Gray matter related proteins are associated with childhood ... · Vaibhav Singh, MSc E. Daniëlle...

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

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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


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


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



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



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)



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)



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



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



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



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

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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

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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



















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20122591929ndash1935












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20061293173ndash3185





7784ndash7795




1989531487ndash1493
















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20122591929ndash1935












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20061293173ndash3185





7784ndash7795




1989531487ndash1493
















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2009391ndash8







580ndash590




2063ndash2075




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Reprints



























(NWO)













68S7ndashS12




887ndash902

























3924ndash3934




20122591929ndash1935












e66117




1576ndash1585









e12442














88753ndash761







20061293173ndash3185





7784ndash7795




1989531487ndash1493
















2547ndash2554


















200511225ndash229












2009391ndash8







580ndash590




2063ndash2075




522043ndash2048

























Reprints
























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20122591929ndash1935












e66117




1576ndash1585









e12442














88753ndash761







20061293173ndash3185





7784ndash7795




1989531487ndash1493
















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200511225ndash229












2009391ndash8







580ndash590




2063ndash2075




522043ndash2048

























Reprints






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2063ndash2075




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Reprints




Gray matter related proteins are associated with childhood ... · Vaibhav Singh, MSc E. Daniëlle...

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Transcript of Gray matter related proteins are associated with childhood ... · Vaibhav Singh, MSc E. Daniëlle...