolumn shows overlap with the work of Schutzer et al who used CIS-MS (n5 9 age 18ndash42 years) and established MS andcontrols (n 5 6 age 31ndash54 years)
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
The highest expression of dickkopf-related protein 3is reported in the brain and spinal cord (table 3)13
Thus the majority of proteins with increased abun-dance in our MS group (compared to the monophasicADS group) were neuronal-related with the excep-tion of 2 immune functionndashrelated proteins (Iggamma-1 chain C region and Ig kappa chain V-IIIregion POM) (table 3)
Our study reports 7 proteins with decreased abun-dance in pediatric-onset MS compared to monopha-sic ADS (table 2 and table e-3) Six of these 7 proteinshave previously been reported as specific componentsof innate immune functions (table 4) Haptoglobinan acute phase protein29 was identified as the mostdistinctive protein of those that were elevated in chil-dren with monophasic ADS (compared to childrenwith MS) (1829 18 significant peptides for a total of29 peptides) Another study in adults showedincreased haptoglobin concentration in patients withNMO compared to adult patients with MS30 C4b-binding protein alpha chain is a crucial componentof complement cascade and inhibits the function ofcomplement component31 Of interest we alsofound monocyte differentiation antigen CD14which is mainly expressed on cells of monocyticlineage (macrophages and monocytes)32 HigherCD14 levels might be linked with increased levelsof cytokines triggering inflammatory processes inchildren with monophasic ADS Monocytic cellssecrete soluble sCD14 (activation product of acti-vated monocytes) so this may affect the enormousmacrophage activation during acute monophasicADS33 Among the 7 identified proteins proteindiaphanous homolog 1 has a role in cell adhesionand is expressed in brain and its variants arerequired for innate immune response to Gram-negative bacterial infection34
Overall in the MS group we found a significantoverrepresentation of proteins associated withchanges in CNS gray matter axons synaptic regula-tion node of Ranvier and brain proteases (table 3)Several of these proteins are part of the axoglial appa-ratus and may relate to disturbances in axoglia inter-action6 (table 3) Two of them (contactin-2 andneurofascin) have been identified as possible axoglialautoantigens in MS20 The overlap of proteinsobserved in previous studies613 (table 3) as part ofthe axoglia apparatus and gray matter provides vali-dation for these proteins
These pathologically relevant proteins (mostlyCNS gray matterndashrelated) that are elevated in theCSF of children with early-onset MS might beinvolved in early disease mechanisms Further insightinto the role of these proteins can be useful for under-standing the disease process Moreover such proteinsmight be useful as a future tool to differentiate
children with MS from children with monophasicADS In addition knowing the start of MS could leadto earlier treatment with disease-modifying therapiesHowever the current research is designated as a dis-covery phase study which serves as a basis for thefollow-up verification and validation phase studiesthat can provide clinically valuable biomarkers Inthe future it would be interesting to further validateour findings with an independent technology and inan independent sample group
The data presented in this study indicate thatmonophasic ADS can be differentiated from MS inchildren primarily by CNS gray matter proteins andimmune-related proteins Our findings point to per-turbed axoglial physiology as a hallmark of the earliestevents of MS pathogenesis
AUTHOR CONTRIBUTIONSThe work was carried out in collaboration among all authors VS
TML and RQH designed the experiment VS and CS performed
the experiment and statistical analysis EDvP IAK and CEC-B
were responsible for sample collection VS EDvP MPS CS
RFN TML and RQH interpreted the results and wrote the article
All authors critically revised and approved the final manuscript
ACKNOWLEDGMENTThe authors thank all the children and their families who participated in
the Dutch Study for Pediatric MS
STUDY FUNDINGThis work was supported by a program grant provided by the EC 7th
frame work programme of Marie Curie Initial Training NetworkmdashThe
United Europeans for the Development of Pharmacogenomics in Mul-
tiple Sclerosis (UEPHAmdashMS grant PITNmdashGAmdash2008212877) Neth-
erlands Multiple Sclerosis Research Foundation to the Rotterdam MS
Centre ErasMS the Netherlands Organization for Scientific Research
(ZONmdashMW RQH) and Hersenstichting Nederland and Zenith grant
93511034 from the Netherlands Organization for Scientific Research
(NWO)
DISCLOSUREV Singh ED van Pelt MP Stoop C Stingl and IA Ketelslegers
report no disclosures RF Neuteboom is on the scientific advisory board
for LAREB CE Catsman-Berrevoets and TM Luider report no disclo-
sures RQ Hintzen is on the advisory board for Biogen Idec Roche and
Sanofi received research support from Biogen Idec Merck-Serono
Roche Genzyme Novartis Dutch MS Society and European Commis-
sion and is on the editorial board for Multiple Sclerosis and Related
Disorders Go to Neurologyorgnn for full disclosure forms
Received April 14 2015 Accepted in final form June 25 2015
REFERENCES1 Krupp LB Banwell B Tenembaum S International Pedi-
atric MSSG Consensus definitions proposed for pediatric
multiple sclerosis and related disorders Neurology 2007
68S7ndashS12
2 Banwell B Ghezzi A Bar-Or A Mikaeloff Y Tardieu M
Multiple sclerosis in children clinical diagnosis therapeu-
tic strategies and future directions Lancet Neurol 20076
887ndash902
3 Banwell B Bar-Or A Arnold DL et al Clinical environ-
mental and genetic determinants of multiple sclerosis in
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
children with acute demyelination a prospective national
cohort study Lancet Neurol 201110436ndash445
4 Neuteboom RF Boon M Catsman Berrevoets CE et al
Prognostic factors after a first attack of inflammatory CNS
demyelination in children Neurology 200871967ndash973
5 Krupp LB Tardieu M Amato MP et al International
Pediatric Multiple Sclerosis Study Group criteria for pedi-
atric multiple sclerosis and immune-mediated central ner-
vous system demyelinating disorders revisions to the 2007
definitions Mult Scler 2013191261ndash1267
6 Dhaunchak AS Becker C Schulman H et al Implication
of perturbed axoglial apparatus in early pediatric multiple
sclerosis Ann Neurol 201271601ndash613
7 Singh V Hintzen RQ Luider TM Stoop MP Proteomics
technologies for biomarker discovery in multiple sclerosis
J Neuroimmunol 201224840ndash47
8 Singh V Stoop MP Stingl C et al Cerebrospinal-fluid-
derived immunoglobulin G of different multiple sclerosis
patients shares mutated sequences in complementarity
determining regions Mol Cell Proteomics 201312
3924ndash3934
9 Ketelslegers IA Catsman-Berrevoets CE Neuteboom RF
et al Incidence of acquired demyelinating syndromes of
the CNS in Dutch children a nationwide study J Neurol
20122591929ndash1935
10 Teunissen CE Petzold A Bennett JL et al A consensus
protocol for the standardization of cerebrospinal fluid col-
lection and biobanking Neurology 2009731914ndash1922
11 Stoop MP Singh V Stingl C et al Effects of natalizumab
treatment on the cerebrospinal fluid proteome of multiple
sclerosis patients J Proteome Res 2013121101ndash1107
12 Team RC R A Language and Environment for Statistical
Computing [online] Available at httpwwwR-project
org Accessed February 4 2014
13 Schutzer SE Angel TE Liu T et al Gray matter is tar-
geted in first-attack multiple sclerosis PLoS One 20138
e66117
14 Stoop MP Dekker LJ Titulaer MK et al Multiple
sclerosis-related proteins identified in cerebrospinal fluid
by advanced mass spectrometry Proteomics 20088
1576ndash1585
15 Stoop MP Dekker LJ Titulaer MK et al Quantitative
matrix-assisted laser desorption ionization-fourier trans-
form ion cyclotron resonance (MALDI-FT-ICR) peptide
profiling and identification of multiple-sclerosis-related
proteins J Proteome Res 200981404ndash1414
16 Stoop MP Singh V Dekker LJ et al Proteomics com-
parison of cerebrospinal fluid of relapsing remitting and
primary progressive multiple sclerosis PLoS One 20105
e12442
17 van Zwam M Samsom JN Nieuwenhuis EE et al Mye-
lin ingestion alters macrophage antigen-presenting func-
tion in vitro and in vivo J Leukoc Biol 201190123ndash132
18 Wasco W Gurubhagavatula S Paradis MD et al Isola-
tion and characterization of APLP2 encoding a homologue
of the Alzheimerrsquos associated amyloid beta protein precur-
sor Nat Genet 1993595ndash100
19 Faivre-Sarrailh C Devaux JJ Neuro-glial interactions at
the nodes of Ranvier implication in health and diseases
Front Cell Neurosci 20137196
20 Derfuss T Linington C Hohlfeld R Meinl E Axo-glial
antigens as targets in multiple sclerosis implications for
axonal and grey matter injury J Mol Med (Berl) 2010
88753ndash761
21 Mathey EK Derfuss T Storch MK et al Neurofascin as a
novel target for autoantibody-mediated axonal injury
J Exp Med 20072042363ndash2372
22 Howell OW Palser A Polito A et al Disruption of neu-
rofascin localization reveals early changes preceding demy-
elination and remyelination in multiple sclerosis Brain
20061293173ndash3185
23 Yamada H Fredette B Shitara K et al The brain chon-
droitin sulfate proteoglycan brevican associates with astro-
cytes ensheathing cerebellar glomeruli and inhibits neurite
outgrowth from granule neurons J Neurosci 199717
7784ndash7795
24 Vilijn MH Das B Kessler JA Fricker LD Cultured as-
trocytes and neurons synthesize and secrete carboxypepti-
dase E a neuropeptide-processing enzyme J Neurochem
1989531487ndash1493
25 Ozkaynak E Abello G Jaegle M et al Adam22 is a major
neuronal receptor for Lgi4-mediated Schwann cell signal-
ing J Neurosci 2010303857ndash3864
26 Scarisbrick IA Yoon H Panos M et al Kallikrein 6 reg-
ulates early CNS demyelination in a viral model of multi-
ple sclerosis Brain Pathol 201222709ndash722
27 Hebb AL Bhan V Wishart AD Moore CS
Robertson GS Human kallikrein 6 cerebrospinal levels
are elevated in multiple sclerosis Curr Drug Discov Tech-
nol 20107137ndash140
28 Latour S Tanaka H Demeure C et al Bidirectional
negative regulation of human T and dendritic cells by
CD47 and its cognate receptor signal-regulator protein-
alpha down-regulation of IL-12 responsiveness and inhi-
bition of dendritic cell activation J Immunol 2001167
2547ndash2554
29 OrsquoDonnell LC Druhan LJ Avalos BR Molecular charac-
terization and expression analysis of leucine-rich alpha2-
glycoprotein a novel marker of granulocytic differentiation
J Leukoc Biol 200272478ndash485
30 Chang KH Tseng MY Ro LS et al Analyses of hapto-
globin level in the cerebrospinal fluid and serum of pa-
tients with neuromyelitis optica and multiple sclerosis
Clin Chim Acta 201341726ndash30
31 Chung LP Reid KB Structural and functional studies on
C4b-binding protein a regulatory component of the
human complement system Biosci Rep 19855855ndash865
32 Kurne A Sayat G Aydin OF et al Lack of association of
the CD14Cmdash159T polymorphism with susceptibility
and progression parameters in Turkish multiple sclerosis
patients J Neuroimmunol 201225083ndash86
33 Kitchens RL Thompson PA Modulatory effects of sCD14
and LBP on LPS-host cell interactions J Endotoxin Res
200511225ndash229
34 Huh JR Foe I Muro I et al The Drosophila inhibitor of
apoptosis (IAP) DIAP2 is dispensable for cell survival
required for the innate immune response to gram-negative
bacterial infection and can be negatively regulated by the
reaperhidgrim family of IAP-binding apoptosis inducers
J Biol Chem 20072822056ndash2068
35 Jacobsen KT Iverfeldt K Amyloid precursor protein and
its homologues a family of proteolysis-dependent recep-
tors Cell Mol Life Sci 2009662299ndash2318
36 Koshimizu H Senatorov V Loh YP Gozes I Neuropro-
tective protein and carboxypeptidase E J Mol Neurosci
2009391ndash8
37 Schafer M Brauer AU Savaskan NE Rathjen FG
Brummendorf T Neurotractinkilon promotes neurite
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
outgrowth and is expressed on reactive astrocytes after
entorhinal cortex lesion Mol Cell Neurosci 200529
580ndash590
38 Stoop MP Coulier L Rosenling T et al Quantitative
proteomics and metabolomics analysis of normal human
cerebrospinal fluid samples Mol Cell Proteomics 20109
2063ndash2075
39 Pepe G Chimienti G Liuzzi GM et al Lipoprotein(a) in
the cerebrospinal fluid of neurological patients with blood-
cerebrospinal fluid barrier dysfunction Clin Chem 2006
522043ndash2048
40 Nielsen MJ Petersen SV Jacobsen C et al Haptoglobin-
related protein is a high-affinity hemoglobin-binding plasma
protein Blood 20061082846ndash2849
10 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000015520152 Neurol Neuroimmunol Neuroinflamm
Vaibhav Singh E Danieumllle van Pelt Marcel P Stoop et al related proteins are associated with childhood-onset multiple sclerosisminusGray matter
This information is current as of September 24 2015
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is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
monophasic ADS and this study suggested dis-turbed axoglial biology during early MSevents6 In the present study we investigatedCSF a body fluid that reflects ongoing CNSpathology7 in a fully unbiased manner Sampleswere analyzed by high-resolution sensitivenano-liquid chromatography Orbitrap massspectrometry (LC-MS)8 Our aim was to findCSF protein markers expressed during a firstevent of CNS demyelination that can help todistinguish children with monophasic ADS(n 5 21) from children with MS (n 5 18)
METHODS Patients Children younger than 18 years old
who presented with a first acquired demyelinating event were
identified by the Dutch Study Group for Pediatric MS which in-
cludes 13 major pediatric neurology centers in the Netherlands
as described earlier9 Children were diagnosed with MS if they
had a second demyelinating attack of the CNS andor MRI evi-
dence of a new lesion at least 1 month after onset1 Clinical fea-
tures and physical examination defined initial clinical phenotypes
of the children This study included 41 children with ADS 22 of
whom had a monophasic disease course and 19 of whom were
diagnosed with MS The CSF samples were collected at first
clinical presentation Children were symptomatic at the time of
sampling Children were not on immunomodulatory treatment at
the time of sampling CSF samples were collected processed and
stored following previously described protocols810
Standard protocol approvals registrations and patientconsents The study was approved by the Clinical Research Eth-ics Board of Erasmus University Written informed consent was
obtained from all patients andor their families
Sample preparation and LC-MS measurements CSF sam-
ples (20 mL) were digested using an in-solution trypsin digestion
protocol as previously described11 Prepared samples were
analyzed by LC-MSMS using an Ultimate 3000 nano RSLC
system (Thermo Fischer Scientific Germering Germany)
online coupled to a hybrid linear ion trapOrbitrap mass
spectrometer (LTQ Orbitrap XL Thermo Fisher Scientific
Bremen Germany) using a data-dependent acquisition method
LC-MS data were analyzed using the Progenesis LC-MS software
package (version 36 Nonlinear Dynamics Ltd Newcastle-upon-
Tyne United Kingdom) Detailed LC-MS measurements and
protein identification and quantification information are
available in the e-Methods at Neurologyorgnn
Statistical analysis The Wilcoxon test (unpaired 2-tailed) was
used to analyze the differences in the abundance of peptides
between children with MS and those with monophasic ADS
p values 001 were considered statistically significant Relative
quantitative differences of peptide abundances between samples
of children with MS and samples of children with monophasic
ADS were calculated as log2 ratio between median abundances of
both groups A set of significantly distinct peptides and proteins
was determined by applying the following stringent criteria (1)
peptides that had at least 15-fold difference in expression at a
p value 001 (2) protein identified by at least 2 peptides and
(3) at least 40 differentially abundant peptides (p 001) per
protein whereby peptides of a given protein were required to
have representation in the same direction (increased or
decreased abundance in MS) In the above dataset we also
excluded those proteins that had only 1 peptide differentially
abundant out of a total 2 To verify these findings and to
determine the statistical background level we performed a
permutation analysis on the entire dataset (2260 peptides)
between samples of children with MS (n 5 18) and samples of
children with monophasic ADS (n 5 21) included with the
abovementioned criteria Through this process we determined
the statistical background level on a set of significantly distinct
peptides The random permutation test was repeated 1000 times
on the dataset with randomized sample group assignment and
the resulting thresholds were saved We used the R software
package for all calculations and graphics (R version 302)12
For other calculations we used SPSS 150 (SPSS Chicago IL)
and Microsoft Excel 2010
RESULTS Patient characteristics We analyzed CSFsamples of 18 children with MS and 21 with mono-phasic ADS One patient with MS and 1 patient withmonophasic ADS were excluded because 200required alignment vectors (weak alignment) werefound during Progenesis LC-MS analysis Of the 18children diagnosed with MS 5 had ON 2 had TM 3had clinical monofocal symptoms and 8 had clinicalpolyfocal symptoms as their presenting symptoms atonset Of the 21 children with monophasic ADS 2had ON 2 had TM 8 had clinical polyfocalsymptoms and 9 had ADEM as their presentingsymptoms at onset No significant differences wereobserved between the 2 groups in terms of sex CSFlevels of total protein and albumin albumin CSFserum quotient CSF leukocyte count and CSFIgG concentration The mean age at onset ofchildren diagnosed with MS (1417 6 15 years)was found to be significantly higher than that ofchildren with monophasic ADS (689 6 49 years)reflecting the epidemiology of these phenotypes Inaddition elevated CSF IgG index and positiveoligoclonal bands were more frequent in childrenwith MS (p 001) Patient characteristics areshown in table 1
Identification of proteins that discriminate MS from
monophasic ADSWe detected 50119 peptide precur-sors from Progenesis label-free analysis LC-MSexperiment from all trypsin-digested protein in CSFsamples A Mascot database search in the humansubset of the Uniprot database resulted into 2260unique peptides that corresponded to 318 proteins(table e-1) The total protein concentrations ofdigested peptide samples quantified (integrated UVarea at 214 nm) during LC-MS measurements didnot show any significant difference (p 5 054)between CSF samples of children with MS andthose of children with monophasic ADS To checktechnical variability we measured 12 reference samples(pooled CSF samples from all patients) at regularintervals Here too the total protein concentrations ofdigested peptide samples quantified (integrated UV
2 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
area at 214 nm) during LC-MS measurements did notshow any difference between reference group 1 (n5 6)and reference group 2 (n 5 6) (reference group 138294 6 15328 reference group 2 42934 6
66190 p 5 03) In addition a number of MSMSfragmentation spectra did not show any significantdifference between samples of children with MS andthose of children with monophasic ADS In particularthe measured MSMS fragmentation spectra for MSand monophasic ADS samples were 16796 6 1795and 16971 6 1153 (p 5 072) respectivelyMoreover the database identified MSMS spectra forMS and monophasic ADS samples as 1607 6 280and 1626 6 256 (p 5 083) respectively
Comparing the abundance of identified peptides(n 5 2260) from CSF samples of children withMS and children with monophasic ADS using thestringent criteria described in the statistical analysiswe found a total of 88 differentially abundant pep-tides (tables e-2 and e-3) Of these 88 peptides 53were significantly increased (table 2 and table e-2) and35 significantly decreased in CSF samples of childrenwith MS (table 2 and table e-3) compared with sam-ples of children with monophasic ADS Peptides withincreased abundance (n 5 53) in the MS group cor-responded to 14 proteins and peptides with decreasedabundance (n 5 35) corresponded to 7 proteins Aninventory of these 21 proteins is given in table 2Moreover fold expression difference between theMS and monophasic ADS groups and statistical signif-icance are plotted simultaneously for our entire dataset(n5 2260 peptides) as a volcano plot (figure 1) Thispermutation analysis resulted in 20 6 41 (median 9)
false-positive peptide markers which indicated thatour observations are not due to chance alone becausemore than 90 of the permutations yielded 0ndash4 sig-nificant hits (ie in 900) Only 4 times out of 1000were more than 88 hits with low p value detected (falsediscovery rate 04) Contrasting this to mere back-ground chance 88 peptides (true hits) were identifiedfrom the actual dataset Therefore comparison of per-mutated data with the real data indicated that theoccurrence of differentially abundant peptides relatedto MS or monophasic ADS was highly significant (p0001) The outcome of the permutation test is shownas a histogram (figure e-1)
Identified proteins with increased abundance(n 5 14) in MS (table 2 and table e-2) were the fol-lowing amyloid-like protein 2 (22 2 significant pep-tides for a total of 2 peptides) neurofascin (33)carboxypeptidase E (23) neuronal growth regulator1 (23) contactin-2 (49) amyloid beta A4 (611)brevican core protein (57) disintegrin and metallopro-teinase domain-containing protein 22 (22) tyrosine-protein phosphatase non-receptor type substrate1 (34) dickkopf-related protein 3 (611) neuronalcell adhesion molecule (918) Ig kappa chain V-IIIregion POM (22) Ig gamma-1 chain C region(511) and kallikrein-6 (47) Proteins identifiedwith decreased abundance (n 5 7) in MS (table 2and table e-3) were the following apolipoproteinB-100 (717) C4b-binding protein alpha chain(24) haptoglobin (1829) haptoglobin-related pro-tein (24) leucine-rich alpha-2-glycoprotein (23)monocyte differentiation antigen CD14 (33) andprotein diaphanous homolog 1 (22) The function of
Table 1 Clinical characteristics of children with multiple sclerosis and monophasic acquired demyelinating syndrome
Monophasic ADS (n 5 21) MS (n 5 18)p Valuea
monophasicADS vs MSMean 6 SD Median Range Mean 6 SD Median Range
Age at onset y 689 6 491 (n 5 21) 581 114ndash1711 1417 6 150 (n 5 18) 1429 1114ndash1621 001
Sex females 714 NA NA 611 NA NA NS
Protein gL 036 6 021 (n 5 21) 03 018ndash115 033 6 014 (n 5 17) 032 019ndash075 NS
Albumin gL 023 6 020 (n 5 11) 016 011ndash079 018 6 007 (n 5 14) 017 008ndash036 NS
Leukocytes 3106 3712 6 3491 (n 5 20) 285 1ndash118 2098 6 2568 (n 5 17) 13 10ndash87 NS
IgG gL 005 6 007 (n 5 11) 003 001ndash027 005 6 003 (n 5 13) 005 003ndash012 NS
IgG index 061 6 010 (n 5 12) 058 05ndash079 136 6 072 (n 5 17) 1 067ndash3 001
Elevated IgGindexb n ()
2 (167) NA NA 16 (941) NA NA 001
Positive OCB n () 2 (125) (n 5 16) NA NA 14 (875) (n 5 16) NA NA 001
Relapsing disease n () 0 (0) NA NA 18 (100) NA NA 001
Abbreviations ADS 5 acquired demyelinating syndrome MS 5 multiple sclerosis NA 5 not applicable NS 5 not significant OCB 5 oligoclonal bandsThis table shows clinical characteristics and routine CSF findings of children with MS (at disease onset) and children with monophasic ADS Data arepresented as the mean 6 SD or median and range In case of missing data the number of patients with available data is indicated in parenthesesaCalculated by Mann-Whitney test and p 001 was considered significantb IgG index considered elevated at 068 or higher4
Neurology Neuroimmunology amp Neuroinflammation 3
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
these 14 proteins and overlap with previous stud-ies613 are summarized in table 3 Among the 14 pro-teins with increased abundance in MS 12 wereassociated with CNS structure and functions (86)especially the gray matter (table 3) whereas only 17of the total identified proteins were related to CNSstructure and functions The 7 proteins with decreasedabundance in MS (relative to the monophasic ADSgroup) were components of the innate immune systemand inflammation (table 4)
For each peptide that passed (53 increased and 35decreased in MS) all stringent criteria (described inthe statistical analysis) the associated protein data-base search identification details p value fold expres-sion difference total number of peptides per proteinnumber of peptides identified below 001 medianabundance and frequencyoccurrence of identifica-tions by MSMS spectra are shown in tables e-2and e-3 Proteins identified in the current study didnot exhibit any myelin-related proteins (eg myelin
Table 2 Identification of proteins differentially abundant in CSF samples of children with MS (n 5 18) andsamples of children with monophasic ADS (n 5 21)
Trend in MSa DescriptionSigntotalb
Fold changemean (minndashmax)c p Valued mean (minndashmax)
Increased abundancein MS (n 5 14)
Amyloid-like protein 2 22 38 (2ndash55) 0006 (0003ndash0008)
Neurofascin 33 25 (21ndash3) 0002 (0001ndash0002)
Carboxypeptidase E 23 24 (21ndash26) 00004 (00002ndash00005)
Neuronal growth regulator 1 23 23 (21ndash25) 00008 (00008ndash00009)
Contactin-2 49 23 (15ndash32) 0002 (00005ndash0003)
Amyloid beta A4 protein 611 22 (18ndash26) 0002 (000003ndash0008)
Brevican core protein 57 21 (17ndash26) 0002 (00001ndash0005)
Disintegrin and metalloproteinasedomain-containing protein 22
22 19 (17ndash21) 00004 (00001ndash00006)
Tyrosine-protein phosphatasenon-receptor type substrate 1
34 19 (15ndash21) 0003 (0001ndash0006)
Dickkopf-related protein 3 611 18 (16ndash2) 0002 (000004ndash0006)
Neuronal cell adhesion molecule 918 18 (16ndash21) 0004 (0001ndash0009)
Ig kappa chain V-III region POM 22 18 (17ndash18) 0002 (00009ndash0003)
Ig gamma-1 chain C region 511 17 (15ndash18) 00016 (00002ndash0005)
Kallikrein-6 47 17 (15ndash19) 0001 (00006ndash0004)
Decreased abundancein MS (n 5 7)
Apolipoprotein B-100 717 661 (35ndash3930) 00026 (000002ndash001)
C4b-binding protein alpha chain 24 81 (29ndash134) 0008 (0007ndash0008)
Haptoglobin 1829 37 (2ndash121) 0002 (000004ndash001)
Haptoglobin-related protein 24 33 (31ndash35) 00002 (000001ndash00005)
Leucine-rich alpha-2-glycoprotein 23 26 (23ndash3) 0008 (0007ndash0009)
Monocyte differentiation antigen CD14 33 19 (18ndash2) 0006 (0004ndash0009)
Protein diaphanous homolog 1 22 18 (17ndash19) 0005 (0001ndash0009)
Abbreviations ADS 5 acquired demyelinating syndrome MS 5 multiple sclerosisThe table shows 21 proteins of which 14 were identified with increased abundance in CSF samples of children with MSand 7 were identified with decreased abundance in CSF samples of children with MS The table includes the direction ofdifference (trend) in MS name of the protein (description) fold expression difference and p value All proteins given in thetable were identified with at least 2 unique peptides differentially abundant peptides (p 001) had at least 15-folddifference in expression (median) between groups and for the same protein 40 of identified peptides were differentiallyabundant with the expression in the same direction (ie either higher or lower in MS) of the same protein Details of eachpeptide of the indicated proteins are listed in tables e-2 and e-3a Protein abundance is either significantly increased or decreased in children with MS compared to children withmonophasic ADSbNumber of differentially identified peptides p 001total number of identified peptides for the same proteinc Fold expression difference calculated based on median abundance from 18 patients with MS and 21 patients with ADSShown is the average of fold difference for all peptides of the same protein Minimum and maximum range for the same isindicateddCalculated by Wilcoxon test Given in the table is the mean p value and range for all differentially abundant peptides of thesame protein
4 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
oligodendrocyte glycoprotein and myelin basicprotein)
We performed a correlation analysis to examinethe influence of age at onset on the abundance ofthe 88 candidate peptides in children with MS and
monophasic ADS We found a mean coefficient ofdetermination (6SD) of 004 6 006 for MS and005 6 005 for monophasic ADS Thus no signif-icant correlation was found between age and peptideabundance for children with MS and monophasicADS for all 88 peptides
DISCUSSION In the current study we used a LC-MS proteomic approach to search for differences inCSF proteome between children with MS andchildren with monophasic ADS A benefit of thisOrbitrap technique is the ability to identifyrelatively vast amounts of different peptides andassess their abundances in a small sample volumeWe observed a striking difference between the 2groups (children with monophasic ADS vs MS)using stringent statistical criteria We searched forthe known functions of the 88 peptides correspondingto 21 distinctive proteins (14 increased and 7decreased in abundance in MS) using biologicaldatabases (wwwgeneontologyorg and wwwnextprotorg) and literature Recently 2 research groups613
demonstrated the identification of axoglial and graymatter proteins using mass spectrometry in CSF ofpatients with MS Similar to our study Dhaunchaket al6 compared CSF of 8 children with MS with CSFof 11 children with monophasic ADS The overlap ofsome proteins in the MS group is noticeable (egcarboxypeptidase E) despite clear differences insample handling such as depletion of abundantproteins with possible carrier function for otherproteins and exclusion of proteins with less than 5 kDaweight (table 3)
Our results show overlap with molecules identi-fied in the CSF of adults with acute-onset MS bySchutzer et al13 who performed mass spectrometryanalysis in CSF of patients with clinically isolatedsyndrome (CIS) vs those with established relapsingremitting MS (RRMS) and controls (table 3) Theyfound proteins that distinguished patients with CISfrom both patients with established RRMS and con-trols For example they found a significant increase inkallikrein-6 and dickkopf-related protein 3 at firstclinical onset compared to patients with establishedRRMS and controls (table 3) They also found a sig-nificant increase in contactin-2 (neuronal membraneprotein) in patients with a first attack of MS relativeto those with established RRMS (table 3)13
Our findings illustrate that the neurodegenerativearm of MS neuropathology is already active at the ear-liest stage of clinical disease also in children Consis-tent with the findings of others and our own previousstudies614ndash16 we again observed a striking lack ofmyelin proteins in these clear-cut cases of acute demy-elination This may not directly imply absence ofsuch free proteins in this type of pathology but rather
Figure 1 Volcano plot
Peptides (n 5 2260) showing distribution of fold change and statistical significance In thisplot each point represents a peptide and shows the ratio between CSF samples of childrenwith multiple sclerosis (MS) (n5 18) and samples of children with monophasic acquire demy-elinating syndromes (ADS) (n5 21) plotted against the level of statistical significance Y-axisshows p values obtained (plotted at log10) from a Wilcoxon test performed between abun-dances of peptides X-axis shows the ratio of the median between MS and monophasic ADSsamples (plotted on log2) (A) Above the dashed horizontal line red points (n 5 53 peptides)were found with increased abundance (right side of the vertical line) and green points (n5 35peptides) with decreased abundance (left side of the vertical line) in the MS group (comparedto the monophasic ADS group) (B) Peptides shown in gray below the dashed horizontal linedid not pass the stringent statistical criteria for identification of a candidate peptide
Neurology Neuroimmunology amp Neuroinflammation 5
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
it may reflect the specific physicochemical propertiesof the hydrophobic myelin components and perhapsdifferent pathways of elimination from the CSF (egvia draining macrophages)17 In any case we must becautious in using the dominant presence of CNS graymatter over white matter proteins as proof that neuro-degeneration is a primary event in MS and wouldprecede demyelination The presence of CNS graymatter may simply represent damage by inflamma-tion and the molecules identified may lead to a better
understanding of this presumed inflammation-induced neurodegeneration It should be stressed thatnot all differentially abundant proteins were overrep-resented in MS some were underrepresented point-ing at more complex mechanisms such as aperturbation in the physiology of the axoglial appara-tus (table 2 and tables e-2 and e-3)6 A confoundingfactor in our study could be the fact that the groupswere not matched according to age because of theoccurrence of monophasic disease at younger ages
Table 3 Function of proteins identified with increased abundance in CSF samples of children with MS compared with samples of children withmonophasic ADS
Protein (accession no) Functions and expressions Dhaunchak et al6 Schutzer et al13
Amyloid-like protein 2 (Q06481) Amyloid precursor protein family memoryprocesses concentrated in synapses18 regulatesneuronal stem cell differentiation during corticaldevelopment
3 variant
Neurofascin (O94856) Synapse formation located at CNS paranodaldomain and expressed by oligodendrocytes20
target for autoantibody-mediated axonal injury inMS21
NS 3
Contactin-2 (Q02246) Axon connection majorly expressed on CNSjuxtaparanodal domain1920
NS
Amyloid beta A4 protein (P05067) Component of amyloid plaques (Alzheimer brains)35
neuronal adhesion3 3
Brevican core protein (Q96GW7) Major proteoglycan in perisynaptic extracellularmatrix of brain inhibits neurite outgrowth fromcerebellar granule neurons23
NS Decreased expression in first-attack CIS-MS relative toestablished RRMS and controls
Carboxypeptidase E (P16870) Found in brain and throughout neuroendocrinesystem involved in synthesis of mostneuropeptides36
Same trend (4 sign21 total p 5 0004)
3
Neuronal growth regulator1 (Q7Z3B1)
Promotes outgrowth and is expressed on reactiveastrocytes after entorhinal cortex lesion (in mice)37
NS 3
Tyrosine-protein phosphatase non-receptor type substrate 1 (P78324)
Supports adhesion of cerebellar neurons neuriteoutgrowth and glial cell attachment28
3 3
Neuronal cell adhesion molecule(Q92823)
Localized at the node of Ranvier and in unmyelinatedaxons paranodal region of CNS axoglial contact19
NS
Disintegrin and metalloproteinasedomain-containing protein 22(Q9P0K1)
Expressed in the juxtaparanodal complex19 Not same trend (2 sign10 total p 5 001 002)
3
Dickkopf-related protein 3 (Q9UBP4) Expressed in the brain and spinal cord synapseformation13
3
Kallikrein-6 (Q92876) Elevated in active MS26 regulates early CNSdemyelination in a viral (mouse) model of MS26
3
Ig gamma-1 chain C region (P01857) Elevated in adult MS and CIS compared tononinflammatory neurologic diseases38
3 3
Ig kappa chain V-III region POM(P01624)
Elevated in adult MS compared to noninflammatoryand inflammatory neurologic diseases38
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis NS 5 not significant RRMS 5
relapsing remitting MS3 5 protein was not identified variant 5 variant of same protein was found with increased expression in first-attack CIS-MS group compared toestablished RRMS and controls 5 increased expression in first-attack CIS-MS group compared to established RRMS and controls 5 protein wasfound with increased expression in first-attack CIS-MS relative to RRMS but with decreased expression in first-attack CIS-MS vs controls NS 5 proteinwas detected in pediatric CSF samples but no statistical difference in their abundance was reported between MS and monophasic ADS group comparisonsame trend (trend is the direction of difference in MS) 5 when abundance of the protein was compared between 2 group (MS vs monophasic ADS)expression of the identified protein showed overlap with our study not same trend 5 when abundance of protein was compared expression of theidentified protein did not show overlap with our studyNodes paranodes juxtaparanodes (begins at the innermost axoglia junction of paranode) and internodes are structural parts of a myelinated axonSummary of the function of differentially abundant increased protein markers in MS and their overlap with previous studies Most of the proteins wereassigned as either neuronal or immune-related molecules based on previous reports and database searches The first column shows the name of the proteinand Uniprot accession number The second column shows the function of the proteins The third column shows comparison with a previous study onchildren with MS (Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourth column showsoverlap with the work of Schutzer et al who used CIS-MS (n 5 9 age 18ndash42 years) and established MS and controls (n 5 6 age 31ndash54 years)
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
We doubt that this influenced the results howeverbecause we did not see an age effect on the abundanceof the 21 identified proteins
Of the 14 proteins with increased abundance inMS (tables 2 and 3) 2 were associated with the amy-loid beta A4 protein family Amyloid-like protein 2 ismainly concentrated at neuronal synapses18 and has arole in memory processes whereas amyloid beta A4protein is associated with neurite growth neuronaladhesion and axonogenesis (table 3) Six proteins(contactin-219 neurofascin20 neuronal growth regu-lator 1 brevican core protein6 neuronal cell adhesionmolecule19 and disintegrin and metalloproteinasedomain-containing protein 22)19 are located at theparanodal and juxtaparanodal region of the CNS ofmyelinated axons (table 3) Contactin-2 is an axonalglycoprotein at the juxtaparanodal domain of myeli-nated axons13 Neurofascin plays a role in the assem-bly of nodes of Ranvier in the CNS21 Two isoformsof neurofascin have been shown to interact withcontactin-associated protein and contactin-1 to forma paranodal junction that attaches the myelin loop tothe axon and helps to separate voltage-gated sodiumchannels at nodal and potassium channels at juxtapar-anodal regions20 Disruption of neurofascin localiza-tion reveals early changes preceding demyelinationand remyelination in MS22 Of interest contactin-2and neurofascin have previously been reported asautoimmune targets in MS20 Neuronal growth
regulator 1 is located at the paranodal region of theCNS and plays a role in axoglia contact at the node ofRanvier Identification of contactin-2 and neurofas-cin in the CSF of children with MS is consistent witha previous study6 however they did not find a sig-nificant difference in the CSF of children with MSand monophasic ADS (tables 2 and 3) Brevican coreprotein is known as a CNS-specific proteoglycan19 atthe surface of neuroglial sheaths where it is enrichedin perisynaptic extracellular matrix23
Among the 14 proteins with increased abun-dance in MS the proteasespeptidases protein car-boxypeptidase E plays a role in the synthesis ofmost neuropeptides24 Disintegrin and metalloprotei-nase domain-containing protein 22 is highly expressedin the brain and localized at the juxtaparanode19 Thismolecule is presumed to be a major neuronal recep-tor25 Another brain-related protease is kallikrein-6which is a secreted serine protease13 It regulates earlyCNS demyelination in a viral model (expression in thebrain and spinal cord of mice) of MS26 In additionelevated levels of kallikrein-6 in CSF have been re-ported in adults with MS compared to neurologic con-trols (table 3)27
Among the other proteins with increased abun-dance in MS tyrosine-protein phosphatase non-receptor type substrate 1 is implicated in neuriteoutgrowth and glia cell attachment and has beenshown to support adhesions of cerebellar neurons28
Table 4 Function of proteins identified with decreased abundance in CSF samples of children with MScompared to samples of children with monophasic ADS
Protein (accession no) FunctionsDhaunchaket al6
Schutzeret al13
Apolipoprotein B-100 (P04114) Innate immune-related not produced inCNS39
3 3
C4b-binding protein alpha chain (P04003) Innate immune defense involved incomplement activity31
3 3
Haptoglobin (P00738) Innate immune defense29 3 3
Haptoglobin-related protein (P00739) Innate immune defense40 3 3
Leucine-rich alpha-2-glycoprotein (P02750) Induced by inflammation and involved incell adhesion high expression in the deepcerebral cortex25 novel marker ofgranulocytic differentiation29
3 3
Monocyte differentiation antigen CD14(P08571)
Main modulator of innate immune system32 3 3
Protein diaphanous homolog 1 (O60610) Coordinates cellular dynamics byregulating microfilament and microtubulefunction role in cellndashmatrix adhesionsvariant related to innate immune function34
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis3 5 Protein was not identifiedSummary of the function of differentially abundant decreased protein markers in MS and their overlap with previousstudies Most of the proteins were assigned as either neuronal or immune-related molecules based on previous reports anddatabase searches The first column shows the name of the protein and Uniprot accession number The second columnshows the function of the proteins The third column shows comparison with a previous study on children with MS(Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourthcolumn shows overlap with the work of Schutzer et al who used CIS-MS (n5 9 age 18ndash42 years) and established MS andcontrols (n 5 6 age 31ndash54 years)
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
The highest expression of dickkopf-related protein 3is reported in the brain and spinal cord (table 3)13
Thus the majority of proteins with increased abun-dance in our MS group (compared to the monophasicADS group) were neuronal-related with the excep-tion of 2 immune functionndashrelated proteins (Iggamma-1 chain C region and Ig kappa chain V-IIIregion POM) (table 3)
Our study reports 7 proteins with decreased abun-dance in pediatric-onset MS compared to monopha-sic ADS (table 2 and table e-3) Six of these 7 proteinshave previously been reported as specific componentsof innate immune functions (table 4) Haptoglobinan acute phase protein29 was identified as the mostdistinctive protein of those that were elevated in chil-dren with monophasic ADS (compared to childrenwith MS) (1829 18 significant peptides for a total of29 peptides) Another study in adults showedincreased haptoglobin concentration in patients withNMO compared to adult patients with MS30 C4b-binding protein alpha chain is a crucial componentof complement cascade and inhibits the function ofcomplement component31 Of interest we alsofound monocyte differentiation antigen CD14which is mainly expressed on cells of monocyticlineage (macrophages and monocytes)32 HigherCD14 levels might be linked with increased levelsof cytokines triggering inflammatory processes inchildren with monophasic ADS Monocytic cellssecrete soluble sCD14 (activation product of acti-vated monocytes) so this may affect the enormousmacrophage activation during acute monophasicADS33 Among the 7 identified proteins proteindiaphanous homolog 1 has a role in cell adhesionand is expressed in brain and its variants arerequired for innate immune response to Gram-negative bacterial infection34
Overall in the MS group we found a significantoverrepresentation of proteins associated withchanges in CNS gray matter axons synaptic regula-tion node of Ranvier and brain proteases (table 3)Several of these proteins are part of the axoglial appa-ratus and may relate to disturbances in axoglia inter-action6 (table 3) Two of them (contactin-2 andneurofascin) have been identified as possible axoglialautoantigens in MS20 The overlap of proteinsobserved in previous studies613 (table 3) as part ofthe axoglia apparatus and gray matter provides vali-dation for these proteins
These pathologically relevant proteins (mostlyCNS gray matterndashrelated) that are elevated in theCSF of children with early-onset MS might beinvolved in early disease mechanisms Further insightinto the role of these proteins can be useful for under-standing the disease process Moreover such proteinsmight be useful as a future tool to differentiate
children with MS from children with monophasicADS In addition knowing the start of MS could leadto earlier treatment with disease-modifying therapiesHowever the current research is designated as a dis-covery phase study which serves as a basis for thefollow-up verification and validation phase studiesthat can provide clinically valuable biomarkers Inthe future it would be interesting to further validateour findings with an independent technology and inan independent sample group
The data presented in this study indicate thatmonophasic ADS can be differentiated from MS inchildren primarily by CNS gray matter proteins andimmune-related proteins Our findings point to per-turbed axoglial physiology as a hallmark of the earliestevents of MS pathogenesis
AUTHOR CONTRIBUTIONSThe work was carried out in collaboration among all authors VS
TML and RQH designed the experiment VS and CS performed
the experiment and statistical analysis EDvP IAK and CEC-B
were responsible for sample collection VS EDvP MPS CS
RFN TML and RQH interpreted the results and wrote the article
All authors critically revised and approved the final manuscript
ACKNOWLEDGMENTThe authors thank all the children and their families who participated in
the Dutch Study for Pediatric MS
STUDY FUNDINGThis work was supported by a program grant provided by the EC 7th
frame work programme of Marie Curie Initial Training NetworkmdashThe
United Europeans for the Development of Pharmacogenomics in Mul-
tiple Sclerosis (UEPHAmdashMS grant PITNmdashGAmdash2008212877) Neth-
erlands Multiple Sclerosis Research Foundation to the Rotterdam MS
Centre ErasMS the Netherlands Organization for Scientific Research
(ZONmdashMW RQH) and Hersenstichting Nederland and Zenith grant
93511034 from the Netherlands Organization for Scientific Research
(NWO)
DISCLOSUREV Singh ED van Pelt MP Stoop C Stingl and IA Ketelslegers
report no disclosures RF Neuteboom is on the scientific advisory board
for LAREB CE Catsman-Berrevoets and TM Luider report no disclo-
sures RQ Hintzen is on the advisory board for Biogen Idec Roche and
Sanofi received research support from Biogen Idec Merck-Serono
Roche Genzyme Novartis Dutch MS Society and European Commis-
sion and is on the editorial board for Multiple Sclerosis and Related
Disorders Go to Neurologyorgnn for full disclosure forms
Received April 14 2015 Accepted in final form June 25 2015
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atric MSSG Consensus definitions proposed for pediatric
multiple sclerosis and related disorders Neurology 2007
68S7ndashS12
2 Banwell B Ghezzi A Bar-Or A Mikaeloff Y Tardieu M
Multiple sclerosis in children clinical diagnosis therapeu-
tic strategies and future directions Lancet Neurol 20076
887ndash902
3 Banwell B Bar-Or A Arnold DL et al Clinical environ-
mental and genetic determinants of multiple sclerosis in
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
children with acute demyelination a prospective national
cohort study Lancet Neurol 201110436ndash445
4 Neuteboom RF Boon M Catsman Berrevoets CE et al
Prognostic factors after a first attack of inflammatory CNS
demyelination in children Neurology 200871967ndash973
5 Krupp LB Tardieu M Amato MP et al International
Pediatric Multiple Sclerosis Study Group criteria for pedi-
atric multiple sclerosis and immune-mediated central ner-
vous system demyelinating disorders revisions to the 2007
definitions Mult Scler 2013191261ndash1267
6 Dhaunchak AS Becker C Schulman H et al Implication
of perturbed axoglial apparatus in early pediatric multiple
sclerosis Ann Neurol 201271601ndash613
7 Singh V Hintzen RQ Luider TM Stoop MP Proteomics
technologies for biomarker discovery in multiple sclerosis
J Neuroimmunol 201224840ndash47
8 Singh V Stoop MP Stingl C et al Cerebrospinal-fluid-
derived immunoglobulin G of different multiple sclerosis
patients shares mutated sequences in complementarity
determining regions Mol Cell Proteomics 201312
3924ndash3934
9 Ketelslegers IA Catsman-Berrevoets CE Neuteboom RF
et al Incidence of acquired demyelinating syndromes of
the CNS in Dutch children a nationwide study J Neurol
20122591929ndash1935
10 Teunissen CE Petzold A Bennett JL et al A consensus
protocol for the standardization of cerebrospinal fluid col-
lection and biobanking Neurology 2009731914ndash1922
11 Stoop MP Singh V Stingl C et al Effects of natalizumab
treatment on the cerebrospinal fluid proteome of multiple
sclerosis patients J Proteome Res 2013121101ndash1107
12 Team RC R A Language and Environment for Statistical
Computing [online] Available at httpwwwR-project
org Accessed February 4 2014
13 Schutzer SE Angel TE Liu T et al Gray matter is tar-
geted in first-attack multiple sclerosis PLoS One 20138
e66117
14 Stoop MP Dekker LJ Titulaer MK et al Multiple
sclerosis-related proteins identified in cerebrospinal fluid
by advanced mass spectrometry Proteomics 20088
1576ndash1585
15 Stoop MP Dekker LJ Titulaer MK et al Quantitative
matrix-assisted laser desorption ionization-fourier trans-
form ion cyclotron resonance (MALDI-FT-ICR) peptide
profiling and identification of multiple-sclerosis-related
proteins J Proteome Res 200981404ndash1414
16 Stoop MP Singh V Dekker LJ et al Proteomics com-
parison of cerebrospinal fluid of relapsing remitting and
primary progressive multiple sclerosis PLoS One 20105
e12442
17 van Zwam M Samsom JN Nieuwenhuis EE et al Mye-
lin ingestion alters macrophage antigen-presenting func-
tion in vitro and in vivo J Leukoc Biol 201190123ndash132
18 Wasco W Gurubhagavatula S Paradis MD et al Isola-
tion and characterization of APLP2 encoding a homologue
of the Alzheimerrsquos associated amyloid beta protein precur-
sor Nat Genet 1993595ndash100
19 Faivre-Sarrailh C Devaux JJ Neuro-glial interactions at
the nodes of Ranvier implication in health and diseases
Front Cell Neurosci 20137196
20 Derfuss T Linington C Hohlfeld R Meinl E Axo-glial
antigens as targets in multiple sclerosis implications for
axonal and grey matter injury J Mol Med (Berl) 2010
88753ndash761
21 Mathey EK Derfuss T Storch MK et al Neurofascin as a
novel target for autoantibody-mediated axonal injury
J Exp Med 20072042363ndash2372
22 Howell OW Palser A Polito A et al Disruption of neu-
rofascin localization reveals early changes preceding demy-
elination and remyelination in multiple sclerosis Brain
20061293173ndash3185
23 Yamada H Fredette B Shitara K et al The brain chon-
droitin sulfate proteoglycan brevican associates with astro-
cytes ensheathing cerebellar glomeruli and inhibits neurite
outgrowth from granule neurons J Neurosci 199717
7784ndash7795
24 Vilijn MH Das B Kessler JA Fricker LD Cultured as-
trocytes and neurons synthesize and secrete carboxypepti-
dase E a neuropeptide-processing enzyme J Neurochem
1989531487ndash1493
25 Ozkaynak E Abello G Jaegle M et al Adam22 is a major
neuronal receptor for Lgi4-mediated Schwann cell signal-
ing J Neurosci 2010303857ndash3864
26 Scarisbrick IA Yoon H Panos M et al Kallikrein 6 reg-
ulates early CNS demyelination in a viral model of multi-
ple sclerosis Brain Pathol 201222709ndash722
27 Hebb AL Bhan V Wishart AD Moore CS
Robertson GS Human kallikrein 6 cerebrospinal levels
are elevated in multiple sclerosis Curr Drug Discov Tech-
nol 20107137ndash140
28 Latour S Tanaka H Demeure C et al Bidirectional
negative regulation of human T and dendritic cells by
CD47 and its cognate receptor signal-regulator protein-
alpha down-regulation of IL-12 responsiveness and inhi-
bition of dendritic cell activation J Immunol 2001167
2547ndash2554
29 OrsquoDonnell LC Druhan LJ Avalos BR Molecular charac-
terization and expression analysis of leucine-rich alpha2-
glycoprotein a novel marker of granulocytic differentiation
J Leukoc Biol 200272478ndash485
30 Chang KH Tseng MY Ro LS et al Analyses of hapto-
globin level in the cerebrospinal fluid and serum of pa-
tients with neuromyelitis optica and multiple sclerosis
Clin Chim Acta 201341726ndash30
31 Chung LP Reid KB Structural and functional studies on
C4b-binding protein a regulatory component of the
human complement system Biosci Rep 19855855ndash865
32 Kurne A Sayat G Aydin OF et al Lack of association of
the CD14Cmdash159T polymorphism with susceptibility
and progression parameters in Turkish multiple sclerosis
patients J Neuroimmunol 201225083ndash86
33 Kitchens RL Thompson PA Modulatory effects of sCD14
and LBP on LPS-host cell interactions J Endotoxin Res
200511225ndash229
34 Huh JR Foe I Muro I et al The Drosophila inhibitor of
apoptosis (IAP) DIAP2 is dispensable for cell survival
required for the innate immune response to gram-negative
bacterial infection and can be negatively regulated by the
reaperhidgrim family of IAP-binding apoptosis inducers
J Biol Chem 20072822056ndash2068
35 Jacobsen KT Iverfeldt K Amyloid precursor protein and
its homologues a family of proteolysis-dependent recep-
tors Cell Mol Life Sci 2009662299ndash2318
36 Koshimizu H Senatorov V Loh YP Gozes I Neuropro-
tective protein and carboxypeptidase E J Mol Neurosci
2009391ndash8
37 Schafer M Brauer AU Savaskan NE Rathjen FG
Brummendorf T Neurotractinkilon promotes neurite
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
outgrowth and is expressed on reactive astrocytes after
entorhinal cortex lesion Mol Cell Neurosci 200529
580ndash590
38 Stoop MP Coulier L Rosenling T et al Quantitative
proteomics and metabolomics analysis of normal human
cerebrospinal fluid samples Mol Cell Proteomics 20109
2063ndash2075
39 Pepe G Chimienti G Liuzzi GM et al Lipoprotein(a) in
the cerebrospinal fluid of neurological patients with blood-
cerebrospinal fluid barrier dysfunction Clin Chem 2006
522043ndash2048
40 Nielsen MJ Petersen SV Jacobsen C et al Haptoglobin-
related protein is a high-affinity hemoglobin-binding plasma
protein Blood 20061082846ndash2849
10 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000015520152 Neurol Neuroimmunol Neuroinflamm
Vaibhav Singh E Danieumllle van Pelt Marcel P Stoop et al related proteins are associated with childhood-onset multiple sclerosisminusGray matter
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area at 214 nm) during LC-MS measurements did notshow any difference between reference group 1 (n5 6)and reference group 2 (n 5 6) (reference group 138294 6 15328 reference group 2 42934 6
66190 p 5 03) In addition a number of MSMSfragmentation spectra did not show any significantdifference between samples of children with MS andthose of children with monophasic ADS In particularthe measured MSMS fragmentation spectra for MSand monophasic ADS samples were 16796 6 1795and 16971 6 1153 (p 5 072) respectivelyMoreover the database identified MSMS spectra forMS and monophasic ADS samples as 1607 6 280and 1626 6 256 (p 5 083) respectively
Comparing the abundance of identified peptides(n 5 2260) from CSF samples of children withMS and children with monophasic ADS using thestringent criteria described in the statistical analysiswe found a total of 88 differentially abundant pep-tides (tables e-2 and e-3) Of these 88 peptides 53were significantly increased (table 2 and table e-2) and35 significantly decreased in CSF samples of childrenwith MS (table 2 and table e-3) compared with sam-ples of children with monophasic ADS Peptides withincreased abundance (n 5 53) in the MS group cor-responded to 14 proteins and peptides with decreasedabundance (n 5 35) corresponded to 7 proteins Aninventory of these 21 proteins is given in table 2Moreover fold expression difference between theMS and monophasic ADS groups and statistical signif-icance are plotted simultaneously for our entire dataset(n5 2260 peptides) as a volcano plot (figure 1) Thispermutation analysis resulted in 20 6 41 (median 9)
false-positive peptide markers which indicated thatour observations are not due to chance alone becausemore than 90 of the permutations yielded 0ndash4 sig-nificant hits (ie in 900) Only 4 times out of 1000were more than 88 hits with low p value detected (falsediscovery rate 04) Contrasting this to mere back-ground chance 88 peptides (true hits) were identifiedfrom the actual dataset Therefore comparison of per-mutated data with the real data indicated that theoccurrence of differentially abundant peptides relatedto MS or monophasic ADS was highly significant (p0001) The outcome of the permutation test is shownas a histogram (figure e-1)
Identified proteins with increased abundance(n 5 14) in MS (table 2 and table e-2) were the fol-lowing amyloid-like protein 2 (22 2 significant pep-tides for a total of 2 peptides) neurofascin (33)carboxypeptidase E (23) neuronal growth regulator1 (23) contactin-2 (49) amyloid beta A4 (611)brevican core protein (57) disintegrin and metallopro-teinase domain-containing protein 22 (22) tyrosine-protein phosphatase non-receptor type substrate1 (34) dickkopf-related protein 3 (611) neuronalcell adhesion molecule (918) Ig kappa chain V-IIIregion POM (22) Ig gamma-1 chain C region(511) and kallikrein-6 (47) Proteins identifiedwith decreased abundance (n 5 7) in MS (table 2and table e-3) were the following apolipoproteinB-100 (717) C4b-binding protein alpha chain(24) haptoglobin (1829) haptoglobin-related pro-tein (24) leucine-rich alpha-2-glycoprotein (23)monocyte differentiation antigen CD14 (33) andprotein diaphanous homolog 1 (22) The function of
Table 1 Clinical characteristics of children with multiple sclerosis and monophasic acquired demyelinating syndrome
Monophasic ADS (n 5 21) MS (n 5 18)p Valuea
monophasicADS vs MSMean 6 SD Median Range Mean 6 SD Median Range
Age at onset y 689 6 491 (n 5 21) 581 114ndash1711 1417 6 150 (n 5 18) 1429 1114ndash1621 001
Sex females 714 NA NA 611 NA NA NS
Protein gL 036 6 021 (n 5 21) 03 018ndash115 033 6 014 (n 5 17) 032 019ndash075 NS
Albumin gL 023 6 020 (n 5 11) 016 011ndash079 018 6 007 (n 5 14) 017 008ndash036 NS
Leukocytes 3106 3712 6 3491 (n 5 20) 285 1ndash118 2098 6 2568 (n 5 17) 13 10ndash87 NS
IgG gL 005 6 007 (n 5 11) 003 001ndash027 005 6 003 (n 5 13) 005 003ndash012 NS
IgG index 061 6 010 (n 5 12) 058 05ndash079 136 6 072 (n 5 17) 1 067ndash3 001
Elevated IgGindexb n ()
2 (167) NA NA 16 (941) NA NA 001
Positive OCB n () 2 (125) (n 5 16) NA NA 14 (875) (n 5 16) NA NA 001
Relapsing disease n () 0 (0) NA NA 18 (100) NA NA 001
Abbreviations ADS 5 acquired demyelinating syndrome MS 5 multiple sclerosis NA 5 not applicable NS 5 not significant OCB 5 oligoclonal bandsThis table shows clinical characteristics and routine CSF findings of children with MS (at disease onset) and children with monophasic ADS Data arepresented as the mean 6 SD or median and range In case of missing data the number of patients with available data is indicated in parenthesesaCalculated by Mann-Whitney test and p 001 was considered significantb IgG index considered elevated at 068 or higher4
Neurology Neuroimmunology amp Neuroinflammation 3
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
these 14 proteins and overlap with previous stud-ies613 are summarized in table 3 Among the 14 pro-teins with increased abundance in MS 12 wereassociated with CNS structure and functions (86)especially the gray matter (table 3) whereas only 17of the total identified proteins were related to CNSstructure and functions The 7 proteins with decreasedabundance in MS (relative to the monophasic ADSgroup) were components of the innate immune systemand inflammation (table 4)
For each peptide that passed (53 increased and 35decreased in MS) all stringent criteria (described inthe statistical analysis) the associated protein data-base search identification details p value fold expres-sion difference total number of peptides per proteinnumber of peptides identified below 001 medianabundance and frequencyoccurrence of identifica-tions by MSMS spectra are shown in tables e-2and e-3 Proteins identified in the current study didnot exhibit any myelin-related proteins (eg myelin
Table 2 Identification of proteins differentially abundant in CSF samples of children with MS (n 5 18) andsamples of children with monophasic ADS (n 5 21)
Trend in MSa DescriptionSigntotalb
Fold changemean (minndashmax)c p Valued mean (minndashmax)
Increased abundancein MS (n 5 14)
Amyloid-like protein 2 22 38 (2ndash55) 0006 (0003ndash0008)
Neurofascin 33 25 (21ndash3) 0002 (0001ndash0002)
Carboxypeptidase E 23 24 (21ndash26) 00004 (00002ndash00005)
Neuronal growth regulator 1 23 23 (21ndash25) 00008 (00008ndash00009)
Contactin-2 49 23 (15ndash32) 0002 (00005ndash0003)
Amyloid beta A4 protein 611 22 (18ndash26) 0002 (000003ndash0008)
Brevican core protein 57 21 (17ndash26) 0002 (00001ndash0005)
Disintegrin and metalloproteinasedomain-containing protein 22
22 19 (17ndash21) 00004 (00001ndash00006)
Tyrosine-protein phosphatasenon-receptor type substrate 1
34 19 (15ndash21) 0003 (0001ndash0006)
Dickkopf-related protein 3 611 18 (16ndash2) 0002 (000004ndash0006)
Neuronal cell adhesion molecule 918 18 (16ndash21) 0004 (0001ndash0009)
Ig kappa chain V-III region POM 22 18 (17ndash18) 0002 (00009ndash0003)
Ig gamma-1 chain C region 511 17 (15ndash18) 00016 (00002ndash0005)
Kallikrein-6 47 17 (15ndash19) 0001 (00006ndash0004)
Decreased abundancein MS (n 5 7)
Apolipoprotein B-100 717 661 (35ndash3930) 00026 (000002ndash001)
C4b-binding protein alpha chain 24 81 (29ndash134) 0008 (0007ndash0008)
Haptoglobin 1829 37 (2ndash121) 0002 (000004ndash001)
Haptoglobin-related protein 24 33 (31ndash35) 00002 (000001ndash00005)
Leucine-rich alpha-2-glycoprotein 23 26 (23ndash3) 0008 (0007ndash0009)
Monocyte differentiation antigen CD14 33 19 (18ndash2) 0006 (0004ndash0009)
Protein diaphanous homolog 1 22 18 (17ndash19) 0005 (0001ndash0009)
Abbreviations ADS 5 acquired demyelinating syndrome MS 5 multiple sclerosisThe table shows 21 proteins of which 14 were identified with increased abundance in CSF samples of children with MSand 7 were identified with decreased abundance in CSF samples of children with MS The table includes the direction ofdifference (trend) in MS name of the protein (description) fold expression difference and p value All proteins given in thetable were identified with at least 2 unique peptides differentially abundant peptides (p 001) had at least 15-folddifference in expression (median) between groups and for the same protein 40 of identified peptides were differentiallyabundant with the expression in the same direction (ie either higher or lower in MS) of the same protein Details of eachpeptide of the indicated proteins are listed in tables e-2 and e-3a Protein abundance is either significantly increased or decreased in children with MS compared to children withmonophasic ADSbNumber of differentially identified peptides p 001total number of identified peptides for the same proteinc Fold expression difference calculated based on median abundance from 18 patients with MS and 21 patients with ADSShown is the average of fold difference for all peptides of the same protein Minimum and maximum range for the same isindicateddCalculated by Wilcoxon test Given in the table is the mean p value and range for all differentially abundant peptides of thesame protein
4 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
oligodendrocyte glycoprotein and myelin basicprotein)
We performed a correlation analysis to examinethe influence of age at onset on the abundance ofthe 88 candidate peptides in children with MS and
monophasic ADS We found a mean coefficient ofdetermination (6SD) of 004 6 006 for MS and005 6 005 for monophasic ADS Thus no signif-icant correlation was found between age and peptideabundance for children with MS and monophasicADS for all 88 peptides
DISCUSSION In the current study we used a LC-MS proteomic approach to search for differences inCSF proteome between children with MS andchildren with monophasic ADS A benefit of thisOrbitrap technique is the ability to identifyrelatively vast amounts of different peptides andassess their abundances in a small sample volumeWe observed a striking difference between the 2groups (children with monophasic ADS vs MS)using stringent statistical criteria We searched forthe known functions of the 88 peptides correspondingto 21 distinctive proteins (14 increased and 7decreased in abundance in MS) using biologicaldatabases (wwwgeneontologyorg and wwwnextprotorg) and literature Recently 2 research groups613
demonstrated the identification of axoglial and graymatter proteins using mass spectrometry in CSF ofpatients with MS Similar to our study Dhaunchaket al6 compared CSF of 8 children with MS with CSFof 11 children with monophasic ADS The overlap ofsome proteins in the MS group is noticeable (egcarboxypeptidase E) despite clear differences insample handling such as depletion of abundantproteins with possible carrier function for otherproteins and exclusion of proteins with less than 5 kDaweight (table 3)
Our results show overlap with molecules identi-fied in the CSF of adults with acute-onset MS bySchutzer et al13 who performed mass spectrometryanalysis in CSF of patients with clinically isolatedsyndrome (CIS) vs those with established relapsingremitting MS (RRMS) and controls (table 3) Theyfound proteins that distinguished patients with CISfrom both patients with established RRMS and con-trols For example they found a significant increase inkallikrein-6 and dickkopf-related protein 3 at firstclinical onset compared to patients with establishedRRMS and controls (table 3) They also found a sig-nificant increase in contactin-2 (neuronal membraneprotein) in patients with a first attack of MS relativeto those with established RRMS (table 3)13
Our findings illustrate that the neurodegenerativearm of MS neuropathology is already active at the ear-liest stage of clinical disease also in children Consis-tent with the findings of others and our own previousstudies614ndash16 we again observed a striking lack ofmyelin proteins in these clear-cut cases of acute demy-elination This may not directly imply absence ofsuch free proteins in this type of pathology but rather
Figure 1 Volcano plot
Peptides (n 5 2260) showing distribution of fold change and statistical significance In thisplot each point represents a peptide and shows the ratio between CSF samples of childrenwith multiple sclerosis (MS) (n5 18) and samples of children with monophasic acquire demy-elinating syndromes (ADS) (n5 21) plotted against the level of statistical significance Y-axisshows p values obtained (plotted at log10) from a Wilcoxon test performed between abun-dances of peptides X-axis shows the ratio of the median between MS and monophasic ADSsamples (plotted on log2) (A) Above the dashed horizontal line red points (n 5 53 peptides)were found with increased abundance (right side of the vertical line) and green points (n5 35peptides) with decreased abundance (left side of the vertical line) in the MS group (comparedto the monophasic ADS group) (B) Peptides shown in gray below the dashed horizontal linedid not pass the stringent statistical criteria for identification of a candidate peptide
Neurology Neuroimmunology amp Neuroinflammation 5
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
it may reflect the specific physicochemical propertiesof the hydrophobic myelin components and perhapsdifferent pathways of elimination from the CSF (egvia draining macrophages)17 In any case we must becautious in using the dominant presence of CNS graymatter over white matter proteins as proof that neuro-degeneration is a primary event in MS and wouldprecede demyelination The presence of CNS graymatter may simply represent damage by inflamma-tion and the molecules identified may lead to a better
understanding of this presumed inflammation-induced neurodegeneration It should be stressed thatnot all differentially abundant proteins were overrep-resented in MS some were underrepresented point-ing at more complex mechanisms such as aperturbation in the physiology of the axoglial appara-tus (table 2 and tables e-2 and e-3)6 A confoundingfactor in our study could be the fact that the groupswere not matched according to age because of theoccurrence of monophasic disease at younger ages
Table 3 Function of proteins identified with increased abundance in CSF samples of children with MS compared with samples of children withmonophasic ADS
Protein (accession no) Functions and expressions Dhaunchak et al6 Schutzer et al13
Amyloid-like protein 2 (Q06481) Amyloid precursor protein family memoryprocesses concentrated in synapses18 regulatesneuronal stem cell differentiation during corticaldevelopment
3 variant
Neurofascin (O94856) Synapse formation located at CNS paranodaldomain and expressed by oligodendrocytes20
target for autoantibody-mediated axonal injury inMS21
NS 3
Contactin-2 (Q02246) Axon connection majorly expressed on CNSjuxtaparanodal domain1920
NS
Amyloid beta A4 protein (P05067) Component of amyloid plaques (Alzheimer brains)35
neuronal adhesion3 3
Brevican core protein (Q96GW7) Major proteoglycan in perisynaptic extracellularmatrix of brain inhibits neurite outgrowth fromcerebellar granule neurons23
NS Decreased expression in first-attack CIS-MS relative toestablished RRMS and controls
Carboxypeptidase E (P16870) Found in brain and throughout neuroendocrinesystem involved in synthesis of mostneuropeptides36
Same trend (4 sign21 total p 5 0004)
3
Neuronal growth regulator1 (Q7Z3B1)
Promotes outgrowth and is expressed on reactiveastrocytes after entorhinal cortex lesion (in mice)37
NS 3
Tyrosine-protein phosphatase non-receptor type substrate 1 (P78324)
Supports adhesion of cerebellar neurons neuriteoutgrowth and glial cell attachment28
3 3
Neuronal cell adhesion molecule(Q92823)
Localized at the node of Ranvier and in unmyelinatedaxons paranodal region of CNS axoglial contact19
NS
Disintegrin and metalloproteinasedomain-containing protein 22(Q9P0K1)
Expressed in the juxtaparanodal complex19 Not same trend (2 sign10 total p 5 001 002)
3
Dickkopf-related protein 3 (Q9UBP4) Expressed in the brain and spinal cord synapseformation13
3
Kallikrein-6 (Q92876) Elevated in active MS26 regulates early CNSdemyelination in a viral (mouse) model of MS26
3
Ig gamma-1 chain C region (P01857) Elevated in adult MS and CIS compared tononinflammatory neurologic diseases38
3 3
Ig kappa chain V-III region POM(P01624)
Elevated in adult MS compared to noninflammatoryand inflammatory neurologic diseases38
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis NS 5 not significant RRMS 5
relapsing remitting MS3 5 protein was not identified variant 5 variant of same protein was found with increased expression in first-attack CIS-MS group compared toestablished RRMS and controls 5 increased expression in first-attack CIS-MS group compared to established RRMS and controls 5 protein wasfound with increased expression in first-attack CIS-MS relative to RRMS but with decreased expression in first-attack CIS-MS vs controls NS 5 proteinwas detected in pediatric CSF samples but no statistical difference in their abundance was reported between MS and monophasic ADS group comparisonsame trend (trend is the direction of difference in MS) 5 when abundance of the protein was compared between 2 group (MS vs monophasic ADS)expression of the identified protein showed overlap with our study not same trend 5 when abundance of protein was compared expression of theidentified protein did not show overlap with our studyNodes paranodes juxtaparanodes (begins at the innermost axoglia junction of paranode) and internodes are structural parts of a myelinated axonSummary of the function of differentially abundant increased protein markers in MS and their overlap with previous studies Most of the proteins wereassigned as either neuronal or immune-related molecules based on previous reports and database searches The first column shows the name of the proteinand Uniprot accession number The second column shows the function of the proteins The third column shows comparison with a previous study onchildren with MS (Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourth column showsoverlap with the work of Schutzer et al who used CIS-MS (n 5 9 age 18ndash42 years) and established MS and controls (n 5 6 age 31ndash54 years)
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
We doubt that this influenced the results howeverbecause we did not see an age effect on the abundanceof the 21 identified proteins
Of the 14 proteins with increased abundance inMS (tables 2 and 3) 2 were associated with the amy-loid beta A4 protein family Amyloid-like protein 2 ismainly concentrated at neuronal synapses18 and has arole in memory processes whereas amyloid beta A4protein is associated with neurite growth neuronaladhesion and axonogenesis (table 3) Six proteins(contactin-219 neurofascin20 neuronal growth regu-lator 1 brevican core protein6 neuronal cell adhesionmolecule19 and disintegrin and metalloproteinasedomain-containing protein 22)19 are located at theparanodal and juxtaparanodal region of the CNS ofmyelinated axons (table 3) Contactin-2 is an axonalglycoprotein at the juxtaparanodal domain of myeli-nated axons13 Neurofascin plays a role in the assem-bly of nodes of Ranvier in the CNS21 Two isoformsof neurofascin have been shown to interact withcontactin-associated protein and contactin-1 to forma paranodal junction that attaches the myelin loop tothe axon and helps to separate voltage-gated sodiumchannels at nodal and potassium channels at juxtapar-anodal regions20 Disruption of neurofascin localiza-tion reveals early changes preceding demyelinationand remyelination in MS22 Of interest contactin-2and neurofascin have previously been reported asautoimmune targets in MS20 Neuronal growth
regulator 1 is located at the paranodal region of theCNS and plays a role in axoglia contact at the node ofRanvier Identification of contactin-2 and neurofas-cin in the CSF of children with MS is consistent witha previous study6 however they did not find a sig-nificant difference in the CSF of children with MSand monophasic ADS (tables 2 and 3) Brevican coreprotein is known as a CNS-specific proteoglycan19 atthe surface of neuroglial sheaths where it is enrichedin perisynaptic extracellular matrix23
Among the 14 proteins with increased abun-dance in MS the proteasespeptidases protein car-boxypeptidase E plays a role in the synthesis ofmost neuropeptides24 Disintegrin and metalloprotei-nase domain-containing protein 22 is highly expressedin the brain and localized at the juxtaparanode19 Thismolecule is presumed to be a major neuronal recep-tor25 Another brain-related protease is kallikrein-6which is a secreted serine protease13 It regulates earlyCNS demyelination in a viral model (expression in thebrain and spinal cord of mice) of MS26 In additionelevated levels of kallikrein-6 in CSF have been re-ported in adults with MS compared to neurologic con-trols (table 3)27
Among the other proteins with increased abun-dance in MS tyrosine-protein phosphatase non-receptor type substrate 1 is implicated in neuriteoutgrowth and glia cell attachment and has beenshown to support adhesions of cerebellar neurons28
Table 4 Function of proteins identified with decreased abundance in CSF samples of children with MScompared to samples of children with monophasic ADS
Protein (accession no) FunctionsDhaunchaket al6
Schutzeret al13
Apolipoprotein B-100 (P04114) Innate immune-related not produced inCNS39
3 3
C4b-binding protein alpha chain (P04003) Innate immune defense involved incomplement activity31
3 3
Haptoglobin (P00738) Innate immune defense29 3 3
Haptoglobin-related protein (P00739) Innate immune defense40 3 3
Leucine-rich alpha-2-glycoprotein (P02750) Induced by inflammation and involved incell adhesion high expression in the deepcerebral cortex25 novel marker ofgranulocytic differentiation29
3 3
Monocyte differentiation antigen CD14(P08571)
Main modulator of innate immune system32 3 3
Protein diaphanous homolog 1 (O60610) Coordinates cellular dynamics byregulating microfilament and microtubulefunction role in cellndashmatrix adhesionsvariant related to innate immune function34
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis3 5 Protein was not identifiedSummary of the function of differentially abundant decreased protein markers in MS and their overlap with previousstudies Most of the proteins were assigned as either neuronal or immune-related molecules based on previous reports anddatabase searches The first column shows the name of the protein and Uniprot accession number The second columnshows the function of the proteins The third column shows comparison with a previous study on children with MS(Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourthcolumn shows overlap with the work of Schutzer et al who used CIS-MS (n5 9 age 18ndash42 years) and established MS andcontrols (n 5 6 age 31ndash54 years)
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
The highest expression of dickkopf-related protein 3is reported in the brain and spinal cord (table 3)13
Thus the majority of proteins with increased abun-dance in our MS group (compared to the monophasicADS group) were neuronal-related with the excep-tion of 2 immune functionndashrelated proteins (Iggamma-1 chain C region and Ig kappa chain V-IIIregion POM) (table 3)
Our study reports 7 proteins with decreased abun-dance in pediatric-onset MS compared to monopha-sic ADS (table 2 and table e-3) Six of these 7 proteinshave previously been reported as specific componentsof innate immune functions (table 4) Haptoglobinan acute phase protein29 was identified as the mostdistinctive protein of those that were elevated in chil-dren with monophasic ADS (compared to childrenwith MS) (1829 18 significant peptides for a total of29 peptides) Another study in adults showedincreased haptoglobin concentration in patients withNMO compared to adult patients with MS30 C4b-binding protein alpha chain is a crucial componentof complement cascade and inhibits the function ofcomplement component31 Of interest we alsofound monocyte differentiation antigen CD14which is mainly expressed on cells of monocyticlineage (macrophages and monocytes)32 HigherCD14 levels might be linked with increased levelsof cytokines triggering inflammatory processes inchildren with monophasic ADS Monocytic cellssecrete soluble sCD14 (activation product of acti-vated monocytes) so this may affect the enormousmacrophage activation during acute monophasicADS33 Among the 7 identified proteins proteindiaphanous homolog 1 has a role in cell adhesionand is expressed in brain and its variants arerequired for innate immune response to Gram-negative bacterial infection34
Overall in the MS group we found a significantoverrepresentation of proteins associated withchanges in CNS gray matter axons synaptic regula-tion node of Ranvier and brain proteases (table 3)Several of these proteins are part of the axoglial appa-ratus and may relate to disturbances in axoglia inter-action6 (table 3) Two of them (contactin-2 andneurofascin) have been identified as possible axoglialautoantigens in MS20 The overlap of proteinsobserved in previous studies613 (table 3) as part ofthe axoglia apparatus and gray matter provides vali-dation for these proteins
These pathologically relevant proteins (mostlyCNS gray matterndashrelated) that are elevated in theCSF of children with early-onset MS might beinvolved in early disease mechanisms Further insightinto the role of these proteins can be useful for under-standing the disease process Moreover such proteinsmight be useful as a future tool to differentiate
children with MS from children with monophasicADS In addition knowing the start of MS could leadto earlier treatment with disease-modifying therapiesHowever the current research is designated as a dis-covery phase study which serves as a basis for thefollow-up verification and validation phase studiesthat can provide clinically valuable biomarkers Inthe future it would be interesting to further validateour findings with an independent technology and inan independent sample group
The data presented in this study indicate thatmonophasic ADS can be differentiated from MS inchildren primarily by CNS gray matter proteins andimmune-related proteins Our findings point to per-turbed axoglial physiology as a hallmark of the earliestevents of MS pathogenesis
AUTHOR CONTRIBUTIONSThe work was carried out in collaboration among all authors VS
TML and RQH designed the experiment VS and CS performed
the experiment and statistical analysis EDvP IAK and CEC-B
were responsible for sample collection VS EDvP MPS CS
RFN TML and RQH interpreted the results and wrote the article
All authors critically revised and approved the final manuscript
ACKNOWLEDGMENTThe authors thank all the children and their families who participated in
the Dutch Study for Pediatric MS
STUDY FUNDINGThis work was supported by a program grant provided by the EC 7th
frame work programme of Marie Curie Initial Training NetworkmdashThe
United Europeans for the Development of Pharmacogenomics in Mul-
tiple Sclerosis (UEPHAmdashMS grant PITNmdashGAmdash2008212877) Neth-
erlands Multiple Sclerosis Research Foundation to the Rotterdam MS
Centre ErasMS the Netherlands Organization for Scientific Research
(ZONmdashMW RQH) and Hersenstichting Nederland and Zenith grant
93511034 from the Netherlands Organization for Scientific Research
(NWO)
DISCLOSUREV Singh ED van Pelt MP Stoop C Stingl and IA Ketelslegers
report no disclosures RF Neuteboom is on the scientific advisory board
for LAREB CE Catsman-Berrevoets and TM Luider report no disclo-
sures RQ Hintzen is on the advisory board for Biogen Idec Roche and
Sanofi received research support from Biogen Idec Merck-Serono
Roche Genzyme Novartis Dutch MS Society and European Commis-
sion and is on the editorial board for Multiple Sclerosis and Related
Disorders Go to Neurologyorgnn for full disclosure forms
Received April 14 2015 Accepted in final form June 25 2015
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ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
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12 Team RC R A Language and Environment for Statistical
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13 Schutzer SE Angel TE Liu T et al Gray matter is tar-
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14 Stoop MP Dekker LJ Titulaer MK et al Multiple
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by advanced mass spectrometry Proteomics 20088
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15 Stoop MP Dekker LJ Titulaer MK et al Quantitative
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16 Stoop MP Singh V Dekker LJ et al Proteomics com-
parison of cerebrospinal fluid of relapsing remitting and
primary progressive multiple sclerosis PLoS One 20105
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17 van Zwam M Samsom JN Nieuwenhuis EE et al Mye-
lin ingestion alters macrophage antigen-presenting func-
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18 Wasco W Gurubhagavatula S Paradis MD et al Isola-
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of the Alzheimerrsquos associated amyloid beta protein precur-
sor Nat Genet 1993595ndash100
19 Faivre-Sarrailh C Devaux JJ Neuro-glial interactions at
the nodes of Ranvier implication in health and diseases
Front Cell Neurosci 20137196
20 Derfuss T Linington C Hohlfeld R Meinl E Axo-glial
antigens as targets in multiple sclerosis implications for
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88753ndash761
21 Mathey EK Derfuss T Storch MK et al Neurofascin as a
novel target for autoantibody-mediated axonal injury
J Exp Med 20072042363ndash2372
22 Howell OW Palser A Polito A et al Disruption of neu-
rofascin localization reveals early changes preceding demy-
elination and remyelination in multiple sclerosis Brain
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23 Yamada H Fredette B Shitara K et al The brain chon-
droitin sulfate proteoglycan brevican associates with astro-
cytes ensheathing cerebellar glomeruli and inhibits neurite
outgrowth from granule neurons J Neurosci 199717
7784ndash7795
24 Vilijn MH Das B Kessler JA Fricker LD Cultured as-
trocytes and neurons synthesize and secrete carboxypepti-
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25 Ozkaynak E Abello G Jaegle M et al Adam22 is a major
neuronal receptor for Lgi4-mediated Schwann cell signal-
ing J Neurosci 2010303857ndash3864
26 Scarisbrick IA Yoon H Panos M et al Kallikrein 6 reg-
ulates early CNS demyelination in a viral model of multi-
ple sclerosis Brain Pathol 201222709ndash722
27 Hebb AL Bhan V Wishart AD Moore CS
Robertson GS Human kallikrein 6 cerebrospinal levels
are elevated in multiple sclerosis Curr Drug Discov Tech-
nol 20107137ndash140
28 Latour S Tanaka H Demeure C et al Bidirectional
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29 OrsquoDonnell LC Druhan LJ Avalos BR Molecular charac-
terization and expression analysis of leucine-rich alpha2-
glycoprotein a novel marker of granulocytic differentiation
J Leukoc Biol 200272478ndash485
30 Chang KH Tseng MY Ro LS et al Analyses of hapto-
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tients with neuromyelitis optica and multiple sclerosis
Clin Chim Acta 201341726ndash30
31 Chung LP Reid KB Structural and functional studies on
C4b-binding protein a regulatory component of the
human complement system Biosci Rep 19855855ndash865
32 Kurne A Sayat G Aydin OF et al Lack of association of
the CD14Cmdash159T polymorphism with susceptibility
and progression parameters in Turkish multiple sclerosis
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33 Kitchens RL Thompson PA Modulatory effects of sCD14
and LBP on LPS-host cell interactions J Endotoxin Res
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34 Huh JR Foe I Muro I et al The Drosophila inhibitor of
apoptosis (IAP) DIAP2 is dispensable for cell survival
required for the innate immune response to gram-negative
bacterial infection and can be negatively regulated by the
reaperhidgrim family of IAP-binding apoptosis inducers
J Biol Chem 20072822056ndash2068
35 Jacobsen KT Iverfeldt K Amyloid precursor protein and
its homologues a family of proteolysis-dependent recep-
tors Cell Mol Life Sci 2009662299ndash2318
36 Koshimizu H Senatorov V Loh YP Gozes I Neuropro-
tective protein and carboxypeptidase E J Mol Neurosci
2009391ndash8
37 Schafer M Brauer AU Savaskan NE Rathjen FG
Brummendorf T Neurotractinkilon promotes neurite
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
outgrowth and is expressed on reactive astrocytes after
entorhinal cortex lesion Mol Cell Neurosci 200529
580ndash590
38 Stoop MP Coulier L Rosenling T et al Quantitative
proteomics and metabolomics analysis of normal human
cerebrospinal fluid samples Mol Cell Proteomics 20109
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39 Pepe G Chimienti G Liuzzi GM et al Lipoprotein(a) in
the cerebrospinal fluid of neurological patients with blood-
cerebrospinal fluid barrier dysfunction Clin Chem 2006
522043ndash2048
40 Nielsen MJ Petersen SV Jacobsen C et al Haptoglobin-
related protein is a high-affinity hemoglobin-binding plasma
protein Blood 20061082846ndash2849
10 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000015520152 Neurol Neuroimmunol Neuroinflamm
Vaibhav Singh E Danieumllle van Pelt Marcel P Stoop et al related proteins are associated with childhood-onset multiple sclerosisminusGray matter
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these 14 proteins and overlap with previous stud-ies613 are summarized in table 3 Among the 14 pro-teins with increased abundance in MS 12 wereassociated with CNS structure and functions (86)especially the gray matter (table 3) whereas only 17of the total identified proteins were related to CNSstructure and functions The 7 proteins with decreasedabundance in MS (relative to the monophasic ADSgroup) were components of the innate immune systemand inflammation (table 4)
For each peptide that passed (53 increased and 35decreased in MS) all stringent criteria (described inthe statistical analysis) the associated protein data-base search identification details p value fold expres-sion difference total number of peptides per proteinnumber of peptides identified below 001 medianabundance and frequencyoccurrence of identifica-tions by MSMS spectra are shown in tables e-2and e-3 Proteins identified in the current study didnot exhibit any myelin-related proteins (eg myelin
Table 2 Identification of proteins differentially abundant in CSF samples of children with MS (n 5 18) andsamples of children with monophasic ADS (n 5 21)
Trend in MSa DescriptionSigntotalb
Fold changemean (minndashmax)c p Valued mean (minndashmax)
Increased abundancein MS (n 5 14)
Amyloid-like protein 2 22 38 (2ndash55) 0006 (0003ndash0008)
Neurofascin 33 25 (21ndash3) 0002 (0001ndash0002)
Carboxypeptidase E 23 24 (21ndash26) 00004 (00002ndash00005)
Neuronal growth regulator 1 23 23 (21ndash25) 00008 (00008ndash00009)
Contactin-2 49 23 (15ndash32) 0002 (00005ndash0003)
Amyloid beta A4 protein 611 22 (18ndash26) 0002 (000003ndash0008)
Brevican core protein 57 21 (17ndash26) 0002 (00001ndash0005)
Disintegrin and metalloproteinasedomain-containing protein 22
22 19 (17ndash21) 00004 (00001ndash00006)
Tyrosine-protein phosphatasenon-receptor type substrate 1
34 19 (15ndash21) 0003 (0001ndash0006)
Dickkopf-related protein 3 611 18 (16ndash2) 0002 (000004ndash0006)
Neuronal cell adhesion molecule 918 18 (16ndash21) 0004 (0001ndash0009)
Ig kappa chain V-III region POM 22 18 (17ndash18) 0002 (00009ndash0003)
Ig gamma-1 chain C region 511 17 (15ndash18) 00016 (00002ndash0005)
Kallikrein-6 47 17 (15ndash19) 0001 (00006ndash0004)
Decreased abundancein MS (n 5 7)
Apolipoprotein B-100 717 661 (35ndash3930) 00026 (000002ndash001)
C4b-binding protein alpha chain 24 81 (29ndash134) 0008 (0007ndash0008)
Haptoglobin 1829 37 (2ndash121) 0002 (000004ndash001)
Haptoglobin-related protein 24 33 (31ndash35) 00002 (000001ndash00005)
Leucine-rich alpha-2-glycoprotein 23 26 (23ndash3) 0008 (0007ndash0009)
Monocyte differentiation antigen CD14 33 19 (18ndash2) 0006 (0004ndash0009)
Protein diaphanous homolog 1 22 18 (17ndash19) 0005 (0001ndash0009)
Abbreviations ADS 5 acquired demyelinating syndrome MS 5 multiple sclerosisThe table shows 21 proteins of which 14 were identified with increased abundance in CSF samples of children with MSand 7 were identified with decreased abundance in CSF samples of children with MS The table includes the direction ofdifference (trend) in MS name of the protein (description) fold expression difference and p value All proteins given in thetable were identified with at least 2 unique peptides differentially abundant peptides (p 001) had at least 15-folddifference in expression (median) between groups and for the same protein 40 of identified peptides were differentiallyabundant with the expression in the same direction (ie either higher or lower in MS) of the same protein Details of eachpeptide of the indicated proteins are listed in tables e-2 and e-3a Protein abundance is either significantly increased or decreased in children with MS compared to children withmonophasic ADSbNumber of differentially identified peptides p 001total number of identified peptides for the same proteinc Fold expression difference calculated based on median abundance from 18 patients with MS and 21 patients with ADSShown is the average of fold difference for all peptides of the same protein Minimum and maximum range for the same isindicateddCalculated by Wilcoxon test Given in the table is the mean p value and range for all differentially abundant peptides of thesame protein
4 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
oligodendrocyte glycoprotein and myelin basicprotein)
We performed a correlation analysis to examinethe influence of age at onset on the abundance ofthe 88 candidate peptides in children with MS and
monophasic ADS We found a mean coefficient ofdetermination (6SD) of 004 6 006 for MS and005 6 005 for monophasic ADS Thus no signif-icant correlation was found between age and peptideabundance for children with MS and monophasicADS for all 88 peptides
DISCUSSION In the current study we used a LC-MS proteomic approach to search for differences inCSF proteome between children with MS andchildren with monophasic ADS A benefit of thisOrbitrap technique is the ability to identifyrelatively vast amounts of different peptides andassess their abundances in a small sample volumeWe observed a striking difference between the 2groups (children with monophasic ADS vs MS)using stringent statistical criteria We searched forthe known functions of the 88 peptides correspondingto 21 distinctive proteins (14 increased and 7decreased in abundance in MS) using biologicaldatabases (wwwgeneontologyorg and wwwnextprotorg) and literature Recently 2 research groups613
demonstrated the identification of axoglial and graymatter proteins using mass spectrometry in CSF ofpatients with MS Similar to our study Dhaunchaket al6 compared CSF of 8 children with MS with CSFof 11 children with monophasic ADS The overlap ofsome proteins in the MS group is noticeable (egcarboxypeptidase E) despite clear differences insample handling such as depletion of abundantproteins with possible carrier function for otherproteins and exclusion of proteins with less than 5 kDaweight (table 3)
Our results show overlap with molecules identi-fied in the CSF of adults with acute-onset MS bySchutzer et al13 who performed mass spectrometryanalysis in CSF of patients with clinically isolatedsyndrome (CIS) vs those with established relapsingremitting MS (RRMS) and controls (table 3) Theyfound proteins that distinguished patients with CISfrom both patients with established RRMS and con-trols For example they found a significant increase inkallikrein-6 and dickkopf-related protein 3 at firstclinical onset compared to patients with establishedRRMS and controls (table 3) They also found a sig-nificant increase in contactin-2 (neuronal membraneprotein) in patients with a first attack of MS relativeto those with established RRMS (table 3)13
Our findings illustrate that the neurodegenerativearm of MS neuropathology is already active at the ear-liest stage of clinical disease also in children Consis-tent with the findings of others and our own previousstudies614ndash16 we again observed a striking lack ofmyelin proteins in these clear-cut cases of acute demy-elination This may not directly imply absence ofsuch free proteins in this type of pathology but rather
Figure 1 Volcano plot
Peptides (n 5 2260) showing distribution of fold change and statistical significance In thisplot each point represents a peptide and shows the ratio between CSF samples of childrenwith multiple sclerosis (MS) (n5 18) and samples of children with monophasic acquire demy-elinating syndromes (ADS) (n5 21) plotted against the level of statistical significance Y-axisshows p values obtained (plotted at log10) from a Wilcoxon test performed between abun-dances of peptides X-axis shows the ratio of the median between MS and monophasic ADSsamples (plotted on log2) (A) Above the dashed horizontal line red points (n 5 53 peptides)were found with increased abundance (right side of the vertical line) and green points (n5 35peptides) with decreased abundance (left side of the vertical line) in the MS group (comparedto the monophasic ADS group) (B) Peptides shown in gray below the dashed horizontal linedid not pass the stringent statistical criteria for identification of a candidate peptide
Neurology Neuroimmunology amp Neuroinflammation 5
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
it may reflect the specific physicochemical propertiesof the hydrophobic myelin components and perhapsdifferent pathways of elimination from the CSF (egvia draining macrophages)17 In any case we must becautious in using the dominant presence of CNS graymatter over white matter proteins as proof that neuro-degeneration is a primary event in MS and wouldprecede demyelination The presence of CNS graymatter may simply represent damage by inflamma-tion and the molecules identified may lead to a better
understanding of this presumed inflammation-induced neurodegeneration It should be stressed thatnot all differentially abundant proteins were overrep-resented in MS some were underrepresented point-ing at more complex mechanisms such as aperturbation in the physiology of the axoglial appara-tus (table 2 and tables e-2 and e-3)6 A confoundingfactor in our study could be the fact that the groupswere not matched according to age because of theoccurrence of monophasic disease at younger ages
Table 3 Function of proteins identified with increased abundance in CSF samples of children with MS compared with samples of children withmonophasic ADS
Protein (accession no) Functions and expressions Dhaunchak et al6 Schutzer et al13
Amyloid-like protein 2 (Q06481) Amyloid precursor protein family memoryprocesses concentrated in synapses18 regulatesneuronal stem cell differentiation during corticaldevelopment
3 variant
Neurofascin (O94856) Synapse formation located at CNS paranodaldomain and expressed by oligodendrocytes20
target for autoantibody-mediated axonal injury inMS21
NS 3
Contactin-2 (Q02246) Axon connection majorly expressed on CNSjuxtaparanodal domain1920
NS
Amyloid beta A4 protein (P05067) Component of amyloid plaques (Alzheimer brains)35
neuronal adhesion3 3
Brevican core protein (Q96GW7) Major proteoglycan in perisynaptic extracellularmatrix of brain inhibits neurite outgrowth fromcerebellar granule neurons23
NS Decreased expression in first-attack CIS-MS relative toestablished RRMS and controls
Carboxypeptidase E (P16870) Found in brain and throughout neuroendocrinesystem involved in synthesis of mostneuropeptides36
Same trend (4 sign21 total p 5 0004)
3
Neuronal growth regulator1 (Q7Z3B1)
Promotes outgrowth and is expressed on reactiveastrocytes after entorhinal cortex lesion (in mice)37
NS 3
Tyrosine-protein phosphatase non-receptor type substrate 1 (P78324)
Supports adhesion of cerebellar neurons neuriteoutgrowth and glial cell attachment28
3 3
Neuronal cell adhesion molecule(Q92823)
Localized at the node of Ranvier and in unmyelinatedaxons paranodal region of CNS axoglial contact19
NS
Disintegrin and metalloproteinasedomain-containing protein 22(Q9P0K1)
Expressed in the juxtaparanodal complex19 Not same trend (2 sign10 total p 5 001 002)
3
Dickkopf-related protein 3 (Q9UBP4) Expressed in the brain and spinal cord synapseformation13
3
Kallikrein-6 (Q92876) Elevated in active MS26 regulates early CNSdemyelination in a viral (mouse) model of MS26
3
Ig gamma-1 chain C region (P01857) Elevated in adult MS and CIS compared tononinflammatory neurologic diseases38
3 3
Ig kappa chain V-III region POM(P01624)
Elevated in adult MS compared to noninflammatoryand inflammatory neurologic diseases38
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis NS 5 not significant RRMS 5
relapsing remitting MS3 5 protein was not identified variant 5 variant of same protein was found with increased expression in first-attack CIS-MS group compared toestablished RRMS and controls 5 increased expression in first-attack CIS-MS group compared to established RRMS and controls 5 protein wasfound with increased expression in first-attack CIS-MS relative to RRMS but with decreased expression in first-attack CIS-MS vs controls NS 5 proteinwas detected in pediatric CSF samples but no statistical difference in their abundance was reported between MS and monophasic ADS group comparisonsame trend (trend is the direction of difference in MS) 5 when abundance of the protein was compared between 2 group (MS vs monophasic ADS)expression of the identified protein showed overlap with our study not same trend 5 when abundance of protein was compared expression of theidentified protein did not show overlap with our studyNodes paranodes juxtaparanodes (begins at the innermost axoglia junction of paranode) and internodes are structural parts of a myelinated axonSummary of the function of differentially abundant increased protein markers in MS and their overlap with previous studies Most of the proteins wereassigned as either neuronal or immune-related molecules based on previous reports and database searches The first column shows the name of the proteinand Uniprot accession number The second column shows the function of the proteins The third column shows comparison with a previous study onchildren with MS (Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourth column showsoverlap with the work of Schutzer et al who used CIS-MS (n 5 9 age 18ndash42 years) and established MS and controls (n 5 6 age 31ndash54 years)
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
We doubt that this influenced the results howeverbecause we did not see an age effect on the abundanceof the 21 identified proteins
Of the 14 proteins with increased abundance inMS (tables 2 and 3) 2 were associated with the amy-loid beta A4 protein family Amyloid-like protein 2 ismainly concentrated at neuronal synapses18 and has arole in memory processes whereas amyloid beta A4protein is associated with neurite growth neuronaladhesion and axonogenesis (table 3) Six proteins(contactin-219 neurofascin20 neuronal growth regu-lator 1 brevican core protein6 neuronal cell adhesionmolecule19 and disintegrin and metalloproteinasedomain-containing protein 22)19 are located at theparanodal and juxtaparanodal region of the CNS ofmyelinated axons (table 3) Contactin-2 is an axonalglycoprotein at the juxtaparanodal domain of myeli-nated axons13 Neurofascin plays a role in the assem-bly of nodes of Ranvier in the CNS21 Two isoformsof neurofascin have been shown to interact withcontactin-associated protein and contactin-1 to forma paranodal junction that attaches the myelin loop tothe axon and helps to separate voltage-gated sodiumchannels at nodal and potassium channels at juxtapar-anodal regions20 Disruption of neurofascin localiza-tion reveals early changes preceding demyelinationand remyelination in MS22 Of interest contactin-2and neurofascin have previously been reported asautoimmune targets in MS20 Neuronal growth
regulator 1 is located at the paranodal region of theCNS and plays a role in axoglia contact at the node ofRanvier Identification of contactin-2 and neurofas-cin in the CSF of children with MS is consistent witha previous study6 however they did not find a sig-nificant difference in the CSF of children with MSand monophasic ADS (tables 2 and 3) Brevican coreprotein is known as a CNS-specific proteoglycan19 atthe surface of neuroglial sheaths where it is enrichedin perisynaptic extracellular matrix23
Among the 14 proteins with increased abun-dance in MS the proteasespeptidases protein car-boxypeptidase E plays a role in the synthesis ofmost neuropeptides24 Disintegrin and metalloprotei-nase domain-containing protein 22 is highly expressedin the brain and localized at the juxtaparanode19 Thismolecule is presumed to be a major neuronal recep-tor25 Another brain-related protease is kallikrein-6which is a secreted serine protease13 It regulates earlyCNS demyelination in a viral model (expression in thebrain and spinal cord of mice) of MS26 In additionelevated levels of kallikrein-6 in CSF have been re-ported in adults with MS compared to neurologic con-trols (table 3)27
Among the other proteins with increased abun-dance in MS tyrosine-protein phosphatase non-receptor type substrate 1 is implicated in neuriteoutgrowth and glia cell attachment and has beenshown to support adhesions of cerebellar neurons28
Table 4 Function of proteins identified with decreased abundance in CSF samples of children with MScompared to samples of children with monophasic ADS
Protein (accession no) FunctionsDhaunchaket al6
Schutzeret al13
Apolipoprotein B-100 (P04114) Innate immune-related not produced inCNS39
3 3
C4b-binding protein alpha chain (P04003) Innate immune defense involved incomplement activity31
3 3
Haptoglobin (P00738) Innate immune defense29 3 3
Haptoglobin-related protein (P00739) Innate immune defense40 3 3
Leucine-rich alpha-2-glycoprotein (P02750) Induced by inflammation and involved incell adhesion high expression in the deepcerebral cortex25 novel marker ofgranulocytic differentiation29
3 3
Monocyte differentiation antigen CD14(P08571)
Main modulator of innate immune system32 3 3
Protein diaphanous homolog 1 (O60610) Coordinates cellular dynamics byregulating microfilament and microtubulefunction role in cellndashmatrix adhesionsvariant related to innate immune function34
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis3 5 Protein was not identifiedSummary of the function of differentially abundant decreased protein markers in MS and their overlap with previousstudies Most of the proteins were assigned as either neuronal or immune-related molecules based on previous reports anddatabase searches The first column shows the name of the protein and Uniprot accession number The second columnshows the function of the proteins The third column shows comparison with a previous study on children with MS(Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourthcolumn shows overlap with the work of Schutzer et al who used CIS-MS (n5 9 age 18ndash42 years) and established MS andcontrols (n 5 6 age 31ndash54 years)
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
The highest expression of dickkopf-related protein 3is reported in the brain and spinal cord (table 3)13
Thus the majority of proteins with increased abun-dance in our MS group (compared to the monophasicADS group) were neuronal-related with the excep-tion of 2 immune functionndashrelated proteins (Iggamma-1 chain C region and Ig kappa chain V-IIIregion POM) (table 3)
Our study reports 7 proteins with decreased abun-dance in pediatric-onset MS compared to monopha-sic ADS (table 2 and table e-3) Six of these 7 proteinshave previously been reported as specific componentsof innate immune functions (table 4) Haptoglobinan acute phase protein29 was identified as the mostdistinctive protein of those that were elevated in chil-dren with monophasic ADS (compared to childrenwith MS) (1829 18 significant peptides for a total of29 peptides) Another study in adults showedincreased haptoglobin concentration in patients withNMO compared to adult patients with MS30 C4b-binding protein alpha chain is a crucial componentof complement cascade and inhibits the function ofcomplement component31 Of interest we alsofound monocyte differentiation antigen CD14which is mainly expressed on cells of monocyticlineage (macrophages and monocytes)32 HigherCD14 levels might be linked with increased levelsof cytokines triggering inflammatory processes inchildren with monophasic ADS Monocytic cellssecrete soluble sCD14 (activation product of acti-vated monocytes) so this may affect the enormousmacrophage activation during acute monophasicADS33 Among the 7 identified proteins proteindiaphanous homolog 1 has a role in cell adhesionand is expressed in brain and its variants arerequired for innate immune response to Gram-negative bacterial infection34
Overall in the MS group we found a significantoverrepresentation of proteins associated withchanges in CNS gray matter axons synaptic regula-tion node of Ranvier and brain proteases (table 3)Several of these proteins are part of the axoglial appa-ratus and may relate to disturbances in axoglia inter-action6 (table 3) Two of them (contactin-2 andneurofascin) have been identified as possible axoglialautoantigens in MS20 The overlap of proteinsobserved in previous studies613 (table 3) as part ofthe axoglia apparatus and gray matter provides vali-dation for these proteins
These pathologically relevant proteins (mostlyCNS gray matterndashrelated) that are elevated in theCSF of children with early-onset MS might beinvolved in early disease mechanisms Further insightinto the role of these proteins can be useful for under-standing the disease process Moreover such proteinsmight be useful as a future tool to differentiate
children with MS from children with monophasicADS In addition knowing the start of MS could leadto earlier treatment with disease-modifying therapiesHowever the current research is designated as a dis-covery phase study which serves as a basis for thefollow-up verification and validation phase studiesthat can provide clinically valuable biomarkers Inthe future it would be interesting to further validateour findings with an independent technology and inan independent sample group
The data presented in this study indicate thatmonophasic ADS can be differentiated from MS inchildren primarily by CNS gray matter proteins andimmune-related proteins Our findings point to per-turbed axoglial physiology as a hallmark of the earliestevents of MS pathogenesis
AUTHOR CONTRIBUTIONSThe work was carried out in collaboration among all authors VS
TML and RQH designed the experiment VS and CS performed
the experiment and statistical analysis EDvP IAK and CEC-B
were responsible for sample collection VS EDvP MPS CS
RFN TML and RQH interpreted the results and wrote the article
All authors critically revised and approved the final manuscript
ACKNOWLEDGMENTThe authors thank all the children and their families who participated in
the Dutch Study for Pediatric MS
STUDY FUNDINGThis work was supported by a program grant provided by the EC 7th
frame work programme of Marie Curie Initial Training NetworkmdashThe
United Europeans for the Development of Pharmacogenomics in Mul-
tiple Sclerosis (UEPHAmdashMS grant PITNmdashGAmdash2008212877) Neth-
erlands Multiple Sclerosis Research Foundation to the Rotterdam MS
Centre ErasMS the Netherlands Organization for Scientific Research
(ZONmdashMW RQH) and Hersenstichting Nederland and Zenith grant
93511034 from the Netherlands Organization for Scientific Research
(NWO)
DISCLOSUREV Singh ED van Pelt MP Stoop C Stingl and IA Ketelslegers
report no disclosures RF Neuteboom is on the scientific advisory board
for LAREB CE Catsman-Berrevoets and TM Luider report no disclo-
sures RQ Hintzen is on the advisory board for Biogen Idec Roche and
Sanofi received research support from Biogen Idec Merck-Serono
Roche Genzyme Novartis Dutch MS Society and European Commis-
sion and is on the editorial board for Multiple Sclerosis and Related
Disorders Go to Neurologyorgnn for full disclosure forms
Received April 14 2015 Accepted in final form June 25 2015
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ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
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15 Stoop MP Dekker LJ Titulaer MK et al Quantitative
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16 Stoop MP Singh V Dekker LJ et al Proteomics com-
parison of cerebrospinal fluid of relapsing remitting and
primary progressive multiple sclerosis PLoS One 20105
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17 van Zwam M Samsom JN Nieuwenhuis EE et al Mye-
lin ingestion alters macrophage antigen-presenting func-
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of the Alzheimerrsquos associated amyloid beta protein precur-
sor Nat Genet 1993595ndash100
19 Faivre-Sarrailh C Devaux JJ Neuro-glial interactions at
the nodes of Ranvier implication in health and diseases
Front Cell Neurosci 20137196
20 Derfuss T Linington C Hohlfeld R Meinl E Axo-glial
antigens as targets in multiple sclerosis implications for
axonal and grey matter injury J Mol Med (Berl) 2010
88753ndash761
21 Mathey EK Derfuss T Storch MK et al Neurofascin as a
novel target for autoantibody-mediated axonal injury
J Exp Med 20072042363ndash2372
22 Howell OW Palser A Polito A et al Disruption of neu-
rofascin localization reveals early changes preceding demy-
elination and remyelination in multiple sclerosis Brain
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23 Yamada H Fredette B Shitara K et al The brain chon-
droitin sulfate proteoglycan brevican associates with astro-
cytes ensheathing cerebellar glomeruli and inhibits neurite
outgrowth from granule neurons J Neurosci 199717
7784ndash7795
24 Vilijn MH Das B Kessler JA Fricker LD Cultured as-
trocytes and neurons synthesize and secrete carboxypepti-
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25 Ozkaynak E Abello G Jaegle M et al Adam22 is a major
neuronal receptor for Lgi4-mediated Schwann cell signal-
ing J Neurosci 2010303857ndash3864
26 Scarisbrick IA Yoon H Panos M et al Kallikrein 6 reg-
ulates early CNS demyelination in a viral model of multi-
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27 Hebb AL Bhan V Wishart AD Moore CS
Robertson GS Human kallikrein 6 cerebrospinal levels
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nol 20107137ndash140
28 Latour S Tanaka H Demeure C et al Bidirectional
negative regulation of human T and dendritic cells by
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alpha down-regulation of IL-12 responsiveness and inhi-
bition of dendritic cell activation J Immunol 2001167
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29 OrsquoDonnell LC Druhan LJ Avalos BR Molecular charac-
terization and expression analysis of leucine-rich alpha2-
glycoprotein a novel marker of granulocytic differentiation
J Leukoc Biol 200272478ndash485
30 Chang KH Tseng MY Ro LS et al Analyses of hapto-
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tients with neuromyelitis optica and multiple sclerosis
Clin Chim Acta 201341726ndash30
31 Chung LP Reid KB Structural and functional studies on
C4b-binding protein a regulatory component of the
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32 Kurne A Sayat G Aydin OF et al Lack of association of
the CD14Cmdash159T polymorphism with susceptibility
and progression parameters in Turkish multiple sclerosis
patients J Neuroimmunol 201225083ndash86
33 Kitchens RL Thompson PA Modulatory effects of sCD14
and LBP on LPS-host cell interactions J Endotoxin Res
200511225ndash229
34 Huh JR Foe I Muro I et al The Drosophila inhibitor of
apoptosis (IAP) DIAP2 is dispensable for cell survival
required for the innate immune response to gram-negative
bacterial infection and can be negatively regulated by the
reaperhidgrim family of IAP-binding apoptosis inducers
J Biol Chem 20072822056ndash2068
35 Jacobsen KT Iverfeldt K Amyloid precursor protein and
its homologues a family of proteolysis-dependent recep-
tors Cell Mol Life Sci 2009662299ndash2318
36 Koshimizu H Senatorov V Loh YP Gozes I Neuropro-
tective protein and carboxypeptidase E J Mol Neurosci
2009391ndash8
37 Schafer M Brauer AU Savaskan NE Rathjen FG
Brummendorf T Neurotractinkilon promotes neurite
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
outgrowth and is expressed on reactive astrocytes after
entorhinal cortex lesion Mol Cell Neurosci 200529
580ndash590
38 Stoop MP Coulier L Rosenling T et al Quantitative
proteomics and metabolomics analysis of normal human
cerebrospinal fluid samples Mol Cell Proteomics 20109
2063ndash2075
39 Pepe G Chimienti G Liuzzi GM et al Lipoprotein(a) in
the cerebrospinal fluid of neurological patients with blood-
cerebrospinal fluid barrier dysfunction Clin Chem 2006
522043ndash2048
40 Nielsen MJ Petersen SV Jacobsen C et al Haptoglobin-
related protein is a high-affinity hemoglobin-binding plasma
protein Blood 20061082846ndash2849
10 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000015520152 Neurol Neuroimmunol Neuroinflamm
Vaibhav Singh E Danieumllle van Pelt Marcel P Stoop et al related proteins are associated with childhood-onset multiple sclerosisminusGray matter
This information is current as of September 24 2015
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent25e155fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2015092425e155DC1
Supplementary material can be found at
References httpnnneurologyorgcontent25e155fullhtmlref-list-1
This article cites 39 articles 10 of which you can access for free at
Citations httpnnneurologyorgcontent25e155fullhtmlotherarticles
This article has been cited by 1 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis
httpnnneurologyorgcgicollectionall_pediatricAll Pediatric
myelitishttpnnneurologyorgcgicollectionacute_disseminated_encephaloAcute disseminated encephalomyelitisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
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is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
oligodendrocyte glycoprotein and myelin basicprotein)
We performed a correlation analysis to examinethe influence of age at onset on the abundance ofthe 88 candidate peptides in children with MS and
monophasic ADS We found a mean coefficient ofdetermination (6SD) of 004 6 006 for MS and005 6 005 for monophasic ADS Thus no signif-icant correlation was found between age and peptideabundance for children with MS and monophasicADS for all 88 peptides
DISCUSSION In the current study we used a LC-MS proteomic approach to search for differences inCSF proteome between children with MS andchildren with monophasic ADS A benefit of thisOrbitrap technique is the ability to identifyrelatively vast amounts of different peptides andassess their abundances in a small sample volumeWe observed a striking difference between the 2groups (children with monophasic ADS vs MS)using stringent statistical criteria We searched forthe known functions of the 88 peptides correspondingto 21 distinctive proteins (14 increased and 7decreased in abundance in MS) using biologicaldatabases (wwwgeneontologyorg and wwwnextprotorg) and literature Recently 2 research groups613
demonstrated the identification of axoglial and graymatter proteins using mass spectrometry in CSF ofpatients with MS Similar to our study Dhaunchaket al6 compared CSF of 8 children with MS with CSFof 11 children with monophasic ADS The overlap ofsome proteins in the MS group is noticeable (egcarboxypeptidase E) despite clear differences insample handling such as depletion of abundantproteins with possible carrier function for otherproteins and exclusion of proteins with less than 5 kDaweight (table 3)
Our results show overlap with molecules identi-fied in the CSF of adults with acute-onset MS bySchutzer et al13 who performed mass spectrometryanalysis in CSF of patients with clinically isolatedsyndrome (CIS) vs those with established relapsingremitting MS (RRMS) and controls (table 3) Theyfound proteins that distinguished patients with CISfrom both patients with established RRMS and con-trols For example they found a significant increase inkallikrein-6 and dickkopf-related protein 3 at firstclinical onset compared to patients with establishedRRMS and controls (table 3) They also found a sig-nificant increase in contactin-2 (neuronal membraneprotein) in patients with a first attack of MS relativeto those with established RRMS (table 3)13
Our findings illustrate that the neurodegenerativearm of MS neuropathology is already active at the ear-liest stage of clinical disease also in children Consis-tent with the findings of others and our own previousstudies614ndash16 we again observed a striking lack ofmyelin proteins in these clear-cut cases of acute demy-elination This may not directly imply absence ofsuch free proteins in this type of pathology but rather
Figure 1 Volcano plot
Peptides (n 5 2260) showing distribution of fold change and statistical significance In thisplot each point represents a peptide and shows the ratio between CSF samples of childrenwith multiple sclerosis (MS) (n5 18) and samples of children with monophasic acquire demy-elinating syndromes (ADS) (n5 21) plotted against the level of statistical significance Y-axisshows p values obtained (plotted at log10) from a Wilcoxon test performed between abun-dances of peptides X-axis shows the ratio of the median between MS and monophasic ADSsamples (plotted on log2) (A) Above the dashed horizontal line red points (n 5 53 peptides)were found with increased abundance (right side of the vertical line) and green points (n5 35peptides) with decreased abundance (left side of the vertical line) in the MS group (comparedto the monophasic ADS group) (B) Peptides shown in gray below the dashed horizontal linedid not pass the stringent statistical criteria for identification of a candidate peptide
Neurology Neuroimmunology amp Neuroinflammation 5
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
it may reflect the specific physicochemical propertiesof the hydrophobic myelin components and perhapsdifferent pathways of elimination from the CSF (egvia draining macrophages)17 In any case we must becautious in using the dominant presence of CNS graymatter over white matter proteins as proof that neuro-degeneration is a primary event in MS and wouldprecede demyelination The presence of CNS graymatter may simply represent damage by inflamma-tion and the molecules identified may lead to a better
understanding of this presumed inflammation-induced neurodegeneration It should be stressed thatnot all differentially abundant proteins were overrep-resented in MS some were underrepresented point-ing at more complex mechanisms such as aperturbation in the physiology of the axoglial appara-tus (table 2 and tables e-2 and e-3)6 A confoundingfactor in our study could be the fact that the groupswere not matched according to age because of theoccurrence of monophasic disease at younger ages
Table 3 Function of proteins identified with increased abundance in CSF samples of children with MS compared with samples of children withmonophasic ADS
Protein (accession no) Functions and expressions Dhaunchak et al6 Schutzer et al13
Amyloid-like protein 2 (Q06481) Amyloid precursor protein family memoryprocesses concentrated in synapses18 regulatesneuronal stem cell differentiation during corticaldevelopment
3 variant
Neurofascin (O94856) Synapse formation located at CNS paranodaldomain and expressed by oligodendrocytes20
target for autoantibody-mediated axonal injury inMS21
NS 3
Contactin-2 (Q02246) Axon connection majorly expressed on CNSjuxtaparanodal domain1920
NS
Amyloid beta A4 protein (P05067) Component of amyloid plaques (Alzheimer brains)35
neuronal adhesion3 3
Brevican core protein (Q96GW7) Major proteoglycan in perisynaptic extracellularmatrix of brain inhibits neurite outgrowth fromcerebellar granule neurons23
NS Decreased expression in first-attack CIS-MS relative toestablished RRMS and controls
Carboxypeptidase E (P16870) Found in brain and throughout neuroendocrinesystem involved in synthesis of mostneuropeptides36
Same trend (4 sign21 total p 5 0004)
3
Neuronal growth regulator1 (Q7Z3B1)
Promotes outgrowth and is expressed on reactiveastrocytes after entorhinal cortex lesion (in mice)37
NS 3
Tyrosine-protein phosphatase non-receptor type substrate 1 (P78324)
Supports adhesion of cerebellar neurons neuriteoutgrowth and glial cell attachment28
3 3
Neuronal cell adhesion molecule(Q92823)
Localized at the node of Ranvier and in unmyelinatedaxons paranodal region of CNS axoglial contact19
NS
Disintegrin and metalloproteinasedomain-containing protein 22(Q9P0K1)
Expressed in the juxtaparanodal complex19 Not same trend (2 sign10 total p 5 001 002)
3
Dickkopf-related protein 3 (Q9UBP4) Expressed in the brain and spinal cord synapseformation13
3
Kallikrein-6 (Q92876) Elevated in active MS26 regulates early CNSdemyelination in a viral (mouse) model of MS26
3
Ig gamma-1 chain C region (P01857) Elevated in adult MS and CIS compared tononinflammatory neurologic diseases38
3 3
Ig kappa chain V-III region POM(P01624)
Elevated in adult MS compared to noninflammatoryand inflammatory neurologic diseases38
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis NS 5 not significant RRMS 5
relapsing remitting MS3 5 protein was not identified variant 5 variant of same protein was found with increased expression in first-attack CIS-MS group compared toestablished RRMS and controls 5 increased expression in first-attack CIS-MS group compared to established RRMS and controls 5 protein wasfound with increased expression in first-attack CIS-MS relative to RRMS but with decreased expression in first-attack CIS-MS vs controls NS 5 proteinwas detected in pediatric CSF samples but no statistical difference in their abundance was reported between MS and monophasic ADS group comparisonsame trend (trend is the direction of difference in MS) 5 when abundance of the protein was compared between 2 group (MS vs monophasic ADS)expression of the identified protein showed overlap with our study not same trend 5 when abundance of protein was compared expression of theidentified protein did not show overlap with our studyNodes paranodes juxtaparanodes (begins at the innermost axoglia junction of paranode) and internodes are structural parts of a myelinated axonSummary of the function of differentially abundant increased protein markers in MS and their overlap with previous studies Most of the proteins wereassigned as either neuronal or immune-related molecules based on previous reports and database searches The first column shows the name of the proteinand Uniprot accession number The second column shows the function of the proteins The third column shows comparison with a previous study onchildren with MS (Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourth column showsoverlap with the work of Schutzer et al who used CIS-MS (n 5 9 age 18ndash42 years) and established MS and controls (n 5 6 age 31ndash54 years)
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
We doubt that this influenced the results howeverbecause we did not see an age effect on the abundanceof the 21 identified proteins
Of the 14 proteins with increased abundance inMS (tables 2 and 3) 2 were associated with the amy-loid beta A4 protein family Amyloid-like protein 2 ismainly concentrated at neuronal synapses18 and has arole in memory processes whereas amyloid beta A4protein is associated with neurite growth neuronaladhesion and axonogenesis (table 3) Six proteins(contactin-219 neurofascin20 neuronal growth regu-lator 1 brevican core protein6 neuronal cell adhesionmolecule19 and disintegrin and metalloproteinasedomain-containing protein 22)19 are located at theparanodal and juxtaparanodal region of the CNS ofmyelinated axons (table 3) Contactin-2 is an axonalglycoprotein at the juxtaparanodal domain of myeli-nated axons13 Neurofascin plays a role in the assem-bly of nodes of Ranvier in the CNS21 Two isoformsof neurofascin have been shown to interact withcontactin-associated protein and contactin-1 to forma paranodal junction that attaches the myelin loop tothe axon and helps to separate voltage-gated sodiumchannels at nodal and potassium channels at juxtapar-anodal regions20 Disruption of neurofascin localiza-tion reveals early changes preceding demyelinationand remyelination in MS22 Of interest contactin-2and neurofascin have previously been reported asautoimmune targets in MS20 Neuronal growth
regulator 1 is located at the paranodal region of theCNS and plays a role in axoglia contact at the node ofRanvier Identification of contactin-2 and neurofas-cin in the CSF of children with MS is consistent witha previous study6 however they did not find a sig-nificant difference in the CSF of children with MSand monophasic ADS (tables 2 and 3) Brevican coreprotein is known as a CNS-specific proteoglycan19 atthe surface of neuroglial sheaths where it is enrichedin perisynaptic extracellular matrix23
Among the 14 proteins with increased abun-dance in MS the proteasespeptidases protein car-boxypeptidase E plays a role in the synthesis ofmost neuropeptides24 Disintegrin and metalloprotei-nase domain-containing protein 22 is highly expressedin the brain and localized at the juxtaparanode19 Thismolecule is presumed to be a major neuronal recep-tor25 Another brain-related protease is kallikrein-6which is a secreted serine protease13 It regulates earlyCNS demyelination in a viral model (expression in thebrain and spinal cord of mice) of MS26 In additionelevated levels of kallikrein-6 in CSF have been re-ported in adults with MS compared to neurologic con-trols (table 3)27
Among the other proteins with increased abun-dance in MS tyrosine-protein phosphatase non-receptor type substrate 1 is implicated in neuriteoutgrowth and glia cell attachment and has beenshown to support adhesions of cerebellar neurons28
Table 4 Function of proteins identified with decreased abundance in CSF samples of children with MScompared to samples of children with monophasic ADS
Protein (accession no) FunctionsDhaunchaket al6
Schutzeret al13
Apolipoprotein B-100 (P04114) Innate immune-related not produced inCNS39
3 3
C4b-binding protein alpha chain (P04003) Innate immune defense involved incomplement activity31
3 3
Haptoglobin (P00738) Innate immune defense29 3 3
Haptoglobin-related protein (P00739) Innate immune defense40 3 3
Leucine-rich alpha-2-glycoprotein (P02750) Induced by inflammation and involved incell adhesion high expression in the deepcerebral cortex25 novel marker ofgranulocytic differentiation29
3 3
Monocyte differentiation antigen CD14(P08571)
Main modulator of innate immune system32 3 3
Protein diaphanous homolog 1 (O60610) Coordinates cellular dynamics byregulating microfilament and microtubulefunction role in cellndashmatrix adhesionsvariant related to innate immune function34
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis3 5 Protein was not identifiedSummary of the function of differentially abundant decreased protein markers in MS and their overlap with previousstudies Most of the proteins were assigned as either neuronal or immune-related molecules based on previous reports anddatabase searches The first column shows the name of the protein and Uniprot accession number The second columnshows the function of the proteins The third column shows comparison with a previous study on children with MS(Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourthcolumn shows overlap with the work of Schutzer et al who used CIS-MS (n5 9 age 18ndash42 years) and established MS andcontrols (n 5 6 age 31ndash54 years)
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
The highest expression of dickkopf-related protein 3is reported in the brain and spinal cord (table 3)13
Thus the majority of proteins with increased abun-dance in our MS group (compared to the monophasicADS group) were neuronal-related with the excep-tion of 2 immune functionndashrelated proteins (Iggamma-1 chain C region and Ig kappa chain V-IIIregion POM) (table 3)
Our study reports 7 proteins with decreased abun-dance in pediatric-onset MS compared to monopha-sic ADS (table 2 and table e-3) Six of these 7 proteinshave previously been reported as specific componentsof innate immune functions (table 4) Haptoglobinan acute phase protein29 was identified as the mostdistinctive protein of those that were elevated in chil-dren with monophasic ADS (compared to childrenwith MS) (1829 18 significant peptides for a total of29 peptides) Another study in adults showedincreased haptoglobin concentration in patients withNMO compared to adult patients with MS30 C4b-binding protein alpha chain is a crucial componentof complement cascade and inhibits the function ofcomplement component31 Of interest we alsofound monocyte differentiation antigen CD14which is mainly expressed on cells of monocyticlineage (macrophages and monocytes)32 HigherCD14 levels might be linked with increased levelsof cytokines triggering inflammatory processes inchildren with monophasic ADS Monocytic cellssecrete soluble sCD14 (activation product of acti-vated monocytes) so this may affect the enormousmacrophage activation during acute monophasicADS33 Among the 7 identified proteins proteindiaphanous homolog 1 has a role in cell adhesionand is expressed in brain and its variants arerequired for innate immune response to Gram-negative bacterial infection34
Overall in the MS group we found a significantoverrepresentation of proteins associated withchanges in CNS gray matter axons synaptic regula-tion node of Ranvier and brain proteases (table 3)Several of these proteins are part of the axoglial appa-ratus and may relate to disturbances in axoglia inter-action6 (table 3) Two of them (contactin-2 andneurofascin) have been identified as possible axoglialautoantigens in MS20 The overlap of proteinsobserved in previous studies613 (table 3) as part ofthe axoglia apparatus and gray matter provides vali-dation for these proteins
These pathologically relevant proteins (mostlyCNS gray matterndashrelated) that are elevated in theCSF of children with early-onset MS might beinvolved in early disease mechanisms Further insightinto the role of these proteins can be useful for under-standing the disease process Moreover such proteinsmight be useful as a future tool to differentiate
children with MS from children with monophasicADS In addition knowing the start of MS could leadto earlier treatment with disease-modifying therapiesHowever the current research is designated as a dis-covery phase study which serves as a basis for thefollow-up verification and validation phase studiesthat can provide clinically valuable biomarkers Inthe future it would be interesting to further validateour findings with an independent technology and inan independent sample group
The data presented in this study indicate thatmonophasic ADS can be differentiated from MS inchildren primarily by CNS gray matter proteins andimmune-related proteins Our findings point to per-turbed axoglial physiology as a hallmark of the earliestevents of MS pathogenesis
AUTHOR CONTRIBUTIONSThe work was carried out in collaboration among all authors VS
TML and RQH designed the experiment VS and CS performed
the experiment and statistical analysis EDvP IAK and CEC-B
were responsible for sample collection VS EDvP MPS CS
RFN TML and RQH interpreted the results and wrote the article
All authors critically revised and approved the final manuscript
ACKNOWLEDGMENTThe authors thank all the children and their families who participated in
the Dutch Study for Pediatric MS
STUDY FUNDINGThis work was supported by a program grant provided by the EC 7th
frame work programme of Marie Curie Initial Training NetworkmdashThe
United Europeans for the Development of Pharmacogenomics in Mul-
tiple Sclerosis (UEPHAmdashMS grant PITNmdashGAmdash2008212877) Neth-
erlands Multiple Sclerosis Research Foundation to the Rotterdam MS
Centre ErasMS the Netherlands Organization for Scientific Research
(ZONmdashMW RQH) and Hersenstichting Nederland and Zenith grant
93511034 from the Netherlands Organization for Scientific Research
(NWO)
DISCLOSUREV Singh ED van Pelt MP Stoop C Stingl and IA Ketelslegers
report no disclosures RF Neuteboom is on the scientific advisory board
for LAREB CE Catsman-Berrevoets and TM Luider report no disclo-
sures RQ Hintzen is on the advisory board for Biogen Idec Roche and
Sanofi received research support from Biogen Idec Merck-Serono
Roche Genzyme Novartis Dutch MS Society and European Commis-
sion and is on the editorial board for Multiple Sclerosis and Related
Disorders Go to Neurologyorgnn for full disclosure forms
Received April 14 2015 Accepted in final form June 25 2015
REFERENCES1 Krupp LB Banwell B Tenembaum S International Pedi-
atric MSSG Consensus definitions proposed for pediatric
multiple sclerosis and related disorders Neurology 2007
68S7ndashS12
2 Banwell B Ghezzi A Bar-Or A Mikaeloff Y Tardieu M
Multiple sclerosis in children clinical diagnosis therapeu-
tic strategies and future directions Lancet Neurol 20076
887ndash902
3 Banwell B Bar-Or A Arnold DL et al Clinical environ-
mental and genetic determinants of multiple sclerosis in
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
children with acute demyelination a prospective national
cohort study Lancet Neurol 201110436ndash445
4 Neuteboom RF Boon M Catsman Berrevoets CE et al
Prognostic factors after a first attack of inflammatory CNS
demyelination in children Neurology 200871967ndash973
5 Krupp LB Tardieu M Amato MP et al International
Pediatric Multiple Sclerosis Study Group criteria for pedi-
atric multiple sclerosis and immune-mediated central ner-
vous system demyelinating disorders revisions to the 2007
definitions Mult Scler 2013191261ndash1267
6 Dhaunchak AS Becker C Schulman H et al Implication
of perturbed axoglial apparatus in early pediatric multiple
sclerosis Ann Neurol 201271601ndash613
7 Singh V Hintzen RQ Luider TM Stoop MP Proteomics
technologies for biomarker discovery in multiple sclerosis
J Neuroimmunol 201224840ndash47
8 Singh V Stoop MP Stingl C et al Cerebrospinal-fluid-
derived immunoglobulin G of different multiple sclerosis
patients shares mutated sequences in complementarity
determining regions Mol Cell Proteomics 201312
3924ndash3934
9 Ketelslegers IA Catsman-Berrevoets CE Neuteboom RF
et al Incidence of acquired demyelinating syndromes of
the CNS in Dutch children a nationwide study J Neurol
20122591929ndash1935
10 Teunissen CE Petzold A Bennett JL et al A consensus
protocol for the standardization of cerebrospinal fluid col-
lection and biobanking Neurology 2009731914ndash1922
11 Stoop MP Singh V Stingl C et al Effects of natalizumab
treatment on the cerebrospinal fluid proteome of multiple
sclerosis patients J Proteome Res 2013121101ndash1107
12 Team RC R A Language and Environment for Statistical
Computing [online] Available at httpwwwR-project
org Accessed February 4 2014
13 Schutzer SE Angel TE Liu T et al Gray matter is tar-
geted in first-attack multiple sclerosis PLoS One 20138
e66117
14 Stoop MP Dekker LJ Titulaer MK et al Multiple
sclerosis-related proteins identified in cerebrospinal fluid
by advanced mass spectrometry Proteomics 20088
1576ndash1585
15 Stoop MP Dekker LJ Titulaer MK et al Quantitative
matrix-assisted laser desorption ionization-fourier trans-
form ion cyclotron resonance (MALDI-FT-ICR) peptide
profiling and identification of multiple-sclerosis-related
proteins J Proteome Res 200981404ndash1414
16 Stoop MP Singh V Dekker LJ et al Proteomics com-
parison of cerebrospinal fluid of relapsing remitting and
primary progressive multiple sclerosis PLoS One 20105
e12442
17 van Zwam M Samsom JN Nieuwenhuis EE et al Mye-
lin ingestion alters macrophage antigen-presenting func-
tion in vitro and in vivo J Leukoc Biol 201190123ndash132
18 Wasco W Gurubhagavatula S Paradis MD et al Isola-
tion and characterization of APLP2 encoding a homologue
of the Alzheimerrsquos associated amyloid beta protein precur-
sor Nat Genet 1993595ndash100
19 Faivre-Sarrailh C Devaux JJ Neuro-glial interactions at
the nodes of Ranvier implication in health and diseases
Front Cell Neurosci 20137196
20 Derfuss T Linington C Hohlfeld R Meinl E Axo-glial
antigens as targets in multiple sclerosis implications for
axonal and grey matter injury J Mol Med (Berl) 2010
88753ndash761
21 Mathey EK Derfuss T Storch MK et al Neurofascin as a
novel target for autoantibody-mediated axonal injury
J Exp Med 20072042363ndash2372
22 Howell OW Palser A Polito A et al Disruption of neu-
rofascin localization reveals early changes preceding demy-
elination and remyelination in multiple sclerosis Brain
20061293173ndash3185
23 Yamada H Fredette B Shitara K et al The brain chon-
droitin sulfate proteoglycan brevican associates with astro-
cytes ensheathing cerebellar glomeruli and inhibits neurite
outgrowth from granule neurons J Neurosci 199717
7784ndash7795
24 Vilijn MH Das B Kessler JA Fricker LD Cultured as-
trocytes and neurons synthesize and secrete carboxypepti-
dase E a neuropeptide-processing enzyme J Neurochem
1989531487ndash1493
25 Ozkaynak E Abello G Jaegle M et al Adam22 is a major
neuronal receptor for Lgi4-mediated Schwann cell signal-
ing J Neurosci 2010303857ndash3864
26 Scarisbrick IA Yoon H Panos M et al Kallikrein 6 reg-
ulates early CNS demyelination in a viral model of multi-
ple sclerosis Brain Pathol 201222709ndash722
27 Hebb AL Bhan V Wishart AD Moore CS
Robertson GS Human kallikrein 6 cerebrospinal levels
are elevated in multiple sclerosis Curr Drug Discov Tech-
nol 20107137ndash140
28 Latour S Tanaka H Demeure C et al Bidirectional
negative regulation of human T and dendritic cells by
CD47 and its cognate receptor signal-regulator protein-
alpha down-regulation of IL-12 responsiveness and inhi-
bition of dendritic cell activation J Immunol 2001167
2547ndash2554
29 OrsquoDonnell LC Druhan LJ Avalos BR Molecular charac-
terization and expression analysis of leucine-rich alpha2-
glycoprotein a novel marker of granulocytic differentiation
J Leukoc Biol 200272478ndash485
30 Chang KH Tseng MY Ro LS et al Analyses of hapto-
globin level in the cerebrospinal fluid and serum of pa-
tients with neuromyelitis optica and multiple sclerosis
Clin Chim Acta 201341726ndash30
31 Chung LP Reid KB Structural and functional studies on
C4b-binding protein a regulatory component of the
human complement system Biosci Rep 19855855ndash865
32 Kurne A Sayat G Aydin OF et al Lack of association of
the CD14Cmdash159T polymorphism with susceptibility
and progression parameters in Turkish multiple sclerosis
patients J Neuroimmunol 201225083ndash86
33 Kitchens RL Thompson PA Modulatory effects of sCD14
and LBP on LPS-host cell interactions J Endotoxin Res
200511225ndash229
34 Huh JR Foe I Muro I et al The Drosophila inhibitor of
apoptosis (IAP) DIAP2 is dispensable for cell survival
required for the innate immune response to gram-negative
bacterial infection and can be negatively regulated by the
reaperhidgrim family of IAP-binding apoptosis inducers
J Biol Chem 20072822056ndash2068
35 Jacobsen KT Iverfeldt K Amyloid precursor protein and
its homologues a family of proteolysis-dependent recep-
tors Cell Mol Life Sci 2009662299ndash2318
36 Koshimizu H Senatorov V Loh YP Gozes I Neuropro-
tective protein and carboxypeptidase E J Mol Neurosci
2009391ndash8
37 Schafer M Brauer AU Savaskan NE Rathjen FG
Brummendorf T Neurotractinkilon promotes neurite
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
outgrowth and is expressed on reactive astrocytes after
entorhinal cortex lesion Mol Cell Neurosci 200529
580ndash590
38 Stoop MP Coulier L Rosenling T et al Quantitative
proteomics and metabolomics analysis of normal human
cerebrospinal fluid samples Mol Cell Proteomics 20109
2063ndash2075
39 Pepe G Chimienti G Liuzzi GM et al Lipoprotein(a) in
the cerebrospinal fluid of neurological patients with blood-
cerebrospinal fluid barrier dysfunction Clin Chem 2006
522043ndash2048
40 Nielsen MJ Petersen SV Jacobsen C et al Haptoglobin-
related protein is a high-affinity hemoglobin-binding plasma
protein Blood 20061082846ndash2849
10 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000015520152 Neurol Neuroimmunol Neuroinflamm
Vaibhav Singh E Danieumllle van Pelt Marcel P Stoop et al related proteins are associated with childhood-onset multiple sclerosisminusGray matter
This information is current as of September 24 2015
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent25e155fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2015092425e155DC1
Supplementary material can be found at
References httpnnneurologyorgcontent25e155fullhtmlref-list-1
This article cites 39 articles 10 of which you can access for free at
Citations httpnnneurologyorgcontent25e155fullhtmlotherarticles
This article has been cited by 1 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis
httpnnneurologyorgcgicollectionall_pediatricAll Pediatric
myelitishttpnnneurologyorgcgicollectionacute_disseminated_encephaloAcute disseminated encephalomyelitisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
it may reflect the specific physicochemical propertiesof the hydrophobic myelin components and perhapsdifferent pathways of elimination from the CSF (egvia draining macrophages)17 In any case we must becautious in using the dominant presence of CNS graymatter over white matter proteins as proof that neuro-degeneration is a primary event in MS and wouldprecede demyelination The presence of CNS graymatter may simply represent damage by inflamma-tion and the molecules identified may lead to a better
understanding of this presumed inflammation-induced neurodegeneration It should be stressed thatnot all differentially abundant proteins were overrep-resented in MS some were underrepresented point-ing at more complex mechanisms such as aperturbation in the physiology of the axoglial appara-tus (table 2 and tables e-2 and e-3)6 A confoundingfactor in our study could be the fact that the groupswere not matched according to age because of theoccurrence of monophasic disease at younger ages
Table 3 Function of proteins identified with increased abundance in CSF samples of children with MS compared with samples of children withmonophasic ADS
Protein (accession no) Functions and expressions Dhaunchak et al6 Schutzer et al13
Amyloid-like protein 2 (Q06481) Amyloid precursor protein family memoryprocesses concentrated in synapses18 regulatesneuronal stem cell differentiation during corticaldevelopment
3 variant
Neurofascin (O94856) Synapse formation located at CNS paranodaldomain and expressed by oligodendrocytes20
target for autoantibody-mediated axonal injury inMS21
NS 3
Contactin-2 (Q02246) Axon connection majorly expressed on CNSjuxtaparanodal domain1920
NS
Amyloid beta A4 protein (P05067) Component of amyloid plaques (Alzheimer brains)35
neuronal adhesion3 3
Brevican core protein (Q96GW7) Major proteoglycan in perisynaptic extracellularmatrix of brain inhibits neurite outgrowth fromcerebellar granule neurons23
NS Decreased expression in first-attack CIS-MS relative toestablished RRMS and controls
Carboxypeptidase E (P16870) Found in brain and throughout neuroendocrinesystem involved in synthesis of mostneuropeptides36
Same trend (4 sign21 total p 5 0004)
3
Neuronal growth regulator1 (Q7Z3B1)
Promotes outgrowth and is expressed on reactiveastrocytes after entorhinal cortex lesion (in mice)37
NS 3
Tyrosine-protein phosphatase non-receptor type substrate 1 (P78324)
Supports adhesion of cerebellar neurons neuriteoutgrowth and glial cell attachment28
3 3
Neuronal cell adhesion molecule(Q92823)
Localized at the node of Ranvier and in unmyelinatedaxons paranodal region of CNS axoglial contact19
NS
Disintegrin and metalloproteinasedomain-containing protein 22(Q9P0K1)
Expressed in the juxtaparanodal complex19 Not same trend (2 sign10 total p 5 001 002)
3
Dickkopf-related protein 3 (Q9UBP4) Expressed in the brain and spinal cord synapseformation13
3
Kallikrein-6 (Q92876) Elevated in active MS26 regulates early CNSdemyelination in a viral (mouse) model of MS26
3
Ig gamma-1 chain C region (P01857) Elevated in adult MS and CIS compared tononinflammatory neurologic diseases38
3 3
Ig kappa chain V-III region POM(P01624)
Elevated in adult MS compared to noninflammatoryand inflammatory neurologic diseases38
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis NS 5 not significant RRMS 5
relapsing remitting MS3 5 protein was not identified variant 5 variant of same protein was found with increased expression in first-attack CIS-MS group compared toestablished RRMS and controls 5 increased expression in first-attack CIS-MS group compared to established RRMS and controls 5 protein wasfound with increased expression in first-attack CIS-MS relative to RRMS but with decreased expression in first-attack CIS-MS vs controls NS 5 proteinwas detected in pediatric CSF samples but no statistical difference in their abundance was reported between MS and monophasic ADS group comparisonsame trend (trend is the direction of difference in MS) 5 when abundance of the protein was compared between 2 group (MS vs monophasic ADS)expression of the identified protein showed overlap with our study not same trend 5 when abundance of protein was compared expression of theidentified protein did not show overlap with our studyNodes paranodes juxtaparanodes (begins at the innermost axoglia junction of paranode) and internodes are structural parts of a myelinated axonSummary of the function of differentially abundant increased protein markers in MS and their overlap with previous studies Most of the proteins wereassigned as either neuronal or immune-related molecules based on previous reports and database searches The first column shows the name of the proteinand Uniprot accession number The second column shows the function of the proteins The third column shows comparison with a previous study onchildren with MS (Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourth column showsoverlap with the work of Schutzer et al who used CIS-MS (n 5 9 age 18ndash42 years) and established MS and controls (n 5 6 age 31ndash54 years)
6 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
We doubt that this influenced the results howeverbecause we did not see an age effect on the abundanceof the 21 identified proteins
Of the 14 proteins with increased abundance inMS (tables 2 and 3) 2 were associated with the amy-loid beta A4 protein family Amyloid-like protein 2 ismainly concentrated at neuronal synapses18 and has arole in memory processes whereas amyloid beta A4protein is associated with neurite growth neuronaladhesion and axonogenesis (table 3) Six proteins(contactin-219 neurofascin20 neuronal growth regu-lator 1 brevican core protein6 neuronal cell adhesionmolecule19 and disintegrin and metalloproteinasedomain-containing protein 22)19 are located at theparanodal and juxtaparanodal region of the CNS ofmyelinated axons (table 3) Contactin-2 is an axonalglycoprotein at the juxtaparanodal domain of myeli-nated axons13 Neurofascin plays a role in the assem-bly of nodes of Ranvier in the CNS21 Two isoformsof neurofascin have been shown to interact withcontactin-associated protein and contactin-1 to forma paranodal junction that attaches the myelin loop tothe axon and helps to separate voltage-gated sodiumchannels at nodal and potassium channels at juxtapar-anodal regions20 Disruption of neurofascin localiza-tion reveals early changes preceding demyelinationand remyelination in MS22 Of interest contactin-2and neurofascin have previously been reported asautoimmune targets in MS20 Neuronal growth
regulator 1 is located at the paranodal region of theCNS and plays a role in axoglia contact at the node ofRanvier Identification of contactin-2 and neurofas-cin in the CSF of children with MS is consistent witha previous study6 however they did not find a sig-nificant difference in the CSF of children with MSand monophasic ADS (tables 2 and 3) Brevican coreprotein is known as a CNS-specific proteoglycan19 atthe surface of neuroglial sheaths where it is enrichedin perisynaptic extracellular matrix23
Among the 14 proteins with increased abun-dance in MS the proteasespeptidases protein car-boxypeptidase E plays a role in the synthesis ofmost neuropeptides24 Disintegrin and metalloprotei-nase domain-containing protein 22 is highly expressedin the brain and localized at the juxtaparanode19 Thismolecule is presumed to be a major neuronal recep-tor25 Another brain-related protease is kallikrein-6which is a secreted serine protease13 It regulates earlyCNS demyelination in a viral model (expression in thebrain and spinal cord of mice) of MS26 In additionelevated levels of kallikrein-6 in CSF have been re-ported in adults with MS compared to neurologic con-trols (table 3)27
Among the other proteins with increased abun-dance in MS tyrosine-protein phosphatase non-receptor type substrate 1 is implicated in neuriteoutgrowth and glia cell attachment and has beenshown to support adhesions of cerebellar neurons28
Table 4 Function of proteins identified with decreased abundance in CSF samples of children with MScompared to samples of children with monophasic ADS
Protein (accession no) FunctionsDhaunchaket al6
Schutzeret al13
Apolipoprotein B-100 (P04114) Innate immune-related not produced inCNS39
3 3
C4b-binding protein alpha chain (P04003) Innate immune defense involved incomplement activity31
3 3
Haptoglobin (P00738) Innate immune defense29 3 3
Haptoglobin-related protein (P00739) Innate immune defense40 3 3
Leucine-rich alpha-2-glycoprotein (P02750) Induced by inflammation and involved incell adhesion high expression in the deepcerebral cortex25 novel marker ofgranulocytic differentiation29
3 3
Monocyte differentiation antigen CD14(P08571)
Main modulator of innate immune system32 3 3
Protein diaphanous homolog 1 (O60610) Coordinates cellular dynamics byregulating microfilament and microtubulefunction role in cellndashmatrix adhesionsvariant related to innate immune function34
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis3 5 Protein was not identifiedSummary of the function of differentially abundant decreased protein markers in MS and their overlap with previousstudies Most of the proteins were assigned as either neuronal or immune-related molecules based on previous reports anddatabase searches The first column shows the name of the protein and Uniprot accession number The second columnshows the function of the proteins The third column shows comparison with a previous study on children with MS(Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourthcolumn shows overlap with the work of Schutzer et al who used CIS-MS (n5 9 age 18ndash42 years) and established MS andcontrols (n 5 6 age 31ndash54 years)
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
The highest expression of dickkopf-related protein 3is reported in the brain and spinal cord (table 3)13
Thus the majority of proteins with increased abun-dance in our MS group (compared to the monophasicADS group) were neuronal-related with the excep-tion of 2 immune functionndashrelated proteins (Iggamma-1 chain C region and Ig kappa chain V-IIIregion POM) (table 3)
Our study reports 7 proteins with decreased abun-dance in pediatric-onset MS compared to monopha-sic ADS (table 2 and table e-3) Six of these 7 proteinshave previously been reported as specific componentsof innate immune functions (table 4) Haptoglobinan acute phase protein29 was identified as the mostdistinctive protein of those that were elevated in chil-dren with monophasic ADS (compared to childrenwith MS) (1829 18 significant peptides for a total of29 peptides) Another study in adults showedincreased haptoglobin concentration in patients withNMO compared to adult patients with MS30 C4b-binding protein alpha chain is a crucial componentof complement cascade and inhibits the function ofcomplement component31 Of interest we alsofound monocyte differentiation antigen CD14which is mainly expressed on cells of monocyticlineage (macrophages and monocytes)32 HigherCD14 levels might be linked with increased levelsof cytokines triggering inflammatory processes inchildren with monophasic ADS Monocytic cellssecrete soluble sCD14 (activation product of acti-vated monocytes) so this may affect the enormousmacrophage activation during acute monophasicADS33 Among the 7 identified proteins proteindiaphanous homolog 1 has a role in cell adhesionand is expressed in brain and its variants arerequired for innate immune response to Gram-negative bacterial infection34
Overall in the MS group we found a significantoverrepresentation of proteins associated withchanges in CNS gray matter axons synaptic regula-tion node of Ranvier and brain proteases (table 3)Several of these proteins are part of the axoglial appa-ratus and may relate to disturbances in axoglia inter-action6 (table 3) Two of them (contactin-2 andneurofascin) have been identified as possible axoglialautoantigens in MS20 The overlap of proteinsobserved in previous studies613 (table 3) as part ofthe axoglia apparatus and gray matter provides vali-dation for these proteins
These pathologically relevant proteins (mostlyCNS gray matterndashrelated) that are elevated in theCSF of children with early-onset MS might beinvolved in early disease mechanisms Further insightinto the role of these proteins can be useful for under-standing the disease process Moreover such proteinsmight be useful as a future tool to differentiate
children with MS from children with monophasicADS In addition knowing the start of MS could leadto earlier treatment with disease-modifying therapiesHowever the current research is designated as a dis-covery phase study which serves as a basis for thefollow-up verification and validation phase studiesthat can provide clinically valuable biomarkers Inthe future it would be interesting to further validateour findings with an independent technology and inan independent sample group
The data presented in this study indicate thatmonophasic ADS can be differentiated from MS inchildren primarily by CNS gray matter proteins andimmune-related proteins Our findings point to per-turbed axoglial physiology as a hallmark of the earliestevents of MS pathogenesis
AUTHOR CONTRIBUTIONSThe work was carried out in collaboration among all authors VS
TML and RQH designed the experiment VS and CS performed
the experiment and statistical analysis EDvP IAK and CEC-B
were responsible for sample collection VS EDvP MPS CS
RFN TML and RQH interpreted the results and wrote the article
All authors critically revised and approved the final manuscript
ACKNOWLEDGMENTThe authors thank all the children and their families who participated in
the Dutch Study for Pediatric MS
STUDY FUNDINGThis work was supported by a program grant provided by the EC 7th
frame work programme of Marie Curie Initial Training NetworkmdashThe
United Europeans for the Development of Pharmacogenomics in Mul-
tiple Sclerosis (UEPHAmdashMS grant PITNmdashGAmdash2008212877) Neth-
erlands Multiple Sclerosis Research Foundation to the Rotterdam MS
Centre ErasMS the Netherlands Organization for Scientific Research
(ZONmdashMW RQH) and Hersenstichting Nederland and Zenith grant
93511034 from the Netherlands Organization for Scientific Research
(NWO)
DISCLOSUREV Singh ED van Pelt MP Stoop C Stingl and IA Ketelslegers
report no disclosures RF Neuteboom is on the scientific advisory board
for LAREB CE Catsman-Berrevoets and TM Luider report no disclo-
sures RQ Hintzen is on the advisory board for Biogen Idec Roche and
Sanofi received research support from Biogen Idec Merck-Serono
Roche Genzyme Novartis Dutch MS Society and European Commis-
sion and is on the editorial board for Multiple Sclerosis and Related
Disorders Go to Neurologyorgnn for full disclosure forms
Received April 14 2015 Accepted in final form June 25 2015
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atric MSSG Consensus definitions proposed for pediatric
multiple sclerosis and related disorders Neurology 2007
68S7ndashS12
2 Banwell B Ghezzi A Bar-Or A Mikaeloff Y Tardieu M
Multiple sclerosis in children clinical diagnosis therapeu-
tic strategies and future directions Lancet Neurol 20076
887ndash902
3 Banwell B Bar-Or A Arnold DL et al Clinical environ-
mental and genetic determinants of multiple sclerosis in
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
children with acute demyelination a prospective national
cohort study Lancet Neurol 201110436ndash445
4 Neuteboom RF Boon M Catsman Berrevoets CE et al
Prognostic factors after a first attack of inflammatory CNS
demyelination in children Neurology 200871967ndash973
5 Krupp LB Tardieu M Amato MP et al International
Pediatric Multiple Sclerosis Study Group criteria for pedi-
atric multiple sclerosis and immune-mediated central ner-
vous system demyelinating disorders revisions to the 2007
definitions Mult Scler 2013191261ndash1267
6 Dhaunchak AS Becker C Schulman H et al Implication
of perturbed axoglial apparatus in early pediatric multiple
sclerosis Ann Neurol 201271601ndash613
7 Singh V Hintzen RQ Luider TM Stoop MP Proteomics
technologies for biomarker discovery in multiple sclerosis
J Neuroimmunol 201224840ndash47
8 Singh V Stoop MP Stingl C et al Cerebrospinal-fluid-
derived immunoglobulin G of different multiple sclerosis
patients shares mutated sequences in complementarity
determining regions Mol Cell Proteomics 201312
3924ndash3934
9 Ketelslegers IA Catsman-Berrevoets CE Neuteboom RF
et al Incidence of acquired demyelinating syndromes of
the CNS in Dutch children a nationwide study J Neurol
20122591929ndash1935
10 Teunissen CE Petzold A Bennett JL et al A consensus
protocol for the standardization of cerebrospinal fluid col-
lection and biobanking Neurology 2009731914ndash1922
11 Stoop MP Singh V Stingl C et al Effects of natalizumab
treatment on the cerebrospinal fluid proteome of multiple
sclerosis patients J Proteome Res 2013121101ndash1107
12 Team RC R A Language and Environment for Statistical
Computing [online] Available at httpwwwR-project
org Accessed February 4 2014
13 Schutzer SE Angel TE Liu T et al Gray matter is tar-
geted in first-attack multiple sclerosis PLoS One 20138
e66117
14 Stoop MP Dekker LJ Titulaer MK et al Multiple
sclerosis-related proteins identified in cerebrospinal fluid
by advanced mass spectrometry Proteomics 20088
1576ndash1585
15 Stoop MP Dekker LJ Titulaer MK et al Quantitative
matrix-assisted laser desorption ionization-fourier trans-
form ion cyclotron resonance (MALDI-FT-ICR) peptide
profiling and identification of multiple-sclerosis-related
proteins J Proteome Res 200981404ndash1414
16 Stoop MP Singh V Dekker LJ et al Proteomics com-
parison of cerebrospinal fluid of relapsing remitting and
primary progressive multiple sclerosis PLoS One 20105
e12442
17 van Zwam M Samsom JN Nieuwenhuis EE et al Mye-
lin ingestion alters macrophage antigen-presenting func-
tion in vitro and in vivo J Leukoc Biol 201190123ndash132
18 Wasco W Gurubhagavatula S Paradis MD et al Isola-
tion and characterization of APLP2 encoding a homologue
of the Alzheimerrsquos associated amyloid beta protein precur-
sor Nat Genet 1993595ndash100
19 Faivre-Sarrailh C Devaux JJ Neuro-glial interactions at
the nodes of Ranvier implication in health and diseases
Front Cell Neurosci 20137196
20 Derfuss T Linington C Hohlfeld R Meinl E Axo-glial
antigens as targets in multiple sclerosis implications for
axonal and grey matter injury J Mol Med (Berl) 2010
88753ndash761
21 Mathey EK Derfuss T Storch MK et al Neurofascin as a
novel target for autoantibody-mediated axonal injury
J Exp Med 20072042363ndash2372
22 Howell OW Palser A Polito A et al Disruption of neu-
rofascin localization reveals early changes preceding demy-
elination and remyelination in multiple sclerosis Brain
20061293173ndash3185
23 Yamada H Fredette B Shitara K et al The brain chon-
droitin sulfate proteoglycan brevican associates with astro-
cytes ensheathing cerebellar glomeruli and inhibits neurite
outgrowth from granule neurons J Neurosci 199717
7784ndash7795
24 Vilijn MH Das B Kessler JA Fricker LD Cultured as-
trocytes and neurons synthesize and secrete carboxypepti-
dase E a neuropeptide-processing enzyme J Neurochem
1989531487ndash1493
25 Ozkaynak E Abello G Jaegle M et al Adam22 is a major
neuronal receptor for Lgi4-mediated Schwann cell signal-
ing J Neurosci 2010303857ndash3864
26 Scarisbrick IA Yoon H Panos M et al Kallikrein 6 reg-
ulates early CNS demyelination in a viral model of multi-
ple sclerosis Brain Pathol 201222709ndash722
27 Hebb AL Bhan V Wishart AD Moore CS
Robertson GS Human kallikrein 6 cerebrospinal levels
are elevated in multiple sclerosis Curr Drug Discov Tech-
nol 20107137ndash140
28 Latour S Tanaka H Demeure C et al Bidirectional
negative regulation of human T and dendritic cells by
CD47 and its cognate receptor signal-regulator protein-
alpha down-regulation of IL-12 responsiveness and inhi-
bition of dendritic cell activation J Immunol 2001167
2547ndash2554
29 OrsquoDonnell LC Druhan LJ Avalos BR Molecular charac-
terization and expression analysis of leucine-rich alpha2-
glycoprotein a novel marker of granulocytic differentiation
J Leukoc Biol 200272478ndash485
30 Chang KH Tseng MY Ro LS et al Analyses of hapto-
globin level in the cerebrospinal fluid and serum of pa-
tients with neuromyelitis optica and multiple sclerosis
Clin Chim Acta 201341726ndash30
31 Chung LP Reid KB Structural and functional studies on
C4b-binding protein a regulatory component of the
human complement system Biosci Rep 19855855ndash865
32 Kurne A Sayat G Aydin OF et al Lack of association of
the CD14Cmdash159T polymorphism with susceptibility
and progression parameters in Turkish multiple sclerosis
patients J Neuroimmunol 201225083ndash86
33 Kitchens RL Thompson PA Modulatory effects of sCD14
and LBP on LPS-host cell interactions J Endotoxin Res
200511225ndash229
34 Huh JR Foe I Muro I et al The Drosophila inhibitor of
apoptosis (IAP) DIAP2 is dispensable for cell survival
required for the innate immune response to gram-negative
bacterial infection and can be negatively regulated by the
reaperhidgrim family of IAP-binding apoptosis inducers
J Biol Chem 20072822056ndash2068
35 Jacobsen KT Iverfeldt K Amyloid precursor protein and
its homologues a family of proteolysis-dependent recep-
tors Cell Mol Life Sci 2009662299ndash2318
36 Koshimizu H Senatorov V Loh YP Gozes I Neuropro-
tective protein and carboxypeptidase E J Mol Neurosci
2009391ndash8
37 Schafer M Brauer AU Savaskan NE Rathjen FG
Brummendorf T Neurotractinkilon promotes neurite
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
outgrowth and is expressed on reactive astrocytes after
entorhinal cortex lesion Mol Cell Neurosci 200529
580ndash590
38 Stoop MP Coulier L Rosenling T et al Quantitative
proteomics and metabolomics analysis of normal human
cerebrospinal fluid samples Mol Cell Proteomics 20109
2063ndash2075
39 Pepe G Chimienti G Liuzzi GM et al Lipoprotein(a) in
the cerebrospinal fluid of neurological patients with blood-
cerebrospinal fluid barrier dysfunction Clin Chem 2006
522043ndash2048
40 Nielsen MJ Petersen SV Jacobsen C et al Haptoglobin-
related protein is a high-affinity hemoglobin-binding plasma
protein Blood 20061082846ndash2849
10 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000015520152 Neurol Neuroimmunol Neuroinflamm
Vaibhav Singh E Danieumllle van Pelt Marcel P Stoop et al related proteins are associated with childhood-onset multiple sclerosisminusGray matter
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We doubt that this influenced the results howeverbecause we did not see an age effect on the abundanceof the 21 identified proteins
Of the 14 proteins with increased abundance inMS (tables 2 and 3) 2 were associated with the amy-loid beta A4 protein family Amyloid-like protein 2 ismainly concentrated at neuronal synapses18 and has arole in memory processes whereas amyloid beta A4protein is associated with neurite growth neuronaladhesion and axonogenesis (table 3) Six proteins(contactin-219 neurofascin20 neuronal growth regu-lator 1 brevican core protein6 neuronal cell adhesionmolecule19 and disintegrin and metalloproteinasedomain-containing protein 22)19 are located at theparanodal and juxtaparanodal region of the CNS ofmyelinated axons (table 3) Contactin-2 is an axonalglycoprotein at the juxtaparanodal domain of myeli-nated axons13 Neurofascin plays a role in the assem-bly of nodes of Ranvier in the CNS21 Two isoformsof neurofascin have been shown to interact withcontactin-associated protein and contactin-1 to forma paranodal junction that attaches the myelin loop tothe axon and helps to separate voltage-gated sodiumchannels at nodal and potassium channels at juxtapar-anodal regions20 Disruption of neurofascin localiza-tion reveals early changes preceding demyelinationand remyelination in MS22 Of interest contactin-2and neurofascin have previously been reported asautoimmune targets in MS20 Neuronal growth
regulator 1 is located at the paranodal region of theCNS and plays a role in axoglia contact at the node ofRanvier Identification of contactin-2 and neurofas-cin in the CSF of children with MS is consistent witha previous study6 however they did not find a sig-nificant difference in the CSF of children with MSand monophasic ADS (tables 2 and 3) Brevican coreprotein is known as a CNS-specific proteoglycan19 atthe surface of neuroglial sheaths where it is enrichedin perisynaptic extracellular matrix23
Among the 14 proteins with increased abun-dance in MS the proteasespeptidases protein car-boxypeptidase E plays a role in the synthesis ofmost neuropeptides24 Disintegrin and metalloprotei-nase domain-containing protein 22 is highly expressedin the brain and localized at the juxtaparanode19 Thismolecule is presumed to be a major neuronal recep-tor25 Another brain-related protease is kallikrein-6which is a secreted serine protease13 It regulates earlyCNS demyelination in a viral model (expression in thebrain and spinal cord of mice) of MS26 In additionelevated levels of kallikrein-6 in CSF have been re-ported in adults with MS compared to neurologic con-trols (table 3)27
Among the other proteins with increased abun-dance in MS tyrosine-protein phosphatase non-receptor type substrate 1 is implicated in neuriteoutgrowth and glia cell attachment and has beenshown to support adhesions of cerebellar neurons28
Table 4 Function of proteins identified with decreased abundance in CSF samples of children with MScompared to samples of children with monophasic ADS
Protein (accession no) FunctionsDhaunchaket al6
Schutzeret al13
Apolipoprotein B-100 (P04114) Innate immune-related not produced inCNS39
3 3
C4b-binding protein alpha chain (P04003) Innate immune defense involved incomplement activity31
3 3
Haptoglobin (P00738) Innate immune defense29 3 3
Haptoglobin-related protein (P00739) Innate immune defense40 3 3
Leucine-rich alpha-2-glycoprotein (P02750) Induced by inflammation and involved incell adhesion high expression in the deepcerebral cortex25 novel marker ofgranulocytic differentiation29
3 3
Monocyte differentiation antigen CD14(P08571)
Main modulator of innate immune system32 3 3
Protein diaphanous homolog 1 (O60610) Coordinates cellular dynamics byregulating microfilament and microtubulefunction role in cellndashmatrix adhesionsvariant related to innate immune function34
3 3
Abbreviations ADS 5 acquired demyelinating syndrome CIS 5 clinically isolated syndrome MS 5 multiple sclerosis3 5 Protein was not identifiedSummary of the function of differentially abundant decreased protein markers in MS and their overlap with previousstudies Most of the proteins were assigned as either neuronal or immune-related molecules based on previous reports anddatabase searches The first column shows the name of the protein and Uniprot accession number The second columnshows the function of the proteins The third column shows comparison with a previous study on children with MS(Dhaunchak et al) using ADS-MS (n 5 8 mean age 12 years) vs monophasic ADS (n 5 11 mean age 10 years) The fourthcolumn shows overlap with the work of Schutzer et al who used CIS-MS (n5 9 age 18ndash42 years) and established MS andcontrols (n 5 6 age 31ndash54 years)
Neurology Neuroimmunology amp Neuroinflammation 7
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
The highest expression of dickkopf-related protein 3is reported in the brain and spinal cord (table 3)13
Thus the majority of proteins with increased abun-dance in our MS group (compared to the monophasicADS group) were neuronal-related with the excep-tion of 2 immune functionndashrelated proteins (Iggamma-1 chain C region and Ig kappa chain V-IIIregion POM) (table 3)
Our study reports 7 proteins with decreased abun-dance in pediatric-onset MS compared to monopha-sic ADS (table 2 and table e-3) Six of these 7 proteinshave previously been reported as specific componentsof innate immune functions (table 4) Haptoglobinan acute phase protein29 was identified as the mostdistinctive protein of those that were elevated in chil-dren with monophasic ADS (compared to childrenwith MS) (1829 18 significant peptides for a total of29 peptides) Another study in adults showedincreased haptoglobin concentration in patients withNMO compared to adult patients with MS30 C4b-binding protein alpha chain is a crucial componentof complement cascade and inhibits the function ofcomplement component31 Of interest we alsofound monocyte differentiation antigen CD14which is mainly expressed on cells of monocyticlineage (macrophages and monocytes)32 HigherCD14 levels might be linked with increased levelsof cytokines triggering inflammatory processes inchildren with monophasic ADS Monocytic cellssecrete soluble sCD14 (activation product of acti-vated monocytes) so this may affect the enormousmacrophage activation during acute monophasicADS33 Among the 7 identified proteins proteindiaphanous homolog 1 has a role in cell adhesionand is expressed in brain and its variants arerequired for innate immune response to Gram-negative bacterial infection34
Overall in the MS group we found a significantoverrepresentation of proteins associated withchanges in CNS gray matter axons synaptic regula-tion node of Ranvier and brain proteases (table 3)Several of these proteins are part of the axoglial appa-ratus and may relate to disturbances in axoglia inter-action6 (table 3) Two of them (contactin-2 andneurofascin) have been identified as possible axoglialautoantigens in MS20 The overlap of proteinsobserved in previous studies613 (table 3) as part ofthe axoglia apparatus and gray matter provides vali-dation for these proteins
These pathologically relevant proteins (mostlyCNS gray matterndashrelated) that are elevated in theCSF of children with early-onset MS might beinvolved in early disease mechanisms Further insightinto the role of these proteins can be useful for under-standing the disease process Moreover such proteinsmight be useful as a future tool to differentiate
children with MS from children with monophasicADS In addition knowing the start of MS could leadto earlier treatment with disease-modifying therapiesHowever the current research is designated as a dis-covery phase study which serves as a basis for thefollow-up verification and validation phase studiesthat can provide clinically valuable biomarkers Inthe future it would be interesting to further validateour findings with an independent technology and inan independent sample group
The data presented in this study indicate thatmonophasic ADS can be differentiated from MS inchildren primarily by CNS gray matter proteins andimmune-related proteins Our findings point to per-turbed axoglial physiology as a hallmark of the earliestevents of MS pathogenesis
AUTHOR CONTRIBUTIONSThe work was carried out in collaboration among all authors VS
TML and RQH designed the experiment VS and CS performed
the experiment and statistical analysis EDvP IAK and CEC-B
were responsible for sample collection VS EDvP MPS CS
RFN TML and RQH interpreted the results and wrote the article
All authors critically revised and approved the final manuscript
ACKNOWLEDGMENTThe authors thank all the children and their families who participated in
the Dutch Study for Pediatric MS
STUDY FUNDINGThis work was supported by a program grant provided by the EC 7th
frame work programme of Marie Curie Initial Training NetworkmdashThe
United Europeans for the Development of Pharmacogenomics in Mul-
tiple Sclerosis (UEPHAmdashMS grant PITNmdashGAmdash2008212877) Neth-
erlands Multiple Sclerosis Research Foundation to the Rotterdam MS
Centre ErasMS the Netherlands Organization for Scientific Research
(ZONmdashMW RQH) and Hersenstichting Nederland and Zenith grant
93511034 from the Netherlands Organization for Scientific Research
(NWO)
DISCLOSUREV Singh ED van Pelt MP Stoop C Stingl and IA Ketelslegers
report no disclosures RF Neuteboom is on the scientific advisory board
for LAREB CE Catsman-Berrevoets and TM Luider report no disclo-
sures RQ Hintzen is on the advisory board for Biogen Idec Roche and
Sanofi received research support from Biogen Idec Merck-Serono
Roche Genzyme Novartis Dutch MS Society and European Commis-
sion and is on the editorial board for Multiple Sclerosis and Related
Disorders Go to Neurologyorgnn for full disclosure forms
Received April 14 2015 Accepted in final form June 25 2015
REFERENCES1 Krupp LB Banwell B Tenembaum S International Pedi-
atric MSSG Consensus definitions proposed for pediatric
multiple sclerosis and related disorders Neurology 2007
68S7ndashS12
2 Banwell B Ghezzi A Bar-Or A Mikaeloff Y Tardieu M
Multiple sclerosis in children clinical diagnosis therapeu-
tic strategies and future directions Lancet Neurol 20076
887ndash902
3 Banwell B Bar-Or A Arnold DL et al Clinical environ-
mental and genetic determinants of multiple sclerosis in
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
children with acute demyelination a prospective national
cohort study Lancet Neurol 201110436ndash445
4 Neuteboom RF Boon M Catsman Berrevoets CE et al
Prognostic factors after a first attack of inflammatory CNS
demyelination in children Neurology 200871967ndash973
5 Krupp LB Tardieu M Amato MP et al International
Pediatric Multiple Sclerosis Study Group criteria for pedi-
atric multiple sclerosis and immune-mediated central ner-
vous system demyelinating disorders revisions to the 2007
definitions Mult Scler 2013191261ndash1267
6 Dhaunchak AS Becker C Schulman H et al Implication
of perturbed axoglial apparatus in early pediatric multiple
sclerosis Ann Neurol 201271601ndash613
7 Singh V Hintzen RQ Luider TM Stoop MP Proteomics
technologies for biomarker discovery in multiple sclerosis
J Neuroimmunol 201224840ndash47
8 Singh V Stoop MP Stingl C et al Cerebrospinal-fluid-
derived immunoglobulin G of different multiple sclerosis
patients shares mutated sequences in complementarity
determining regions Mol Cell Proteomics 201312
3924ndash3934
9 Ketelslegers IA Catsman-Berrevoets CE Neuteboom RF
et al Incidence of acquired demyelinating syndromes of
the CNS in Dutch children a nationwide study J Neurol
20122591929ndash1935
10 Teunissen CE Petzold A Bennett JL et al A consensus
protocol for the standardization of cerebrospinal fluid col-
lection and biobanking Neurology 2009731914ndash1922
11 Stoop MP Singh V Stingl C et al Effects of natalizumab
treatment on the cerebrospinal fluid proteome of multiple
sclerosis patients J Proteome Res 2013121101ndash1107
12 Team RC R A Language and Environment for Statistical
Computing [online] Available at httpwwwR-project
org Accessed February 4 2014
13 Schutzer SE Angel TE Liu T et al Gray matter is tar-
geted in first-attack multiple sclerosis PLoS One 20138
e66117
14 Stoop MP Dekker LJ Titulaer MK et al Multiple
sclerosis-related proteins identified in cerebrospinal fluid
by advanced mass spectrometry Proteomics 20088
1576ndash1585
15 Stoop MP Dekker LJ Titulaer MK et al Quantitative
matrix-assisted laser desorption ionization-fourier trans-
form ion cyclotron resonance (MALDI-FT-ICR) peptide
profiling and identification of multiple-sclerosis-related
proteins J Proteome Res 200981404ndash1414
16 Stoop MP Singh V Dekker LJ et al Proteomics com-
parison of cerebrospinal fluid of relapsing remitting and
primary progressive multiple sclerosis PLoS One 20105
e12442
17 van Zwam M Samsom JN Nieuwenhuis EE et al Mye-
lin ingestion alters macrophage antigen-presenting func-
tion in vitro and in vivo J Leukoc Biol 201190123ndash132
18 Wasco W Gurubhagavatula S Paradis MD et al Isola-
tion and characterization of APLP2 encoding a homologue
of the Alzheimerrsquos associated amyloid beta protein precur-
sor Nat Genet 1993595ndash100
19 Faivre-Sarrailh C Devaux JJ Neuro-glial interactions at
the nodes of Ranvier implication in health and diseases
Front Cell Neurosci 20137196
20 Derfuss T Linington C Hohlfeld R Meinl E Axo-glial
antigens as targets in multiple sclerosis implications for
axonal and grey matter injury J Mol Med (Berl) 2010
88753ndash761
21 Mathey EK Derfuss T Storch MK et al Neurofascin as a
novel target for autoantibody-mediated axonal injury
J Exp Med 20072042363ndash2372
22 Howell OW Palser A Polito A et al Disruption of neu-
rofascin localization reveals early changes preceding demy-
elination and remyelination in multiple sclerosis Brain
20061293173ndash3185
23 Yamada H Fredette B Shitara K et al The brain chon-
droitin sulfate proteoglycan brevican associates with astro-
cytes ensheathing cerebellar glomeruli and inhibits neurite
outgrowth from granule neurons J Neurosci 199717
7784ndash7795
24 Vilijn MH Das B Kessler JA Fricker LD Cultured as-
trocytes and neurons synthesize and secrete carboxypepti-
dase E a neuropeptide-processing enzyme J Neurochem
1989531487ndash1493
25 Ozkaynak E Abello G Jaegle M et al Adam22 is a major
neuronal receptor for Lgi4-mediated Schwann cell signal-
ing J Neurosci 2010303857ndash3864
26 Scarisbrick IA Yoon H Panos M et al Kallikrein 6 reg-
ulates early CNS demyelination in a viral model of multi-
ple sclerosis Brain Pathol 201222709ndash722
27 Hebb AL Bhan V Wishart AD Moore CS
Robertson GS Human kallikrein 6 cerebrospinal levels
are elevated in multiple sclerosis Curr Drug Discov Tech-
nol 20107137ndash140
28 Latour S Tanaka H Demeure C et al Bidirectional
negative regulation of human T and dendritic cells by
CD47 and its cognate receptor signal-regulator protein-
alpha down-regulation of IL-12 responsiveness and inhi-
bition of dendritic cell activation J Immunol 2001167
2547ndash2554
29 OrsquoDonnell LC Druhan LJ Avalos BR Molecular charac-
terization and expression analysis of leucine-rich alpha2-
glycoprotein a novel marker of granulocytic differentiation
J Leukoc Biol 200272478ndash485
30 Chang KH Tseng MY Ro LS et al Analyses of hapto-
globin level in the cerebrospinal fluid and serum of pa-
tients with neuromyelitis optica and multiple sclerosis
Clin Chim Acta 201341726ndash30
31 Chung LP Reid KB Structural and functional studies on
C4b-binding protein a regulatory component of the
human complement system Biosci Rep 19855855ndash865
32 Kurne A Sayat G Aydin OF et al Lack of association of
the CD14Cmdash159T polymorphism with susceptibility
and progression parameters in Turkish multiple sclerosis
patients J Neuroimmunol 201225083ndash86
33 Kitchens RL Thompson PA Modulatory effects of sCD14
and LBP on LPS-host cell interactions J Endotoxin Res
200511225ndash229
34 Huh JR Foe I Muro I et al The Drosophila inhibitor of
apoptosis (IAP) DIAP2 is dispensable for cell survival
required for the innate immune response to gram-negative
bacterial infection and can be negatively regulated by the
reaperhidgrim family of IAP-binding apoptosis inducers
J Biol Chem 20072822056ndash2068
35 Jacobsen KT Iverfeldt K Amyloid precursor protein and
its homologues a family of proteolysis-dependent recep-
tors Cell Mol Life Sci 2009662299ndash2318
36 Koshimizu H Senatorov V Loh YP Gozes I Neuropro-
tective protein and carboxypeptidase E J Mol Neurosci
2009391ndash8
37 Schafer M Brauer AU Savaskan NE Rathjen FG
Brummendorf T Neurotractinkilon promotes neurite
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
outgrowth and is expressed on reactive astrocytes after
entorhinal cortex lesion Mol Cell Neurosci 200529
580ndash590
38 Stoop MP Coulier L Rosenling T et al Quantitative
proteomics and metabolomics analysis of normal human
cerebrospinal fluid samples Mol Cell Proteomics 20109
2063ndash2075
39 Pepe G Chimienti G Liuzzi GM et al Lipoprotein(a) in
the cerebrospinal fluid of neurological patients with blood-
cerebrospinal fluid barrier dysfunction Clin Chem 2006
522043ndash2048
40 Nielsen MJ Petersen SV Jacobsen C et al Haptoglobin-
related protein is a high-affinity hemoglobin-binding plasma
protein Blood 20061082846ndash2849
10 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000015520152 Neurol Neuroimmunol Neuroinflamm
Vaibhav Singh E Danieumllle van Pelt Marcel P Stoop et al related proteins are associated with childhood-onset multiple sclerosisminusGray matter
This information is current as of September 24 2015
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent25e155fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2015092425e155DC1
Supplementary material can be found at
References httpnnneurologyorgcontent25e155fullhtmlref-list-1
This article cites 39 articles 10 of which you can access for free at
Citations httpnnneurologyorgcontent25e155fullhtmlotherarticles
This article has been cited by 1 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis
httpnnneurologyorgcgicollectionall_pediatricAll Pediatric
myelitishttpnnneurologyorgcgicollectionacute_disseminated_encephaloAcute disseminated encephalomyelitisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
The highest expression of dickkopf-related protein 3is reported in the brain and spinal cord (table 3)13
Thus the majority of proteins with increased abun-dance in our MS group (compared to the monophasicADS group) were neuronal-related with the excep-tion of 2 immune functionndashrelated proteins (Iggamma-1 chain C region and Ig kappa chain V-IIIregion POM) (table 3)
Our study reports 7 proteins with decreased abun-dance in pediatric-onset MS compared to monopha-sic ADS (table 2 and table e-3) Six of these 7 proteinshave previously been reported as specific componentsof innate immune functions (table 4) Haptoglobinan acute phase protein29 was identified as the mostdistinctive protein of those that were elevated in chil-dren with monophasic ADS (compared to childrenwith MS) (1829 18 significant peptides for a total of29 peptides) Another study in adults showedincreased haptoglobin concentration in patients withNMO compared to adult patients with MS30 C4b-binding protein alpha chain is a crucial componentof complement cascade and inhibits the function ofcomplement component31 Of interest we alsofound monocyte differentiation antigen CD14which is mainly expressed on cells of monocyticlineage (macrophages and monocytes)32 HigherCD14 levels might be linked with increased levelsof cytokines triggering inflammatory processes inchildren with monophasic ADS Monocytic cellssecrete soluble sCD14 (activation product of acti-vated monocytes) so this may affect the enormousmacrophage activation during acute monophasicADS33 Among the 7 identified proteins proteindiaphanous homolog 1 has a role in cell adhesionand is expressed in brain and its variants arerequired for innate immune response to Gram-negative bacterial infection34
Overall in the MS group we found a significantoverrepresentation of proteins associated withchanges in CNS gray matter axons synaptic regula-tion node of Ranvier and brain proteases (table 3)Several of these proteins are part of the axoglial appa-ratus and may relate to disturbances in axoglia inter-action6 (table 3) Two of them (contactin-2 andneurofascin) have been identified as possible axoglialautoantigens in MS20 The overlap of proteinsobserved in previous studies613 (table 3) as part ofthe axoglia apparatus and gray matter provides vali-dation for these proteins
These pathologically relevant proteins (mostlyCNS gray matterndashrelated) that are elevated in theCSF of children with early-onset MS might beinvolved in early disease mechanisms Further insightinto the role of these proteins can be useful for under-standing the disease process Moreover such proteinsmight be useful as a future tool to differentiate
children with MS from children with monophasicADS In addition knowing the start of MS could leadto earlier treatment with disease-modifying therapiesHowever the current research is designated as a dis-covery phase study which serves as a basis for thefollow-up verification and validation phase studiesthat can provide clinically valuable biomarkers Inthe future it would be interesting to further validateour findings with an independent technology and inan independent sample group
The data presented in this study indicate thatmonophasic ADS can be differentiated from MS inchildren primarily by CNS gray matter proteins andimmune-related proteins Our findings point to per-turbed axoglial physiology as a hallmark of the earliestevents of MS pathogenesis
AUTHOR CONTRIBUTIONSThe work was carried out in collaboration among all authors VS
TML and RQH designed the experiment VS and CS performed
the experiment and statistical analysis EDvP IAK and CEC-B
were responsible for sample collection VS EDvP MPS CS
RFN TML and RQH interpreted the results and wrote the article
All authors critically revised and approved the final manuscript
ACKNOWLEDGMENTThe authors thank all the children and their families who participated in
the Dutch Study for Pediatric MS
STUDY FUNDINGThis work was supported by a program grant provided by the EC 7th
frame work programme of Marie Curie Initial Training NetworkmdashThe
United Europeans for the Development of Pharmacogenomics in Mul-
tiple Sclerosis (UEPHAmdashMS grant PITNmdashGAmdash2008212877) Neth-
erlands Multiple Sclerosis Research Foundation to the Rotterdam MS
Centre ErasMS the Netherlands Organization for Scientific Research
(ZONmdashMW RQH) and Hersenstichting Nederland and Zenith grant
93511034 from the Netherlands Organization for Scientific Research
(NWO)
DISCLOSUREV Singh ED van Pelt MP Stoop C Stingl and IA Ketelslegers
report no disclosures RF Neuteboom is on the scientific advisory board
for LAREB CE Catsman-Berrevoets and TM Luider report no disclo-
sures RQ Hintzen is on the advisory board for Biogen Idec Roche and
Sanofi received research support from Biogen Idec Merck-Serono
Roche Genzyme Novartis Dutch MS Society and European Commis-
sion and is on the editorial board for Multiple Sclerosis and Related
Disorders Go to Neurologyorgnn for full disclosure forms
Received April 14 2015 Accepted in final form June 25 2015
REFERENCES1 Krupp LB Banwell B Tenembaum S International Pedi-
atric MSSG Consensus definitions proposed for pediatric
multiple sclerosis and related disorders Neurology 2007
68S7ndashS12
2 Banwell B Ghezzi A Bar-Or A Mikaeloff Y Tardieu M
Multiple sclerosis in children clinical diagnosis therapeu-
tic strategies and future directions Lancet Neurol 20076
887ndash902
3 Banwell B Bar-Or A Arnold DL et al Clinical environ-
mental and genetic determinants of multiple sclerosis in
8 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
children with acute demyelination a prospective national
cohort study Lancet Neurol 201110436ndash445
4 Neuteboom RF Boon M Catsman Berrevoets CE et al
Prognostic factors after a first attack of inflammatory CNS
demyelination in children Neurology 200871967ndash973
5 Krupp LB Tardieu M Amato MP et al International
Pediatric Multiple Sclerosis Study Group criteria for pedi-
atric multiple sclerosis and immune-mediated central ner-
vous system demyelinating disorders revisions to the 2007
definitions Mult Scler 2013191261ndash1267
6 Dhaunchak AS Becker C Schulman H et al Implication
of perturbed axoglial apparatus in early pediatric multiple
sclerosis Ann Neurol 201271601ndash613
7 Singh V Hintzen RQ Luider TM Stoop MP Proteomics
technologies for biomarker discovery in multiple sclerosis
J Neuroimmunol 201224840ndash47
8 Singh V Stoop MP Stingl C et al Cerebrospinal-fluid-
derived immunoglobulin G of different multiple sclerosis
patients shares mutated sequences in complementarity
determining regions Mol Cell Proteomics 201312
3924ndash3934
9 Ketelslegers IA Catsman-Berrevoets CE Neuteboom RF
et al Incidence of acquired demyelinating syndromes of
the CNS in Dutch children a nationwide study J Neurol
20122591929ndash1935
10 Teunissen CE Petzold A Bennett JL et al A consensus
protocol for the standardization of cerebrospinal fluid col-
lection and biobanking Neurology 2009731914ndash1922
11 Stoop MP Singh V Stingl C et al Effects of natalizumab
treatment on the cerebrospinal fluid proteome of multiple
sclerosis patients J Proteome Res 2013121101ndash1107
12 Team RC R A Language and Environment for Statistical
Computing [online] Available at httpwwwR-project
org Accessed February 4 2014
13 Schutzer SE Angel TE Liu T et al Gray matter is tar-
geted in first-attack multiple sclerosis PLoS One 20138
e66117
14 Stoop MP Dekker LJ Titulaer MK et al Multiple
sclerosis-related proteins identified in cerebrospinal fluid
by advanced mass spectrometry Proteomics 20088
1576ndash1585
15 Stoop MP Dekker LJ Titulaer MK et al Quantitative
matrix-assisted laser desorption ionization-fourier trans-
form ion cyclotron resonance (MALDI-FT-ICR) peptide
profiling and identification of multiple-sclerosis-related
proteins J Proteome Res 200981404ndash1414
16 Stoop MP Singh V Dekker LJ et al Proteomics com-
parison of cerebrospinal fluid of relapsing remitting and
primary progressive multiple sclerosis PLoS One 20105
e12442
17 van Zwam M Samsom JN Nieuwenhuis EE et al Mye-
lin ingestion alters macrophage antigen-presenting func-
tion in vitro and in vivo J Leukoc Biol 201190123ndash132
18 Wasco W Gurubhagavatula S Paradis MD et al Isola-
tion and characterization of APLP2 encoding a homologue
of the Alzheimerrsquos associated amyloid beta protein precur-
sor Nat Genet 1993595ndash100
19 Faivre-Sarrailh C Devaux JJ Neuro-glial interactions at
the nodes of Ranvier implication in health and diseases
Front Cell Neurosci 20137196
20 Derfuss T Linington C Hohlfeld R Meinl E Axo-glial
antigens as targets in multiple sclerosis implications for
axonal and grey matter injury J Mol Med (Berl) 2010
88753ndash761
21 Mathey EK Derfuss T Storch MK et al Neurofascin as a
novel target for autoantibody-mediated axonal injury
J Exp Med 20072042363ndash2372
22 Howell OW Palser A Polito A et al Disruption of neu-
rofascin localization reveals early changes preceding demy-
elination and remyelination in multiple sclerosis Brain
20061293173ndash3185
23 Yamada H Fredette B Shitara K et al The brain chon-
droitin sulfate proteoglycan brevican associates with astro-
cytes ensheathing cerebellar glomeruli and inhibits neurite
outgrowth from granule neurons J Neurosci 199717
7784ndash7795
24 Vilijn MH Das B Kessler JA Fricker LD Cultured as-
trocytes and neurons synthesize and secrete carboxypepti-
dase E a neuropeptide-processing enzyme J Neurochem
1989531487ndash1493
25 Ozkaynak E Abello G Jaegle M et al Adam22 is a major
neuronal receptor for Lgi4-mediated Schwann cell signal-
ing J Neurosci 2010303857ndash3864
26 Scarisbrick IA Yoon H Panos M et al Kallikrein 6 reg-
ulates early CNS demyelination in a viral model of multi-
ple sclerosis Brain Pathol 201222709ndash722
27 Hebb AL Bhan V Wishart AD Moore CS
Robertson GS Human kallikrein 6 cerebrospinal levels
are elevated in multiple sclerosis Curr Drug Discov Tech-
nol 20107137ndash140
28 Latour S Tanaka H Demeure C et al Bidirectional
negative regulation of human T and dendritic cells by
CD47 and its cognate receptor signal-regulator protein-
alpha down-regulation of IL-12 responsiveness and inhi-
bition of dendritic cell activation J Immunol 2001167
2547ndash2554
29 OrsquoDonnell LC Druhan LJ Avalos BR Molecular charac-
terization and expression analysis of leucine-rich alpha2-
glycoprotein a novel marker of granulocytic differentiation
J Leukoc Biol 200272478ndash485
30 Chang KH Tseng MY Ro LS et al Analyses of hapto-
globin level in the cerebrospinal fluid and serum of pa-
tients with neuromyelitis optica and multiple sclerosis
Clin Chim Acta 201341726ndash30
31 Chung LP Reid KB Structural and functional studies on
C4b-binding protein a regulatory component of the
human complement system Biosci Rep 19855855ndash865
32 Kurne A Sayat G Aydin OF et al Lack of association of
the CD14Cmdash159T polymorphism with susceptibility
and progression parameters in Turkish multiple sclerosis
patients J Neuroimmunol 201225083ndash86
33 Kitchens RL Thompson PA Modulatory effects of sCD14
and LBP on LPS-host cell interactions J Endotoxin Res
200511225ndash229
34 Huh JR Foe I Muro I et al The Drosophila inhibitor of
apoptosis (IAP) DIAP2 is dispensable for cell survival
required for the innate immune response to gram-negative
bacterial infection and can be negatively regulated by the
reaperhidgrim family of IAP-binding apoptosis inducers
J Biol Chem 20072822056ndash2068
35 Jacobsen KT Iverfeldt K Amyloid precursor protein and
its homologues a family of proteolysis-dependent recep-
tors Cell Mol Life Sci 2009662299ndash2318
36 Koshimizu H Senatorov V Loh YP Gozes I Neuropro-
tective protein and carboxypeptidase E J Mol Neurosci
2009391ndash8
37 Schafer M Brauer AU Savaskan NE Rathjen FG
Brummendorf T Neurotractinkilon promotes neurite
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
outgrowth and is expressed on reactive astrocytes after
entorhinal cortex lesion Mol Cell Neurosci 200529
580ndash590
38 Stoop MP Coulier L Rosenling T et al Quantitative
proteomics and metabolomics analysis of normal human
cerebrospinal fluid samples Mol Cell Proteomics 20109
2063ndash2075
39 Pepe G Chimienti G Liuzzi GM et al Lipoprotein(a) in
the cerebrospinal fluid of neurological patients with blood-
cerebrospinal fluid barrier dysfunction Clin Chem 2006
522043ndash2048
40 Nielsen MJ Petersen SV Jacobsen C et al Haptoglobin-
related protein is a high-affinity hemoglobin-binding plasma
protein Blood 20061082846ndash2849
10 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000015520152 Neurol Neuroimmunol Neuroinflamm
Vaibhav Singh E Danieumllle van Pelt Marcel P Stoop et al related proteins are associated with childhood-onset multiple sclerosisminusGray matter
This information is current as of September 24 2015
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent25e155fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2015092425e155DC1
Supplementary material can be found at
References httpnnneurologyorgcontent25e155fullhtmlref-list-1
This article cites 39 articles 10 of which you can access for free at
Citations httpnnneurologyorgcontent25e155fullhtmlotherarticles
This article has been cited by 1 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis
httpnnneurologyorgcgicollectionall_pediatricAll Pediatric
myelitishttpnnneurologyorgcgicollectionacute_disseminated_encephaloAcute disseminated encephalomyelitisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
children with acute demyelination a prospective national
cohort study Lancet Neurol 201110436ndash445
4 Neuteboom RF Boon M Catsman Berrevoets CE et al
Prognostic factors after a first attack of inflammatory CNS
demyelination in children Neurology 200871967ndash973
5 Krupp LB Tardieu M Amato MP et al International
Pediatric Multiple Sclerosis Study Group criteria for pedi-
atric multiple sclerosis and immune-mediated central ner-
vous system demyelinating disorders revisions to the 2007
definitions Mult Scler 2013191261ndash1267
6 Dhaunchak AS Becker C Schulman H et al Implication
of perturbed axoglial apparatus in early pediatric multiple
sclerosis Ann Neurol 201271601ndash613
7 Singh V Hintzen RQ Luider TM Stoop MP Proteomics
technologies for biomarker discovery in multiple sclerosis
J Neuroimmunol 201224840ndash47
8 Singh V Stoop MP Stingl C et al Cerebrospinal-fluid-
derived immunoglobulin G of different multiple sclerosis
patients shares mutated sequences in complementarity
determining regions Mol Cell Proteomics 201312
3924ndash3934
9 Ketelslegers IA Catsman-Berrevoets CE Neuteboom RF
et al Incidence of acquired demyelinating syndromes of
the CNS in Dutch children a nationwide study J Neurol
20122591929ndash1935
10 Teunissen CE Petzold A Bennett JL et al A consensus
protocol for the standardization of cerebrospinal fluid col-
lection and biobanking Neurology 2009731914ndash1922
11 Stoop MP Singh V Stingl C et al Effects of natalizumab
treatment on the cerebrospinal fluid proteome of multiple
sclerosis patients J Proteome Res 2013121101ndash1107
12 Team RC R A Language and Environment for Statistical
Computing [online] Available at httpwwwR-project
org Accessed February 4 2014
13 Schutzer SE Angel TE Liu T et al Gray matter is tar-
geted in first-attack multiple sclerosis PLoS One 20138
e66117
14 Stoop MP Dekker LJ Titulaer MK et al Multiple
sclerosis-related proteins identified in cerebrospinal fluid
by advanced mass spectrometry Proteomics 20088
1576ndash1585
15 Stoop MP Dekker LJ Titulaer MK et al Quantitative
matrix-assisted laser desorption ionization-fourier trans-
form ion cyclotron resonance (MALDI-FT-ICR) peptide
profiling and identification of multiple-sclerosis-related
proteins J Proteome Res 200981404ndash1414
16 Stoop MP Singh V Dekker LJ et al Proteomics com-
parison of cerebrospinal fluid of relapsing remitting and
primary progressive multiple sclerosis PLoS One 20105
e12442
17 van Zwam M Samsom JN Nieuwenhuis EE et al Mye-
lin ingestion alters macrophage antigen-presenting func-
tion in vitro and in vivo J Leukoc Biol 201190123ndash132
18 Wasco W Gurubhagavatula S Paradis MD et al Isola-
tion and characterization of APLP2 encoding a homologue
of the Alzheimerrsquos associated amyloid beta protein precur-
sor Nat Genet 1993595ndash100
19 Faivre-Sarrailh C Devaux JJ Neuro-glial interactions at
the nodes of Ranvier implication in health and diseases
Front Cell Neurosci 20137196
20 Derfuss T Linington C Hohlfeld R Meinl E Axo-glial
antigens as targets in multiple sclerosis implications for
axonal and grey matter injury J Mol Med (Berl) 2010
88753ndash761
21 Mathey EK Derfuss T Storch MK et al Neurofascin as a
novel target for autoantibody-mediated axonal injury
J Exp Med 20072042363ndash2372
22 Howell OW Palser A Polito A et al Disruption of neu-
rofascin localization reveals early changes preceding demy-
elination and remyelination in multiple sclerosis Brain
20061293173ndash3185
23 Yamada H Fredette B Shitara K et al The brain chon-
droitin sulfate proteoglycan brevican associates with astro-
cytes ensheathing cerebellar glomeruli and inhibits neurite
outgrowth from granule neurons J Neurosci 199717
7784ndash7795
24 Vilijn MH Das B Kessler JA Fricker LD Cultured as-
trocytes and neurons synthesize and secrete carboxypepti-
dase E a neuropeptide-processing enzyme J Neurochem
1989531487ndash1493
25 Ozkaynak E Abello G Jaegle M et al Adam22 is a major
neuronal receptor for Lgi4-mediated Schwann cell signal-
ing J Neurosci 2010303857ndash3864
26 Scarisbrick IA Yoon H Panos M et al Kallikrein 6 reg-
ulates early CNS demyelination in a viral model of multi-
ple sclerosis Brain Pathol 201222709ndash722
27 Hebb AL Bhan V Wishart AD Moore CS
Robertson GS Human kallikrein 6 cerebrospinal levels
are elevated in multiple sclerosis Curr Drug Discov Tech-
nol 20107137ndash140
28 Latour S Tanaka H Demeure C et al Bidirectional
negative regulation of human T and dendritic cells by
CD47 and its cognate receptor signal-regulator protein-
alpha down-regulation of IL-12 responsiveness and inhi-
bition of dendritic cell activation J Immunol 2001167
2547ndash2554
29 OrsquoDonnell LC Druhan LJ Avalos BR Molecular charac-
terization and expression analysis of leucine-rich alpha2-
glycoprotein a novel marker of granulocytic differentiation
J Leukoc Biol 200272478ndash485
30 Chang KH Tseng MY Ro LS et al Analyses of hapto-
globin level in the cerebrospinal fluid and serum of pa-
tients with neuromyelitis optica and multiple sclerosis
Clin Chim Acta 201341726ndash30
31 Chung LP Reid KB Structural and functional studies on
C4b-binding protein a regulatory component of the
human complement system Biosci Rep 19855855ndash865
32 Kurne A Sayat G Aydin OF et al Lack of association of
the CD14Cmdash159T polymorphism with susceptibility
and progression parameters in Turkish multiple sclerosis
patients J Neuroimmunol 201225083ndash86
33 Kitchens RL Thompson PA Modulatory effects of sCD14
and LBP on LPS-host cell interactions J Endotoxin Res
200511225ndash229
34 Huh JR Foe I Muro I et al The Drosophila inhibitor of
apoptosis (IAP) DIAP2 is dispensable for cell survival
required for the innate immune response to gram-negative
bacterial infection and can be negatively regulated by the
reaperhidgrim family of IAP-binding apoptosis inducers
J Biol Chem 20072822056ndash2068
35 Jacobsen KT Iverfeldt K Amyloid precursor protein and
its homologues a family of proteolysis-dependent recep-
tors Cell Mol Life Sci 2009662299ndash2318
36 Koshimizu H Senatorov V Loh YP Gozes I Neuropro-
tective protein and carboxypeptidase E J Mol Neurosci
2009391ndash8
37 Schafer M Brauer AU Savaskan NE Rathjen FG
Brummendorf T Neurotractinkilon promotes neurite
Neurology Neuroimmunology amp Neuroinflammation 9
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
outgrowth and is expressed on reactive astrocytes after
entorhinal cortex lesion Mol Cell Neurosci 200529
580ndash590
38 Stoop MP Coulier L Rosenling T et al Quantitative
proteomics and metabolomics analysis of normal human
cerebrospinal fluid samples Mol Cell Proteomics 20109
2063ndash2075
39 Pepe G Chimienti G Liuzzi GM et al Lipoprotein(a) in
the cerebrospinal fluid of neurological patients with blood-
cerebrospinal fluid barrier dysfunction Clin Chem 2006
522043ndash2048
40 Nielsen MJ Petersen SV Jacobsen C et al Haptoglobin-
related protein is a high-affinity hemoglobin-binding plasma
protein Blood 20061082846ndash2849
10 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000015520152 Neurol Neuroimmunol Neuroinflamm
Vaibhav Singh E Danieumllle van Pelt Marcel P Stoop et al related proteins are associated with childhood-onset multiple sclerosisminusGray matter
This information is current as of September 24 2015
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent25e155fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2015092425e155DC1
Supplementary material can be found at
References httpnnneurologyorgcontent25e155fullhtmlref-list-1
This article cites 39 articles 10 of which you can access for free at
Citations httpnnneurologyorgcontent25e155fullhtmlotherarticles
This article has been cited by 1 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis
httpnnneurologyorgcgicollectionall_pediatricAll Pediatric
myelitishttpnnneurologyorgcgicollectionacute_disseminated_encephaloAcute disseminated encephalomyelitisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
outgrowth and is expressed on reactive astrocytes after
entorhinal cortex lesion Mol Cell Neurosci 200529
580ndash590
38 Stoop MP Coulier L Rosenling T et al Quantitative
proteomics and metabolomics analysis of normal human
cerebrospinal fluid samples Mol Cell Proteomics 20109
2063ndash2075
39 Pepe G Chimienti G Liuzzi GM et al Lipoprotein(a) in
the cerebrospinal fluid of neurological patients with blood-
cerebrospinal fluid barrier dysfunction Clin Chem 2006
522043ndash2048
40 Nielsen MJ Petersen SV Jacobsen C et al Haptoglobin-
related protein is a high-affinity hemoglobin-binding plasma
protein Blood 20061082846ndash2849
10 Neurology Neuroimmunology amp Neuroinflammation
ordf 2015 American Academy of Neurology Unauthorized reproduction of this article is prohibited
DOI 101212NXI000000000000015520152 Neurol Neuroimmunol Neuroinflamm
Vaibhav Singh E Danieumllle van Pelt Marcel P Stoop et al related proteins are associated with childhood-onset multiple sclerosisminusGray matter
This information is current as of September 24 2015
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent25e155fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2015092425e155DC1
Supplementary material can be found at
References httpnnneurologyorgcontent25e155fullhtmlref-list-1
This article cites 39 articles 10 of which you can access for free at
Citations httpnnneurologyorgcontent25e155fullhtmlotherarticles
This article has been cited by 1 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis
httpnnneurologyorgcgicollectionall_pediatricAll Pediatric
myelitishttpnnneurologyorgcgicollectionacute_disseminated_encephaloAcute disseminated encephalomyelitisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
DOI 101212NXI000000000000015520152 Neurol Neuroimmunol Neuroinflamm
Vaibhav Singh E Danieumllle van Pelt Marcel P Stoop et al related proteins are associated with childhood-onset multiple sclerosisminusGray matter
This information is current as of September 24 2015
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent25e155fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2015092425e155DC1
Supplementary material can be found at
References httpnnneurologyorgcontent25e155fullhtmlref-list-1
This article cites 39 articles 10 of which you can access for free at
Citations httpnnneurologyorgcontent25e155fullhtmlotherarticles
This article has been cited by 1 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis
httpnnneurologyorgcgicollectionall_pediatricAll Pediatric
myelitishttpnnneurologyorgcgicollectionacute_disseminated_encephaloAcute disseminated encephalomyelitisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm
ServicesUpdated Information amp
httpnnneurologyorgcontent25e155fullhtmlincluding high resolution figures can be found at
Supplementary Material httpnnneurologyorgcontentsuppl2015092425e155DC1
Supplementary material can be found at
References httpnnneurologyorgcontent25e155fullhtmlref-list-1
This article cites 39 articles 10 of which you can access for free at
Citations httpnnneurologyorgcontent25e155fullhtmlotherarticles
This article has been cited by 1 HighWire-hosted articles
Subspecialty Collections
httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis
httpnnneurologyorgcgicollectionall_pediatricAll Pediatric
myelitishttpnnneurologyorgcgicollectionacute_disseminated_encephaloAcute disseminated encephalomyelitisfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
2015 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy
is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm