Na u e Men al Heal h | Volume 1 | Augus 2023 | 596–605 596
na u e men al heal h
A icle h ps://doi.o g/10.1038/s44220-023-00103-2
Plasma p o eomics disco e y o men al
heal h isk bioma ke s in adolescen s
Izaque de Sousa Maciel 1,8, Aino-Kaisa Pii onen 1,8, Alexey M. A onin1,8,
Ma iia I ano a1, A o Ala alo 1, Kaus ubh Kisho Jadha 1, Jo di Jul ez2,3,4,
Ma ia Fo as e 3,4,5,6, I ene an Kamp7 & Ka ja M. Kanninen1
An es ima ed 10–20% o adolescen s expe ience men al heal h condi ions,
and mos o hem emain unde diagnosed and unde ea ed. Disco e ing
new suscep ibili y bioma ke s is he e o e impo an o iden i ying
indi iduals a high isk o de eloping men al heal h p oblems, and o
imp o ing ea ly p e en ion. He e we aimed o disco e plasma p o ein-
based suscep ibili y bioma ke s in child en/adolescen s aged 11–16 yea s
a isk o de eloping men al heal h issues. Risk was e alua ed on he basis
o sel - epo ed S eng hs and Di icul ies Ques ionnai e (SDQ) sco es,
and plasma p o eomic da a we e ob ained o indi iduals pa icipa ing in
he Spanish WALNUTs coho s udy by liquid ch oma og aphy– andem
mass spec ome y. Bioin o ma ic analyses we e pe o med o iden i y
he biological p ocesses and pa hways in which he iden i ied bioma ke
candida es a e in ol ed; 58 p o eins we e signi ican ly associa ed wi h he
SDQ sco e. The mos p ominen en iched pa hways ela ed o hese p o eins
included immune esponses, blood coagula ion, neu ogenesis and neu onal
degene a ion. This explo a o y s udy e ealed se e al al e a ions o plasma
p o eins associa ed wi h he SDQ sco e in adolescen s, which opens a
new a enue o de elop no el suscep ibili y bioma ke s o imp o e ea ly
iden i ica ion o indi iduals a isk o men al heal h p oblems.
Adolescence is a pe iod o li e o p o ound changes in he biologi-
cal, psychosocial, cogni i e and emo ional domains
1–3
. The dynamic
b ain de elopmen du ing you h opens a c i ical window o cogni i e
imp o emen , bu also he onse and de elopmen o men al diso -
de s4,5. Se e al men al diso de s, such as a en ion de ici hype ac i i y
diso de , phobias, obsessi e compulsi e diso de , ea ing diso de ,
subs ance use, mood and social anxie y diso de , begin be o e he
indi idual eaches adul hood, wi h a peak age o onse o 14 yea s
5–7
.
Men al diso de s nega i ely impac adolescen de elopmen , leading
o mo bidi y, mo ali y and dys unc ion in la e li e5,8. The e o e, iden-
i ying adolescen s wi h a high isk o de eloping men al heal h issues
and imp o ing ea ly diagnos ics could imp o e he clinical ou comes
and dec ease he socio-economic impac .
Globally he p e alence o men al heal h condi ions in adolescen s
is es ima ed o be be ween 10–20%, and mos cases emain unde diag-
nosed and unde ea ed
9,10
. The social s igma o men al diso de s, he
adolescen and pa en pe cep ion o men al heal h ca e needs, and he
lack o men al heal h esou ces a e some ac o s ha con ibu e o he
numbe o adolescen s wi hou p ope diagnosis and ea men
11,12
.
Fu he mo e, misdiagnosis o o e diagnosis could expose he ado-
lescen o unnecessa y ea men
13
. Diagnos ics o men al diso de s
a e based on he In e na ional Classi ica ion o Diseases (ICD) and he
Recei ed: 22 Decembe 2022
Accep ed: 29 June 2023
Published online: 31 July 2023
Check o upda es
1A.I. Vi anen Ins i u e o Molecula Sciences, Uni e si y o Eas e n Finland, Kuopio, Finland. 2Clinical and Epidemiological Neu oscience (Neu oÈpia),
Ins i u d’In es igació Sani à ia Pe e Vi gili (IISPV), Reus, Spain. 3ISGlobal, Ba celona, Spain. 4CIBER Epidemiología y Salud Pública (CIBEREsp), Mad id,
Spain. 5Uni e si a Pompeu Fab a (UPF), Ba celona, Spain. 6PHAGEX Resea ch G oup, Blanque na School o Heal h Science, Uni e si a Ramon Llull
(URL), Ba celona, Spain. 7Cen e o Sus ainabili y, En i onmen and Heal h, Na ional Ins i u e o Public Heal h and he En i onmen , Bil ho en,
he Ne he lands. 8These au ho s con ibu ed equally: Izaque de Sousa Maciel, Aino-Kaisa Pii onen, Alexey M. A onin. e-mail: ka ja.kanninen@ue . i
Na u e Men al Heal h | Volume 1 | Augus 2023 | 596–605 597
A icle h ps://doi.o g/10.1038/s44220-023-00103-2
Plasma samples and beha io al ou come
The pe iphe al blood plasma samples we e ob ained and analyzed
om a subsample o 91 adolescen s, aged 11–16 yea s, o he WALNUTs
egional Spanish s udy (Table 1). The samples we e collec ed in 2016 a
app oxima ely he same ime ha he pa icipan s illed ou he SDQ.
This baseline subsample wi hou any die a y in e en ion was selec ed
on he basis o he a ailabili y o blood samples and illed SDQ ques-
ionnai es, as well as he o al sco es o he SDQ ques ionnai e. Based
on he sel - epo ed SDQ sco e, he plasma samples we e ca ego ized
in o lowe (SDQ = 0–14) and aised (SDQ = 15–25) g oups
35
. The plasma
samples we e s o ed undis u bed a −80 °C un il hey we e hawed in
2021 o p o ein deple ion and subsequen p o eomic analysis. The
s udies we e e iewed and app o ed by he CEIC Pa c Salu Ma Clini-
cal Resea ch E hics Commi ee (app o al nos. 2015/6026, WALNUTs;
2020/9688, Equal-Li e). W i en in o med consen o pa icipa e in
he o iginal WALNUTs s udy was p o ided by he pa icipan s’ legal
gua dian/nex o kin.
Plasma samples we e p e-p ocessed and analyzed using liquid
ch oma og aphy elec osp ay ioniza ion andem mass spec ome y,
which was pe o med a he Tu ku P o eomics Facili y and suppo ed
by Biocen e Finland. The linea associa ions be ween he SDQ sco e
and he p o ein abundances we e in es iga ed using linea modeling
wi h DeqMS36. To cha ac e ize he biological p ocesses and pa hways
ela ed o he iden i ied p o eins, signi ican ly di e en ly abundan
p o eins (adjus ed P- alue ≤ 0.05) associa ed wi h he SDQ sco e
we e used in u he bioin o ma ic da a analyses. See he Me hods o
mo e de ails.
Resul s
P o ein iden i ica ion
Using mass spec ome y-based p o eomics we success ully iden i ied
1,485 p o eins in he WALNUTs plasma samples (N = 91; mean o 1,228
p o eins pe sample; s anda d e o = 117). The ull lis o he p o eins
is p esen ed in he Supplemen a y In o ma ion. Ou o hese, 77 we e
iden i ied as con aminan s, and we e emo ed. A e ha , 983 p o eins
we e de ec ed in a leas 80% o he samples, and he e o e hese
p o eins we e used o subsequen analysis.
The sex and age a iables we e added o he linea model o
co ec o he possible e ec s. In he analysis, 67 p o eins a had
linea ela ionship wi h he SDQ sco e, ou which 48 we e posi i ely
co ela ed wi h he SDQ sco e, and 19 we e nega i ely co ela ed
(Fig. 1b). The p o eins associa ed wi h he SDQ sco e a e p esen ed
in Table 2.
All he signi ican ly al e ed p o eins we e used o c ea e a hea map
(Fig. 1b) ha shows he p o ein abundances (z-sco es) in ela ion o
he SDQ sco e.
En iched pa hways and biological p ocesses
O he highly abundan p o eins ha we e deple ed om he plasma
samples be o e he mass spec ome y analysis, nine we e ound in
he da a; hese we e conside ed as a possible sou ce o bias and we e
Diagnos ic and S a is ical Manual (DSM) classi ica ions p o ided by he
Wo ld Heal h O ganiza ion (WHO) and he Ame ican Psychia ic Asso-
cia ion, espec i ely14. Clinical in e iews and alida ed ques ionnai es
used o symp om assessmen ( o example, he Beck Dep ession In en-
o y15) ha e a majo ole in men al heal h diagnos ics. The complexi y
o adolescen beha io and he o e lap o symp oma ology o se e al
men al diso de s complica e he p ecise and objec i e diagnosis o
diseases in you h. Fu he mo e, he di icul y in de ining no ma i e o
a ypical expec ed beha io al de elopmen in adolescence16, and lack
o access o p o essional expe ise, con ibu e o inaccu a e judgmen
and p ecise de ini ion o men al heal h condi ions in adolescen s17,18.
The S eng hs and Di icul ies Ques ionnai e (SDQ) is a sc eening
ques ionnai e o emo ional and beha io al p oblems in child en and
young people ha assesses he impac o di icul ies on he child’s li e,
including (1) emo ional symp oms, (2) conduc p oblems, (3) hype -
ac i i y/ina en ion, (4) pee ela ionship p oblems and (5) p osocial
beha io
19–21
. Pas alida ion s udies ha e shown ha he o al SDQ
sco e can be conside ed as a p edic i e ac o o men al heal h diso -
de s, as child en wi h high SDQ sco es ha e an inc eased p obabili y o
clinical diso de s
22,23
. Di e ences in he o al SDQ sco e seem o e lec
he di e ences in p e alence o men al heal h diso de s, al hough
c oss-na ional di e ences exis
22,24
. Thus, de eloping addi ional ools
such as biological measu emen s o assessing men al heal h issues
could imp o e iden i ica ion o adolescen s a high isk o men al
heal h dys unc ion, and enhance mo e p ecise diagnos ics.
Al hough s udying human b ain issue may be he mos e eal-
ing me hod o measu ing al e a ions ela ed o men al diso de s,
i poses se e al se e e limi a ions, including issue access
25
and high
cos in he case o neu oimaging25. By con as , biological luids such
as blood o u ine a e easie o access and a e ou inely used o clini-
cal diagnos ics. Al e a ions in he gene exp ession le els, p o eins
abundance and biological ac i i y can se e as in e nal indica o s
p esen in biological luids (bioma ke s), o pa hogenic p ocesses o
esponses o an ex e nal exposome25,26. The blood connec s he b ain
and pe iphe y, and changes in plasma componen s such as p o eins
can e lec al e a ions in he b ain associa ed wi h men al diso de s
due o he wo-way communica ion be ween he cen al ne ous sys-
em (CNS) and pe iphe al ci cula ion27,28. Pas s udies ha e shown he
plasma p o eomic changes associa ed wi h men al diso de s29–32. Fo
example, signi ican educ ions in glia ma u a ion ac o be a and
b ain-de i ed neu o ophic ac o we e obse ed in pa ien s wi h
schizoph enia (SCZ) when compa ed wi h heal hy olun ee s
29
. Thus,
pe iphe al blood plasma is a sui able biological luid o in es iga ing
molecula al e a ions ha e lec hose associa ed wi h men al heal h
issues, and o p o iding new unde s anding on he bidi ec ional com-
munica ion be ween he b ain and body27–30.
Limi ed knowledge exis s on whe he al e a ions in plasma p o-
eins could se e as ea ly suscep ibili y bioma ke s o p edic he isk
o men al heal h issues, leading o p ope clinical in e en ions be o e
disease onse , e en hough al e a ions in pa hways and molecules
ela ed o ho mone signaling, ene gy me abolism, g ow h ac o s,
in lamma ion, oxida ion/ educ ion and p o ein syn hesis ha e been
commonly associa ed wi h psychia ic diso de s28. Howe e , s udies
by Mongan e al.33 and English e al.34 sugges ha adolescen s a high
isk o psychosis could be iden i ied on he basis o he changes in
he blood p o eome se e al yea s be o e he psycho ic expe iences
mani es . The numbe o s udies ocusing on disco e ing new suscep-
ibili y o p edic i e plasma bioma ke s o men al heal h diseases in
adolescen s is so a limi ed.
This explo a i e s udy aims o iden i y and cha ac e ize al e a-
ions o plasma p o eins in adolescen s a high isk o de eloping
men al heal h issues. We iden i ied 67 plasma p o eins wi h abundances
signi ican ly associa ed wi h he SDQ sco e, o e ing new insigh in o
using p o eins as suscep ibili y bioma ke s o ea ly iden i ica ion o
adolescen s a isk o men al heal h p oblems.
Table 1 | Sample cha ac e is ics
G oup Low Raised
SDQ = 0–14 SDQ = 15–25
Sex Male Female Male Female
SDQ sco es
(mean ± s.d.) 3.50 ± 1.41 3.50 ± 1.47 16.92 ± 2.01 18.00 ± 2.42
Numbe (n (%)) 24 (57.1) 18 (42.9) 27 (55.1) 22 (44.9)
Age
(mean ± s.d.) 13.54 ± 1.07 13.53 ± 0.73 13.93 ± 1.03 14.37 ± 1.13
n, numbe o samples (% o each g oup); s.d., s anda d de ia ion.
Na u e Men al Heal h | Volume 1 | Augus 2023 | 596–605 598
A icle h ps://doi.o g/10.1038/s44220-023-00103-2
excluded om hese analyses, lea ing 58 p o eins signi ican ly associ-
a ed wi h he SDQ sco e.
Those p o eins we e used o a clus e ing analysis o he di e -
en ially abundan p o eins iden i ied in ou s udy using he STRING
da abase
37
. The clus e ing analysis yielded h ee g oups o p o eins, as
shown in Fig. 2a. Clus e 1 con ained up- and down- egula ed p o eins
in ol ed in neu on g ow h, synap ic unc ion, glial cell mig a ion and
choles e ol anspo . The second clus e con ained only up- egula ed
p o eins mos ly in ol ed in he complemen and coagula ion cascades.
Clus e 3 con ained h ee down- egula ed p o eins in ol ed in he
ol ac o y sys em and h ee up- egula ed p o eins in ol ed in p o ein
deg ada ion.
We also pe o med an analysis o en iched pa hways wi h
Reac ome
38
, using all o he iden i ied p o eins as he gene backg ound.
En iched pa hways we e ela ed o immune sys em, coagula ion,
complemen cascade, and pos - ansla ional p o ein modi ica ion
(Fig. 2b). In o al, hi een pa hways we e signi ican ly ( alse disco e y
a e (FDR)-adjus ed P- alue < 0.05) en iched in he pa hway analysis
(Supplemen a y Table 1). The signaling pa hways analysis in ingenui y
pa hway analysis (IPA) e ealed ha canonical pa hways we e associ-
a ed wi h immune esponses, coagula ion, complemen cascade and
signaling, as in he Reac ome analysis (Supplemen a y Table 2).
P edic i e models gene a ion
The ela i ely la ge numbe o samples made i possible o employ
mode n s a egies o de e mine po en ially p edic i e bioma ke s o
he low e sus aised SDQ sco e g oups. A no el QLa ice algo i hm39
was used o c ea e models con aining p edic i e bioma ke s ha bes
sepa a e he wo g oups wi h low and aised SDQ sco es. The Bayesian
in o ma ion c i e ion (BIC) was used o ensu e ha he esul ing models
gene alize well om he aining o es se . We pe o med i e old
c oss- alida ion o unning logis ical eg ession model wi h
QLa ice on di e en pa i ions o he da a keeping he lowes
BIC-sco ing model om each pa i ion. The ecei e ope a o cha ac-
e is ic (ROC) cu es and a ea unde he ROC cu e (AUC) o each
o he models a e p esen ed in Supplemen a y Fig. 2.
Fi e di e se models we e c ea ed using a i e old c oss- alida ion
scheme. These models b ing simila —albei complemen a y—insigh s,
as he whole da ase was spli in o aining and alida ion se s i e
imes, and each ound con ained di e en sub-samples o he da a.
The i e unique models (Table 3) con ained ele en p o eins in o al
(Supplemen a y Table 3). Fou o he i e models con ained p o eins
wi h a p e iously shown connec ion o he CNS. The i s model con-
ained h ee such p o eins: amyloid be a p ecu so -like p o ein 1
(APLP1) (P51693), calcium/calmodulin dependen p o ein kinase II be a
(CAMK2B) (Q13554/Q13555) and Re iculon 4 (RTN4; Q9NQC3), he ROC
pa ame e s o he models a e shown in he Supplemen a y Fig. 2. Only
he i h model con ained no p o eins, p e iously connec ed o b ain
de elopmen . The p o eins p esen in he models can be in es iga ed
u he as po en ial bioma ke s.
Discussion
Plasma p o eomic bioma ke s udies in men al heal h diseases a e a
no el ield. Inc easing e idence shows al e a ions in plasma p o eins
associa ed wi h di e en men al diso de s such as dep ession (MDD),
SCZ, psycho ic diso de s and bipola diso de s
24,25,32
. Mos al e ed pa h-
ways in men al diso de s (such as complemen cascade and signaling
by in e leukins) seem o be common o he abo e-men ioned majo
psychia ic diso de s32. He e we epo plasma p o ein al e a ions
ela ed o immune esponses, blood coagula ion, complemen cascade,
neu onal degene a ion and neu ogenesis in adolescen s a high isk o
men al dys unc ion, which was e alua ed based on he sel - epo ed
SDQ sco e. I should be kep in mind ha assessing he isk o men al
heal h p oblems in adolescen s is associa ed wi h e hical issues, which
should be app op ia ely conside ed.
In his s udy we used he o al SDQ sco e as an indica o o
men al heal h dys unc ion and p edisposi ion o men al heal h
issues in adolescen s. Becke and co-wo ke s ha e shown he p edic-
i e alue o he sel - epo ed SDQ in clinical diagnos ics, especially
combined wi h pa en and/o eache e sions40. Fu he mo e,
he sel - epo ed SDQ was shown o be a eliable and alid me hod
o he assessmen o beha io al p oblems in child en and adoles-
cen s
40
. Goodman e al. ha e shown ha mul i-in o man (pa en s,
eache s, olde child en) SDQs in communi y samples can iden i y
child en and adolescen s wi h a psychia ic diagnosis wi h a speci-
ici y o 94.6% and a sensi i i y o 63.3%; SDQ sco es success ully
iden i ied o e 70% o indi iduals wi h conduc , hype ac i i y,
dep essi e and some anxie y diso de s
19
. The SDQ pe o ms well
as a sc eening ool, bu i is no in ended o be used as a psychia ic
diagnos ic ins umen as such41. I is he e o e conside ed a use ul
and alid ool o sc eening child en and adolescen s a a high isk
o men al diso de s19,41,42.
This s udy e ealed 58 plasma p o ein al e a ions associa ed wi h
he SDQ sco e in adolescen s. The abundances o 39 p o eins we e
enhanced in he aised SDQ sco e g oup, whe eas 19 we e educed. We
iden i ied al e ed p o eins such as clus e in, i onec in, complemen
C2 and coagula ion ac o XI ha ha e also been epo ed o be al e ed
in pas blood p o eomics s udies33,43,44.
0
1
2
–2 –1 0 1
log2FC
–log10(adjus ed P- alue)
Adjus ed P- alue ≥ 0.05
Adjus ed P- alue < 0.05
–4
0
4
0
25
SDQ
b
a
Fig. 1 | Signi ican ly al e ed p o eins iden i ied in mass spec ome y-
based p o eomic analysis. a, Volcano plo o p o eins associa ed wi h he
SDQ sco e; 58 p o eins we e signi ican ly changed, indica ed in blue (adjus ed
P- alue < 0.05). The P- alues we e calcula ed using DeqMS wi h SDQ as a
con inuous a iable, and adjus ed using he Benjamini–Hochbe g me hod.
b, A hea map o p o ein abundances (z-sco es) o p o eins signi ican ly
associa ed wi h he SDQ sco e. The uppe g oup ep esen s nega i ely co ela ed
p o eins (n = 19), and he lowe g oup posi i ely co ela ed p o eins (n = 48).
SDQ sco es a e shown as he g adien a he bo om o he hea map, g een (pink)
indica es indi iduals wi h a low ( aised) SDQ sco e.
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Table 2 | Plasma p o eins wi h abundance changes associa ed wi h he SDQ sco e
P o ein ID Gene names P o ein names E ec size log2FC Adjus ed P- alue
P13796 LCP1 Lymphocy e cy osolic p o ein 1 0.024393435 0.28230175 0.00266891423429772
Q5XPI4 RNF123 Ring inge p o ein 123 0.030214587 0.407569734 0.00266891423429772
P02763 ORM1 O osomucoid 1* 0.041472001 0.582318552 0.00266891423429772
P00450 CP Ce uloplasmin 0.021579105 0.242648946 0.00266891423429772
P01833 PIGR Polyme ic immunoglobulin ecep o −0.051686633 −0.707557707 0.00406569633896016
P35542 SAA4 Se um amyloid A4, cons i u i e 0.031761716 0.354177466 0.00444900848111389
P08697 SERPINF2 Se pin amily F membe 2 0.022213284 0.281759883 0.00444900848111389
P05155 SERPING1 Se pin amily G membe 1 0.019806854 0.24302875 0.00444900848111389
P02675 FGB Fib inogen β chain* 0.026409456 0.431154772 0.00444900848111389
P02679 FGG Fib inogen gamma chain* 0.025721779 0.423203306 0.00444900848111389
P01008 SERPINC1 Se pin amily C membe 1 0.018137241 0.199816818 0.00444900848111389
P25786 PSMA1 P o easome 20S subuni α1 −0.095693078 −1.302777999 0.00444900848111389
Q9UK55 SERPINA10 Se pin amily A membe 10 0.023632886 0.301366666 0.00444900848111389
P05154 SERPINA5 Se pin amily A membe 5 0.027008252 0.324609321 0.00688379748291161
P04180 LCAT Leci hin-choles e ol acyl ans e ase 0.020279571 0.221513014 0.00688379748291161
P02787 TF T ans e in* 0.024014254 0.335644745 0.00840623855309166
P02671 FGA Fib inogen α chain* 0.021692839 0.350696404 0.00972440642891591
O95445 APOM Apolipop o ein M 0.022035032 0.246726784 0.01311860384508440
P08571 CD14 CD14 molecule 0.018772876 0.212669508 0.01311860384508440
P43251 BTD Bio inidase 0.022100724 0.267028414 0.01311860384508440
Q15485 FCN2 Ficolin 2 0.037120727 0.44342002 0.01314547997572250
P08603 CFH Complemen ac o H 0.018284096 0.191087977 0.01561410991073270
P03951 F11 Coagula ion ac o XI 0.024018263 0.287092562 0.01608758697162620
P06681 C2 Complemen C2 0.01678681 0.18562205 0.01826102103619690
P51693 APLP1 Amyloid β p ecu so -like p o ein 1 −0.048592355 −0.725496494 0.01826102103619690
P19652 ORM2 O osomucoid 2* 0.027306639 0.329227308 0.01885805215970600
P01019 AGT Angio ensinogen 0.018588708 0.250511329 0.02185375824202300
P00734 F2 Coagula ion ac o II, h ombin 0.0178514 0.16799325 0.02216801017152710
P26992 CNTFR Cilia y neu o ophic ac o ecep o −0.030575485 −0.400276076 0.02216801017152710
A6NE52 KIAA1875 WD epea domain 97 −0.069097379 −0.878591149 0.02216801017152710
P02768 ALB Albumin* 0.018244928 0.269527276 0.02216801017152710
P11171 EPB41 E y h ocy e memb ane p o ein band 4.1 −0.048815886 −0.659499259 0.02306083695884880
P01042 KNG1 Kininogen 1 0.016314731 0.160500404 0.02306083695884880
P29622 SERPINA4 Se pin amily A membe 4 0.018413978 0.240780219 0.02306083695884880
Q13554;Q13555 CAMK2B, CAMK2G Calcium/calmodulin-dependen p o ein
kinase ype II subuni β 0.051385424 0.751103187 0.02306083695884880
P06276 BCHE Bu y ylcholines e ase 0.020235784 0.247353801 0.02402185464772990
P62873 P62873 G p o ein subuni β 1 −0.054612995 −0.688126035 0.02402185464772990
P00751 CFB Complemen ac o B 0.019090843 0.210533723 0.02402185464772990
P00742 F10 Coagula ion ac o X 0.017052085 0.1825767 0.02402185464772990
Q58FF3 HSP90B2P Pu a i e endoplasmin-like p o ein −0.09637649 −1.287683106 0.02668279775737110
P04217 A1BG α1-B glycop o ein* 0.019130571 0.194414326 0.02898562281342810
P05156 CFI Complemen ac o I 0.018729496 0.217624798 0.02898562281342810
Q9UGM5 FETUB Fe uin B 0.023263795 0.240217283 0.02898562281342810
P02652 APOA2 Apolipop o ein A2 0.020753485 0.191480134 0.02898562281342810
P11166 SLC2A1 Solu e ca ie amily 2 membe 1 −0.046363787 −0.577758688 0.02898562281342810
P00736 C1R Complemen C1 subcomponen 0.015778503 0.134858762 0.02898562281342810
P08185 SERPINA6 Se pin amily A membe 6 0.017945936 0.219458651 0.02898562281342810
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Blood coagula ion and immune esponses, including he com-
plemen cascade, we e he mos en iched pa hways al e ed among
p o eins signi ican ly associa ed wi h he SDQ sco e. Ou clus e ing
analysis e ealed up- egula ion o complemen and blood coagula ion
cascades. Pas s udies ha e also shown associa ions be ween ea ly
changes in complemen and coagula ion cascades and inc eased isk o
psycho ic diso de s in adolescen s33,34,45. Changes in immune esponses
and blood coagula ion ha e also been epo ed in SCZ, MDD and
P o ein ID Gene names P o ein names E ec size log2FC Adjus ed P- alue
P07477;P07478;Q8NHM4 PRSS1, PRSS3P2,
PRSS3P2 Se ine p o ease 1 −0.100752815 −1.377624809 0.03115142391195680
Q6IF82 OR4A47 Ol ac o y ecep o amily 4 sub amily A
membe 47 −0.093345139 −0.983920911 0.03159089421977260
P04004 VTN Vi onec in 0.015083377 0.135248758 0.03608129999454210
Q8N3T6 TMEM132C T ansmemb ane p o ein 132C −0.068568409 −0.868793155 0.03921664831857450
P02647 APOA1 Apolipop o ein A1* 0.017379495 0.156907997 0.03921664831857450
P22792 CPN2 Ca boxypep idase N subuni 2 0.022272689 0.254526745 0.04145715624086360
O95998 IL18BP In e leukin 18 binding p o ein −0.044789476 −0.588596594 0.04145715624086360
P13611 VCAN Ve sican −0.039810664 −0.586620961 0.04145715624086360
O95274 LYPD3 LY6/PLAUR domain-con aining 3 −0.107072994 −1.564572013 0.04145715624086360
Q9NZP8 C1RL Complemen C1 subcomponen -like 0.016939083 0.17007371 0.04145715624086360
P12955 PEPD Pep idase D 0.020336146 0.335635822 0.04145715624086360
P01023 A2M α2-mac oglobulin 0.01942573 0.193716524 0.04145715624086360
P55287 CDH11 Cadhe in 11 −0.149776496 −2.237629717 0.04145715624086360
Q96RD9 FCRL5 Fc ecep o -like 5 −0.034069414 −0.527369507 0.04145715624086360
P21926 CD9 CD9 molecule −0.056132135 −0.78939602 0.04145715624086360
P02746 C1QB Complemen C1q subcomponen
subuni B 0.013951103 0.134418819 0.04145715624086360
Q9NQC3 RTN4 Re iculon 4 −0.122514211 −1.617124146 0.04162111693323930
P10909 CLU Clus e in 0.014330575 0.146386665 0.04293539920884360
P11279 LAMP1 Lysosome-associa ed memb ane
glycop o ein 1 0.013986417 0.153178004 0.04321300399755200
Q96PD5 PGLYRP2 N-ace ylmu amoyl-l-alanine amidase 0.014620832 0.141416175 0.04742114492279560
P o eins a e p esen ed wi h hei UniP o accession numbe and co esponding p o ein name. The as e isks indica e he highly abundan p o eins ha we e deple ed in he p e-p ocessing
s age. The e ec size indica es he log2- old-change in exp ession ha esul s om a uni change in SDQ. The P- alues we e calcula ed using DeqMS SDQ sco e as con inuous a iable and
adjus ed using he Benjamini–Hochbe g me hod. log2FC, log2- old-change ( a io o means).
G alpha (q) signalling e en s
MyD88:MAL(TIRAP) cascade ini ia ed on plasma me...
Pos - ansla ional p o ein phospho yla ion
Regula ion o insulin-like g ow h ac o (IGF) ...
Complemen cascade
Regula ion o complemen cascade
Hemos asis
Common pa hway o ib in clo o ma ion
Pla ele ac i a ion, signaling and agg ega ion
Pla ele deg anula ion
In insic pa hway o ib in clo o ma ion
Fo ma ion o ib in clo (clo ing cascade)
0 1 2 3
Clo ing and hemos asis
Complemen cascade
Regula ion and signalling
log10 adj. P- alue
1
2
3
a b
FCRL5
PIGR
VTN
A2M
CLU
LCAT
A1BG
CP
APOA1
APOM
CD9
BTD
EPB41
LYPD3
LAMP1
SLC2A1
LCP1
PGLYRP2
APLP1
GNB1
OR4A47
PSMA1
PEPD
CPN2
F2
CD14
CNTFR
TMEM132C
RNF123
ALB VCAN CDH11
RTN4 C1QB
CFI AGT
SERPING1 APOA2
FGA
SAA4
SERPINA10
F11
SERPINA5
SERPINA4
SERPINA6
KNG1
FGB
ORM2
ORM1
SERPINC1
SERPINF2
C1RL
C1R
TF
FETUB
FGG
CFB
F10
CFH
FCN2
C2
BCHE
Fig. 2 | En iched biological p ocesses and pa hways. a, The esul s o he
STRINGdb clus e ing analysis; he p o eins posi i ely co ela ed wi h he
SDQ sco e a e highligh ed g een, whe eas nega i ely co ela ed p o eins a e
highligh ed ed. The numbe and colo s o he lines ep esen he e idence
o he p o ein connec ion acco ding o he de aul STRING da abase scheme.
b, Signi ican ly en iched Reac ome pa hways. The colo s indica e unc ional
g ouping. The en ichmen was pe o med using he en ichPa hway unc ion o
he Reac omePA package, which uses he hype geome ic model. The P- alue was
adjus ed using he Benjamini–Hochbe g me hod.
Table 2 (con inued) | Plasma p o eins wi h abundance changes associa ed wi h he SDQ sco e
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bipola diso de pa ien s in se e al blood p o eomic s udies43,44,46. We
ound posi i e co ela ions wi h he SDQ sco e in coagula ion ac o XI,
coagula ion ac o X and coagula ion ac o II ( h ombin)—all o which
a e in ol ed in blood coagula ion. Inc eased le els o p o h ombin
and se e al coagula ion ac o s (F5, F9, F12, F13A1) we e also ound by
English e al. in adolescen s who la e de eloped a psycho ic diso de
34
.
Se e al complemen componen s and ac o s such as C6, C1S, and
CFI we e al e ed (mos ly inc eased) in high- isk psycho ic diso de
adolescen s
33,34
and i s -episode SCZ-pa ien s
47
. In ou s udy, com-
plemen p o eins such as complemen C1q and C1 subcomponen s,
complemen ac o I, complemen ac o H and complemen C2 we e
signi ican ly and mainly posi i ely associa ed wi h he SDQ sco e. Jiang
e al.
48
sugges ed ha complemen ac i a ion oge he wi h me abolic
up- egula ion can inc ease oxida i e s ess, which can induce p o ein
damage and cell apop osis, and hus con ibu e o he de elopmen
o SCZ. Al oge he , ou esul s a e in line wi h he cu en unde s and-
ing o he ole o al e ed immune esponses and blood coagula ion
in pa hophysiology o men al diso de s. Fu he mo e, ou indings
suppo he inc easing e idence on ea ly changes in coagula ion and
complemen cascades in p edisposi ion o—and de elopmen o —men-
al heal h issues in adolescen s.
A symbolic- eg ession-based algo i hm, QLa ice, was used o
gain an insigh in o he po en ial o p o eins o p edic he SDQ s a us.
The o med models comp ised ele en p o eins, ou o which ha e a
p e iously epo ed connec ion o he CNS, neu ogenesis o men al
heal h. O he ele en p o eins, eigh we e epo ed o belong o he
i s clus e acco ding o STRING da abase analysis. O he p o eins
no p e iously connec ed o he CNS, he LYPD3 p o ein was epo ed
o be an amyloid p ecu so p o ein in e ac o 49, which can explain i s
coincidence wi h APLP1, CAMK2B and CD9 in ou p edic ed models.
APLP1 is a p o ein esiding p edominan ly in b ain issue, and i has
been shown o be in ol ed in b ain de elopmen
50
and synap ogenesis
du ing pos -na al de elopmen in mice51. We iden i ied a signi ican
nega i e associa ion be ween APLP1 and he SDQ sco e. Pandol o and
colleagues ha e sugges ed ha amyloid could be a ma ke o cogni-
i e impai men and al e ed neu ode elopmen in men al diseases.
Dec eased β-amyloid p o eins in CSF ha e been epo ed in pa ien s
wi h SCZ and MDD, and al e ed amyloid p ecu so p o ein me abolism
in pa ien s wi h bipola diso de s
52
. Howe e , al hough he ole o
APLP1 in he pa hogenesis o men al diso de s is s ill unknown, his
p o ein—on he basis o ou da a—wa an s u he in es iga ion in
he con ex o adolescen men al heal h.
Two o he p o eins—RTN4
53,54
and CAMK2B—ha e been epo ed
o be connec ed o neu onal de elopmen and neu oplas ici y
55,56
.
CAMK2B was posi i ely associa ed wi h he SDQ sco e. I is a p o ein
connec ed o dend i ic spine and synapse o ma ion, neu onal plas ic
-
i y and egula ion o sa coplasmic e iculum Ca2+ anspo in skele al
muscle57. The be a subuni was epo ed o be b ain speci ic55, ye li le
is known o i s in ol emen in men al diso de s. Ano he p o ein
p esen in he op model was RTN4, which was nega i ely associa ed
wi h he SDQ sco e, and has been p e iously shown o be associa ed
wi h SCZ53,58. RTN4 is a memb ane shaping p o ein in he endoplasmic
e iculum in ol ed in he main enance o he endoplasmic e iculum
memb ane ubula in eg i y. Impai men in he RTN4 p ocess has been
connec ed o neu odegene a ion. The RTN4A-sub ype, also known as
Nogo-A, is localized in he CNS and has a ole in neu onal g ow h and
ma u a ion du ing ne ous sys em de elopmen
59
. Fu he mo e, RTN4
was shown o be connec ed wi h social beha io and spa ial cogni ion
in a s udy using mice wi h a missense mu a ion in he RTN4 ecep o 59,60.
The in ol emen o RTN4 in adolescen men al heal h emains a opic
wo hy o de ailed in es iga ion. The ou h p o ein wi h epo ed
connec ion o CNS was Cadhe in 11 (P55287), which has been shown
o be en iched in se e al b ain a eas du ing dend i e o ma ion and
synap ogenesis
61–63
. Gi en ha he p o eins epo ed as p obable bio-
ma ke s belonged o he same clus e acco ding o STRING da abase,
and ha ou o he p o eins we e shown o be connec ed o he CNS
and neu onal de elopmen , i is concei able ha all o he p o eins
om he clus e a e connec ed o one p ocess. Fu he in es iga ion
o his ne wo k migh shed new ligh on he nuances o b ain de elop-
men and men al heal h in adolescen s.
Gi ls in he aised SDQ sco e (>15) g oup epo ed sligh ly ea lie
pube y changes compa ed wi h he lowe SDQ sco e (<14) g oup,
whe eas in boys, he sel - epo ed pube y changes be ween he lowe
and he aised SDQ sco e g oups seemed o be he opposi e (Supple-
men a y Table 4); howe e , di e ences in pube y changes we e mino
and sex was added as con ounding ac o in ou linea models. We also
examined o he possible con ounding ac o s a ailable, including he
educa ion le el o each pa en , he le els o media consump ion, le els
o social media engagemen , d ug and alcohol use, and physical ac i -
i y. The esul s showed no signi ican di e ences among he g oups
(P- alue > 0.05). Fu he mo e, we used he in o ma ion on he school
o he subjec s a ended as a andom a iable in linea modeling. No
di e ences in he numbe o signi ican p o eins we e ound. We hus
concluded ha hese ac o s we e no likely o con ound he in es iga-
ion o he connec ion o SDQ o he p o ein abundance le els.
As he main limi a ion in his s udy, he sample numbe is low in
ela ion o he iden i ied p o eins. Linea modeling was pe o med
along wi h some g oup-based compa isons o s eng hen he s a is ical
powe o he analysis, and we managed o de ec s a is ically signi ican
al e a ions wi h hese sample numbe s. Simila N-numbe s ha e also
been used in pas s udies on se um and plasma p o ein bioma ke s in
SCZ, MDD and bipola diso de pa ien s
45
. Fu he mo e, o e nigh
as ing samples a e p e e ed o p o eomics analysis as ood in ake
can in luence he p o ein composi ion and concen a ions in blood
64
.
The plasma samples used in he cu en s udy we e non- as ing samples
due o he p ac ical and e hical issues ela ed o he implemen a ion o
he WALNUTs s udy as blood samples we e d awn om he adolescen s
a school in he a e noon.
Conclusion
In his explo a i e s udy, we iden i ied p o ein-based suscep ibili y
bioma ke candida es associa ed wi h he sel - epo ed SDQ sco e in
Table 3 | The models e u ned by he QLa ice wi h lowes BIC sco e
Model numbe Model unc ional o m genes Model unc ional o m accessions BIC T aining se AUC
1APLP1 + CAMK2B x RTN4 P51693 + Q13554 x Q9NQC3 49.33 0.95
2PIGR + SERPINA4 + CDH11 P01833 + P29622 + P55287 54.25 0.95
3LYPD3 + SERPING1+CAMK2B O95274 + P05155 + Q13554;Q13555 49.88 0.95
4LYPD3 + BTD + APLP1 O95274 + P43251 + P51693 53.85 0.93
5LYPD3 + LCP1 + CD9 O95274 + P13796 + P21926 48.46 0.94
P o eins in he models a e po en ially p edic i e bioma ke s o he aised SDQ sco e in adolescen s. The model o each p o ein con ains gene names and p o ein accession numbe s. T aining
se AUC pe o mances and he BIC sco es o each model a e compa able. The i s model con ained non-linea in e ac ing p o eins CAMK2B and RTN4, whe eas all o he o he in e ac ions
we e linea . The p o ein names and hei ela ion o he accession codes a e p esen ed in Table 1.
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A icle h ps://doi.o g/10.1038/s44220-023-00103-2
adolescen s e lec ing a isk o de eloping men al heal h dys unc ion.
Signi ican al e a ions we e ound in p o eins in ol ed in he immune
esponse, blood coagula ion and hemos asis, neu onal degene a ion
and neu ogenesis. Fu he s udies a e needed o con i m and alida e
hese bioma ke candida es in la ge coho s, as well as ollow-up da a
and s udies o e alua e whe he hese bioma ke s a e associa ed wi h
he isk o ansi ion o he clinical s a e and men al diso de s.
Me hods
Pa icipan ec ui men and sample collec ion
The s udies we e e iewed and app o ed by CEIC Pa c Salu Ma Clini-
cal Resea ch E hics Commi ee (app o al nos. 2015/6026 WALNUTs
and 2020/9688–Equal-li e). W i en in o med consen o pa icipa e
in he o iginal WALNUTs s udy was p o ided by he pa icipan s’ legal
gua dian/nex o kin. No addi ional consen was needed o his s udy,
all o he pa icipan s we e o e ed ee icke s o he science museum
o Ba celona. The speci ics o he WALNUTs coho o ma ion we e
desc ibed in p e ious publica ions21,65. The cu en manusc ip used
a subse o 372 baseline blood samples be o e any die a y in e en ion
o iginally desc ibed in a p e ious wo k
65
. Fo his s udy, a sub-g oup o
91 samples was used o pe o m he p o eomics analysis. These samples
we e selec ed on he basis o he SDQ sco es: 42 wi h he lowes SDQ
sco e (SDQ = 0–14) and 49 wi h he highes SDQ sco e (SDQ = 15–25).
Samples we e d awn by a nu se using K2EDTA plus ubes, es ed o 1 h
and hen cen i uged a 2,500 × g o 20 min a 20 °C, e ige a ed a
4 °C, and ozen o −80 °C wi hin 4 h a e ex ac ion
65
, s o ed a –80 °C,
and we e no hawed un il he p o ein deple ion was pe o med be o e
he p o eomics analysis.
High-abundance p o ein deple ion
Albumin and IgG ep esen mo e han 70% o o al p o ein le els in
human plasma samples. The deple ion o high-abundan p o eins is
he e o e essen ial o he iden i ica ion and analysis o low-abundan
p o eins. A comme cial ki (High Selec Top14 Abundan P o ein Deple-
ion Mini Spin Columns, ca alogue no. A36370, The moScien i ic) was
used o deple e he 14 mos abundan p o eins om plasma be o e
he p o eomic analyses. The deple ed p o eins we e human se um
albumin, albumin, IgG, IgA, IgM, IgD, IgE, kappa and lambda ligh
chains, α1-acidglycop o ein, α1-an i ypsin, α2-mac oglobulin, apoli-
pop o ein A1, ib inogen, hap oglobin and ans e in, acco ding o
manu ac u e ’s manual. B ie ly, 10 µl o o al plasma was added o he
mini spin columns and incuba ed o 10 min while o a ing, ollowed
by cen i uga ion o he columns (1,000 × g) o 2 min. The il a e was
collec ed in 2 ml plas ic ubes and s o ed a −20 °C un il p epa a ion o
mass spec ome y p o eomic analyses, which we e pe o med a he
Tu ku P o eomics Facili y suppo ed by Biocen e Finland.
P o ein p ecipi a ion and diges ion
Samples we e ace one p ecipi a ed and subjec ed o in-solu ion diges-
ion. Sho ly, ou olumes o ice-cold ace one we e used o p ecipi a e
p o eins. P ecipi a ed p o eins we e esuspended o 8 M U ea, 50 mM
T is-HCl o p o ein dena u a ion, educed wi h 5 mM di hio h ei ol
and alkyla ed wi h 13 mM iodoace amide. P o eins we e diges ed o
pep ides wi h ypsin (P omega) (enzyme:p o ein a io 1:30) a 37 °C
o e nigh . A e diges ion he pep ides we e desal ed wi h a Sep-Pak
C18 96-well pla e (Wa e s), e apo a ed and s o ed a −20 °C.
Mass spec ome y analysis
Diges ed pep ide samples we e dissol ed in 0.1% o mic acid and pep-
ide concen a ions we e de e mined wi h a NanoD op de ice. Samples
we e spiked wi h iRT pep ides (Biognosys) o e en ion ime calib a-
ion. Equal amoun s o samples we e analyzed on a nano low HPLC sys-
em (Easy-nLC1200, The mo Fishe Scien i ic) coupled o he Q Exac i e
HF O bi ap mass spec ome e (The mo Fishe Scien i ic) equipped
wi h a nano-elec osp ay ioniza ion sou ce. Pep ides we e i s loaded
on o a apping column and subsequen ly sepa a ed in-line on a 15 cm
C18 column (75 µm × 15 cm, Rep oSil-Pu 3 µm 120 Å C18-AQ, D . Maisch
HPLC GmbH). The mobile phase consis ed o wa e wi h 0.1% o mic
acid (sol en A) o ace oni ile/wa e (80:20 ( / )) wi h 0.1% o mic acid
(sol en B). A 100 min g adien was used o elu e pep ides (50 min om
5% o 21% sol en B ollowed by 40 min om 21% o 36 min sol en B).
Mass spec ome y da a we e acqui ed au oma ically by using The mo
Xcalibu .4.1 so wa e (ca alog no. OPTON-30965; The mo Scien i ic).
In a da a-independen acquisi ion (DIA) me hod, a du y cycle con ained
one ull scan (400–1,000 m/z) and 40 DIA MS/MS scans co e ing he
mass ange 400–1,000 wi h a iable wid h isola ion windows.
P o ein iden i ica ion and quan i ica ion analysis
Da a analysis consis ed o p o ein iden i ica ions and label- ee
quan i ica ions o p o ein abundances. The da a we e analyzed using
he Spec onau so wa e (Biognosys; .17.1.221229). The di ec DIA
app oach was used o iden i y p o eins and label- ee quan i ica ions
we e pe o med wi h he MaxLFQ algo i hm in Spec onau . The main
da a analysis pa ame e s in Spec onau we e: (1) enzyme (T ypsin/P);
(2) up o wo missed clea ages; (3) ixed modi ica ion (ca bamidome-
hyl (cys eine)); (4) a iable modi ica ions (ace yl (p o ein N- e minus)
and oxida ion (me hionine)); (5) he p ecu so FDR cu o (0.01); (6) he
p o ein FDR cu o (0.01); (7) he quan i ica ion MS le el (MS2); (8) he
quan i ica ion ype (a ea unde he cu e wi hin in eg a ion bounda-
ies o each a ge ed ion); (9) he p o ein da abase (Swiss-P o 2022_05
Homo Sapiens66 and Uni e sal P o ein Con aminan da abase67); and
(10) no maliza ion (global median no maliza ion). All o he pep ides
we e used o quan i ica ion.
S a is ical analysis
Da a p e-p ocessing and s a is ical analyses we e pe o med using
R ( .4.2.1). P incipal componen analysis was pe o med o assess
he gene al quali y o he da ase (Supplemen a y Fig. 1). Iden i ied
p o eins wi h mo e han 20% missing alues we e excluded om he
analysis. Sample no maliza ion was pe o med using he medianCen-
e ing me hod om he p oBa ch me hod
68
. Missing alues emaining
in he da ase we e inpu using he sample minimum me hod69. We
ha e compa ed he low SDQ and aised SDQ g oups o he ollowing
a iables: he educa ion le el o each pa en , he le els o media con-
sump ion, le els o social media engagemen , d ug and alcohol use,
and physical ac i i y. We es ed whe he he e a e di e ences o he
con ounding ac o s be ween he g oups using he one-way ANOVA
es . A e co ec ing o mul iple compa isons, no socio-economic,
sociodemog aphic o o he ac o s showed any signi ican di e ence
be ween he g oups, meaning ha he analyzed g oups do no ha e
signi ican di e ences in be ween he mean alues o hose ac o s.
Bioin o ma ic da a analysis
The DeqMS ( .1.16.0) package was used o he di e en ial abundance
analysis36, wi h SDQ sco e used as a con inuous a iable. The sex and
age o he adolescen s we e included in o he linea model o ensu e
ha he p o eins epo ed a e associa ed wi h SDQ and we e no in lu
-
enced by con ounding ac o s. The di e ences in p o ein abundances
we e exp essed as log2- old-change ( he a io o he means o aised
(nume a o ) and low (denomina o ) SDQ g oups). The P- alues we e
adjus ed using Benjamini–Hochbe g p ocedu e.
Plasma p o eomic da ase s om adolescen s ep esen a e y low
numbe o all he p o eomic da ase s70, so o be e in es iga e he unc-
ional en ichmen he ull lis o all p o eins ound in his s udy was used
as he backg ound gene lis in he en ichmen analyses. To cha ac e ize
he en iched pa hways ela ed o he iden i ied p o eins, signi ican ly
di e en ly abundan p o eins (P ≤ 0.05) associa ed wi h he SDQ sco e
we e used in u he bioin o ma ic da a analyses. P o eins deple ed
be o e mass spec ome y analysis ha showed signi ican di e -
ences be ween g oups we e conside ed a possible sou ce o bias and
Na u e Men al Heal h | Volume 1 | Augus 2023 | 596–605 603
A icle h ps://doi.o g/10.1038/s44220-023-00103-2
hus we e excluded. The Reac ome pa hways we e in es iga ed using
he Reac omePA ( .1.9.4) R package
38
. The en ichmen was pe o med
using he en ichPa hway unc ion o he Reac omePA package, which
uses he hype geome ic model. The P- alue was adjus ed using he
Benjamini–Hochbe g me hod. We used IPA (Ingenui y Sys ems) o
he u he en ichmen analyses In IPA co e analysis, de aul so wa e
pa ame e s we e used ( e e ence se : ingenui y knowledge base—genes
only). The z-sco e alues we e used o iden i y canonical pa hways ha
we e expec ed o be changed by hei ac i i y. The STRINGdb package
was used o ge he p o ein–p o ein in e ac ion in o ma ion o he sig-
ni ican ly di e en ially abundan p o eins om he STRING da abase
( .11.5)37. The as g eedy clus e ing unc ion was used o ex ac gene
clus e s wi h s ong associa ions. A no el symbolic- eg ession-based
algo i hm, QLa ice, which is pa o he Feyn package ( .3.0.3), was
used o gene a e models combining p o eins wi h he bes p edic i e
powe o he SDQ sco e based on p o ein bioma ke s39. The algo i hm
was used o ind he models combining p o eins wi h he bes p edic-
i e powe . Possible bioma ke s we e sea ched among he 58 p o eins
signi ican ly associa ed wi h he SDQ sco e, wi h i e ounds o c oss-
alida ion. The esul ing models we e iden i ied as he op models in
each o he c oss- alida ion. Resul isualiza ions we e pe o med
using ggplo 2 ( .3.4.0)71 and ComplexHea map72 ( .2.14.0) packages.
Repo ing summa y
Fu he in o ma ion on esea ch design is a ailable in he Na u e Po -
olio Repo ing Summa y linked o his a icle.
Da a a ailabili y
The da a analyzed in his s udy a e subjec o he ollowing licenses/
es ic ions: he WALNUTs da a a no publicly a ailable due o he
es ic ions o in o med consen . The da a con ain pe sonal in o ma-
ion on child en and, acco ding o he e hical app o al, hey should be
kep con iden ial. Da a a e a ailable om he co esponding au ho on
easonable eques o esea che s who mee he c i e ia o access o
con iden ial da a. A da a-use/ ans e ag eemen is needed o ensu e
he p o ec ion o p i acy and compliance wi h na ional da a p o ec ion
legisla ion, he con en and speci ic clauses o which will depend on
he na u e o he eques ed da a.
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