Ci a ion: Almodó a -Payá, C.;
Gua diola-Ripoll, M.; Gi al -López,
M.; Gallego, C.; Salgado-Pineda, P.;
Mi e , S.; Sal ado , R.; Muñoz, M.J.;
Láza o, L.; Gue e o-Ped aza, A.;
e al. NRN1 Gene as a Po en ial
Ma ke o Ea ly-Onse Schizoph enia:
E idence om Gene ic and
Neu oimaging App oaches. In . J.
Mol. Sci. 2022,23, 7456. h ps://
doi.o g/10.3390/ijms23137456
Academic Edi o : Ma co Fio e
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In e na ional Jou nal o
Molecula Sciences
A icle
NRN1 Gene as a Po en ial Ma ke o Ea ly-Onse Schizoph enia:
E idence om Gene ic and Neu oimaging App oaches
Ca men Almodó a -Payá1,2,†, Ma ia Gua diola-Ripoll 1,2,† , Ma ia Gi al -López 3,4, Ca me Gallego 5,
Pila Salgado-Pineda 1,2, Sal ado Mi e 2,6,7 , Raymond Sal ado 1,2, Ma ía J. Muñoz 8, Luisa Láza o 2,9,10,11 ,
Amalia Gue e o-Ped aza 1,8 , Ma a Pa ellada 2,12,13,14, Ma ía I. Ca ión15, Manuel J. Cues a 16,17,
Te esa Ma is any 18, Sal ado Sa ó1,2 , Lou des Fañanás2,19,20, Luis F. Callado 2,21,22 , Bá ba a A ias 2,19,20 ,
Edi h Poma ol-Clo e 1,2,* and Ma Fa jó-Vilas 1,2,19,*
1FIDMAG Ge manes Hospi alà ies Resea ch Founda ion, 08830 San Boi de Llob ega , Ba celona, Spain;
[email p o ec ed] (C.A.-P.); [email p o ec ed] (M.G.-R.); [email p o ec ed] (P.S.-P.);
[email p o ec ed] (R.S.); ague e [email p o ec ed] (A.G.-P.); [email p o ec ed] (S.S.)
2Ins i u o de Salud Ca los III, Biomedical Resea ch Ne wo k in Men al Heal h (CIBERSAM),
28029 Mad id, Mad id, Spain; [email p o ec ed] (S.M.); llaza [email p o ec ed] (L.L.); pa [email p o ec ed] (M.P.);
[email p o ec ed] (L.F.); [email p o ec ed] (L.F.C.); [email p o ec ed] (B.A.)
3Depa amen de Psiquia ia, Hospi al Uni e si a i Ge mans T ias i Pujol (HUGTP),
08916 Badalona, Ba celona, Spain; [email p o ec ed]
4Depa amen de Psiquia ia i Medicina Legal, Uni e si a Au ònoma de Ba celona (UAB),
08193 Bella e a, Ba celona, Spain
5Depa men o Cell Biology, Molecula Biology Ins i u e o Ba celona (IBMB-CSIC),
08028 Ba celona, Ba celona, Spain; [email p o ec ed]
6Cen e de Salu Men al d’Adul s de Lleida, Se ei de Psiquia ia, Salu Men al i Addiccions,
Hospi al Uni e si a i San a Ma ia de Lleida, 25198 Lleida, Lleida, Spain
7Ins i u de Rece ca Biomèdica (IRB), 25198 Lleida, Lleida, Spain
8Complex Assis encial en Salu Men al Beni o Menni, 08830 San Boi de Llob ega , Ba celona, Spain;
[email p o ec ed]
9Depa men o Child and Adolescen Psychia y and Psychology, Ins i u e o Neu osciences,
Hospi al Clinic de Ba celona, 08036 Ba celona, Ba celona, Spain
10 Depa amen de Medicina, Uni e si a de Ba celona (UB), 08036 Ba celona, Ba celona, Spain
11 Ins i u d’In es igacions Biomèdiques Augus Pi i Sunye (IDIBAPS), 08036 Ba celona, Ba celona, Spain
12 Se icio de Psiquia ía del Niño y del Adolescen e, Hospi al Gene al Uni e si a io G ego io Ma añón,
28007 Mad id, Mad id, Spain
13 Ins i u o de In es igación Sani a ia del Hospi al G ego io Ma añón (IiSGM), 28007 Mad id, Mad id, Spain
14
Depa amen o de Psiquia ía, Facul ad de Medicina, Uni e sidad Complu ense, 28040 Mad id, Mad id, Spain
15 Hospi al San Ra ael, 08035 Ba celona, Ba celona, Spain; [email p o ec ed]
16 Se icio de Psiquia ía, Hospi al Uni e si a io de Na a a, 31008 Pamplona, Na a a, Spain;
[email p o ec ed]
17 Ins i u o de In es igación Sani a ia de Na a a (IdiSNA), 31008 Pamplona, Na a a, Spain
18 Depa amen de Diagnòs ic pe la Ima ge, Hospi al San Joan de Déu Fundacióde Rece ca,
08950 Esplugues de Llob ega , Ba celona, Spain; ma is any@sjdhospi alba celona.o g
19 Depa amen de Biologia E olu i a, Ecología i Ciències Ambien als, Uni e si a de Ba celona (UB),
08028 Ba celona, Ba celona, Spain
20 Ins i u de Biomedicina de la Uni e si a de Ba celona (IBUB), 08028 Ba celona, Ba celona, Spain
21 Depa men o Pha macology, Uni e si y o he Basque Coun y, UPV/EHU, 48940 Leioa, Bizkaia, Spain
22 Bioc uces Bizkaia Heal h Resea ch Ins i u e, 48903 Ba akaldo, Bizkaia, Spain
*Co espondence: [email p o ec ed] (E.P.-C.); [email p o ec ed] (M.F.-V.)
† These au ho s con ibu ed equally o his wo k.
Abs ac :
Included in he neu o ophins amily, he Neu i in 1 gene (NRN1) has eme ged as an
a ac i e candida e gene o schizoph enia (SZ) since i has been associa ed wi h he isk o he
diso de and gene al cogni i e pe o mance. In his wo k, we aimed o u he in es iga e he
associa ion o NRN1 wi h SZ by explo ing i s ole on age a onse and i s b ain ac i i y co ela es.
Fi s , we de eloped wo gene ic associa ion analyses using a amily-based sample (80 ea ly-onse (EO)
ios (o sp ing onse
≤
18 yea s) and 71 adul -onse (AO) ios) and an independen case–con ol
sample (120 heal hy subjec s (HS), 87 EO and 138 AO pa ien s). Second, we explo ed he e ec
o NRN1 on b ain ac i i y du ing a wo king memo y ask (N-back ask; 39 HS, 39 EO and 39 AO;
In . J. Mol. Sci. 2022,23, 7456. h ps://doi.o g/10.3390/ijms23137456 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2022,23, 7456 2 o 18
ma ched by age, sex and es ima ed IQ). Di e en haplo ypes encompassing he same h ee Single
Nucleo ide Polymo phisms(SNPs, s3763180– s10484320– s4960155) we e associa ed wi h EO in he
wo samples (GCT, TCC and GTT). Besides, he GTT haplo ype was associa ed wi h wo se N-back
ask pe o mance in EO and was linked o an ine icien do sola e al p e on al co ex ac i i y in
subjec s wi h EO compa ed o HS. Ou esul s show con e gen e idence on he NRN1 associa ion
wi h EO bo h om gene ic and neu oimaging app oaches, highligh ing he ole o neu o ophins in
he pa hophysiology o SZ.
Keywo ds:
schizoph enia-spec um diso de s; NRN1; age a onse ; wo king memo y; unc ional
magne ic esonance imaging ( MRI)
1. In oduc ion
Subs an ial e idence highligh s he impo ance o he gene ic componen in he ae iol-
ogy o schizoph enia (SZ), wi h an es ima ed he i abili y o a ound 65–79% [
1
,
2
]. Indeed,
genome-wide associa ion s udies (GWAS) ha e con i med SZ’s polygenic a chi ec u e,
esul ing om he agg ega ed e ec o low impac a ian s and epo ing an SNP-based
he i abili y o 24% [
3
]. Mo eo e , genomic da a con e ge in o iden i iable biological pa h-
ways in ol ed in neu ode elopmen , pa icula ly highligh ing he mechanism o synap ic
plas ici y [3–5].
Howe e , in he sea ch o speci ic gene ic ac o s ela ed o SZ, s udies ace se e al
challenges ha a ise om he gene ic and pheno ypic complexi y o he diso de [
6
]. Then,
i has been sugges ed ha combining complemen a y designs, such as amily-based and
case–con ol, would clea he way o dissec he gene ic in luences o he diso de [
7
]. In
his sense, amily-based gene ic associa ion designs ha e he ad an age o educing he
p oblem o s a i ica ion and spu ious associa ion when compa ed o case–con ol s udies,
while he la e usually allow o la ge sample sizes [8].
In addi ion o he design, he app oaches o he pheno ypic complexi y o SZ ha e also
been conside ed using na owe pheno ypes wi h pa icula ae iological signi icance o
educe he he e ogenei y and iden i y speci ic gene ic ac o s associa ed wi h he diso de .
One o hese pheno ypes is he age a onse , which shows a he i abili y o a ound 33% [
9
].
No ably, ea ly age a onse (EO) has cap u ed much a en ion because i is conside ed a
ma ke o a highe gene ic liabili y han adul -onse (AO) [
10
,
11
]. The EO e m includes
cases wi h onse up o 18 yea s o age and, despi e being a bi a y, oughly co esponds
wi h he uppe age cu -o in mos published s udies o child and adolescen psychosis [
12
].
In his suppo , EO subjec s show a highe amilial agg ega ion o SZ and o he men al
diso de s [
13
], poo e p emo bid adjus men [
14
] and neu ocogni i e pe o mance [
15
],
mo e se e e ou comes [
16
,
17
] and mo e p ominen al e a ions in neu ode elopmen al
ajec o ies han AO o ms [
18
]. The ew GWAS ocused on sea ching o gene ic loci
associa ed wi h age a onse in SZ ha e con i med ha some a ian s o e lap wi h hose
con e ing isk o SZ, while o he s a e pu e modi ie s [
19
–
22
]. Rema kably, EO pa ien s
p esen highe SZ polygenic isk sco es han hei siblings, wi h he sco es e ec i ely
p edic ing an ea lie age a onse [
23
]. In e es ingly, he a ian s associa ed wi h an ea lie
age a onse con e ge in o molecula ne wo ks ela ed o ne ous sys em de elopmen , he
egula ion o axon ex ension, modula ion o glial p oli e a ion, molecula anspo , and
cell- o-cell signalling and in e ac ions [20,22].
Among genes wi h pi o al oles h ough all s ages o he b ain’s o ma ion, he e is
he Neu i in 1 gene (NRN1, 6p25.1) (see e iew [
24
]), which is highly exp essed in he
hippocampus, he ce eb al co ex and he ce ebellum [
25
,
26
] in an ac i i y-dependen
manne [
27
,
28
]. Al hough he N n1 ecep o and i s downs eam signalling e ec o s a e
s ill being s udied, i seems ha N n1 egula es synap ic exci abili y h ough he ac i a ion
o he insulin ecep o (IR) and i s downs eam signalling pa hways [
29
,
30
]. Consequen ly,
inadequa e N n1 sus enance could ansla e in o he abno mal o ma ion o synapses, a
In . J. Mol. Sci. 2022,23, 7456 3 o 18
educed capaci y o pe o m adap i e esponses and, in u n, a highe isk o de eloping
a men al diso de . In ac , he in e es in he ole o N n1 in SZ has been mo i a ed by
se e al s udies, which ha e e idenced i s impac on cogni i e unc ion h ough synap ic
plas ici y mechanisms. F om cell- and animal-based app oaches, i has been shown ha
he i al-media ed o e exp ession o NRN1 in di e en models (unp edic able s ess-
induced a dep ession model, mice exposed o low- equency elec omagne ic ields and
an Alzheime ’s disease model Tg2576 mouse) p e en s he a ophy o dend i es and spines
and imp o es associa ed beha iou s, such as anxie y, dep ession, de ici s in no el objec
ecogni ion, lea ning and memo y [
31
–
33
]. Addi ionally, he exp ession o NRN1 has been
shown o inc ease in he hippocampus o mice exposed o elec ocon ulsi e he apy and
luoxe ine adminis a ion [
32
,
34
]. These s udies highligh he po en ial he apeu ic use o
NRN1 in diso de s associa ed wi h loss o cogni i e unc ion, such as SZ, and appeal o
a be e unde s anding o i s molecula mechanisms. F om human-based s udies, NRN1
has been al eady de ined as a candida e gene o SZ since speci ic allelic a ian s ha e
been associa ed wi h an inc emen ed isk o de eloping he diso de . Mo eo e , NRN1 has
also been desc ibed as a modi ie o he SZ pheno ype due o i s associa ion wi h pa ien s’
gene al cogni i e abili y and age a onse [
35
,
36
]. This sugges s ha NRN1 may be in ol ed
in c i ical mechanisms o b ain de elopmen , pa icula ly in hose mos suscep ible o he
ea lie onse o he symp oms.
Neu oimaging da a can p o ide e idence on how he gene ic ac o s unde lying an
ea lie age a onse con ibu e o he neu obiology o he diso de [
37
]. In his sense,
unc ional neu oimaging s udies ocused on explo ing he b ain ac i i y du ing wo king
memo y (WM) asks ( ela ed o he capaci y o e ain and use men al i ems du ing a
sho pe iod) a e o pa icula in e es . Sub le WM de iances ha e been desc ibed in he
heal hy siblings o subjec s wi h SZ compa ed o heal hy subjec s (HS) in s udies ocused on
cogni ion [
38
], b ain ac i i y [
39
] and connec i i y [
40
]. This sugges s ha WM al e a ions
in SZ a e gene ically in luenced. Indeed, disabili ies in his cogni i e domain a e conside ed
o be a co e ea u e o SZ [
41
] and ha e been epo ed o be e en mo e se e e in EO
pa ien s [15].
Se e al s udies based on unc ional magne ic esonance neu oimaging ( MRI) and
explo ing b ain ne wo ks suppo ing WM ha e consis en ly desc ibed on opa ie al di e -
ences in indi iduals wi h SZ when compa ed o HS. Mos o hese s udies desc ibed he
dec eased ac i i y o he do sola e al p e on al co ex (DLPFC), he en ola e al p e on al
co ex (VLPFC) and an e io cingula e co ex (ACC) as a key mechanism o WM dys unc-
ion [
42
]. The ew unc ional neu oimaging s udies speci ically ocused on indi iduals wi h
EO ha e epo ed simila pa e ns o abno mal ac i a ions in hese egions o he p e on al
co ex (e.g., VLPFC, DLPFC, and ACC) plus some limbic and empo al egions [
43
–
48
].
Howe e , hose s udies a e sca ce, in pa , due o he low a e o EO, which ep esen s only
abou 8% among indi iduals wi h SZ [
49
], and hey ha e epo ed inconsis en indings
ega ding he di ec ion o he esul s. In his con ex , he s udy o he gene ic WM co ela es
in indi iduals wi h EO o ms is pa icula ly pe inen since i could o e insigh s in o he
impac o gene ic a chi ec u e on b ain ac i i y and, ul ima ely, on he clinical mani es a ion
o SZ.
Conside ing all he abo e-ci ed e idence, we hypo hesised ha he polymo phic
a iabili y o NRN1 would be di e en ially associa ed wi h he isk o de eloping EO
o ms o SZ compa ed o AO. We de eloped his s udy by combining di e en designs
( amily-based and case–con ol sample app oaches) o p o ide obus ness o ou indings.
Addi ionally, we hypo hesised ha hose gene ic a ian s con e ing isk o EO would
di e en ially impac WM- ela ed b ain ac i i y.
In . J. Mol. Sci. 2022,23, 7456 4 o 18
2. Resul s
2.1. Gene ic Associa ion Analyses
2.1.1. Family-Based
The geno ypes/alleles coun s and equencies o EO/AO o sp ing and pa en s a e
lis ed in Supplemen a y Table S1. As shown in Table 1, wi hin EO amilies, he GCT
haplo ype including SNP6, SNP7 and SNP8 (HAP678) was signi ican ly unde - ansmi ed
om pa en s o a ec ed o sp ing (p
pe m
= 0.03). Ou analyses did no e eal any associa ion
be ween he gene ic a iabili y a NRN1 wi h he isk o AO SSD, nei he in he allelic,
geno ypic o haplo ype app oach.
Table 1.
Signi ican gene ic associa ion esul s wi hin ea ly-onse (EO) amily-based and EO case–
con ol samples. A he op ow, o he amily-based app oach, he e a e he ansmi ed and
no - ansmi ed haplo ype (HAP) coun s om he e ozygous pa en s o a ec ed o sp ing o amily-
based analyses. Below, o he case–con ol app oach, he equency (%) in heal hy subjec s (HS)
and schizoph enia spec um diso de s (SSD) a e gi en wi h he isk geno ype placed las . The odds
a io (OR) associa ed wi h he geno ype and he con idence in e al (CI 95%) a e also epo ed.
The empi ical p- alues ob ained a e 10,000 pe mu a ion p ocedu es (p
pe m
) o he T ansmission
Disequilib ium Tes (TDT) o he logis ic eg ession (addi i e model) a e shown.
SNPs Haplo ype T ansmi ed EO SSD No T ansmi ed EO SSD OR (CI 95%) TDT; ppe m
HAP678 GCT 13 27 0.48 (0.25–0.93) 4.90; 0.03
SNPs Geno ypes
Haplo ypes F equency EO SSD F equency HS OR (CI 95%) Wald; ppe m
SNP6 TT/TG/GG 11 (0.13)/40 (0.48)/33 (0.39) 31 (0.26)/58 (0.49)/30 (0.25) 1.68 (1.01–2.57) 2.39; 0.02 a,b
SNP7 CC/CT/TT 43 (0.50)/35 (0.41)/8 (0.09) 78 (0.66)/35 (0.29)/6 (0.05) 1.69 (1.06–2.71) 2.19; 0.03 b
SNP8 CC/CT/TT 14 (0.17)/41 (0.49)/29 (0.35) 33 (0.29)/58 (0.51)/23 (0.20) 1.66 (1.08–2.56) 2.31; 0.02 a,c
HAP678 TCC 0.37 0.50 0.59 (0.39–0.89) 6.44; 0.01
HAP678 GTT 0.30 0.20 1.70 (1.08–2.67) 5.28; 0.02
a
The geno ypic model was also signi ican (p
pe m
< 0.05).
b
The dominan model was also signi ican (p
pe m
< 0.05).
cThe ecessi e model was also signi ican (ppe m < 0.05).
2.1.2. Case–Con ol
The dis ibu ion o he geno ypes/alleles in HS, EO and AO subjec s is epo ed in
Supplemen a y Table S2. As exposed in Table 1, we obse ed a signi ican associa ion o
SNP6 G allele (p
pe m
= 0.02), SNP7 T allele (p
pe m
= 0.03) and SNP8 T allele (p
pe m
= 0.02)
wi h EO SSD unde an addi i e model. We also iden i ied an associa ion o wo haplo ypes
including SNP6, SNP7 and SNP8 (HAP678) wi h he isk o EO SSD, which was in line
wi h he SNP-based esul s. The GTT haplo ype was signi ican ly mo e equen in subjec s
wi h EO SSD han in HS (p
pe m
= 0.02), while he TCC was mo e equen in HS (p
pe m
= 0.01).
O he 2-SNP and 4-SNP haplo ypes con aining hese same a ian s we e also associa ed
wi h he isk o EO (Supplemen a y Table S3). Ou analyses did no e eal any e ec o
gene ic a iabili y a NRN1 on he isk o AO SSD, in any o he es ed models (allelic,
geno ypic and haplo ypic).
2.2. Neu oimaging Gene ic Associa ion Analyses
2.2.1. N-Back Func ional Response
The h ee g oups (HS, EO and AO) showed ypical WM- ela ed ac i a ion and deac i-
a ion pa e ns (Supplemen a y Figu es S1–S3). In addi ion, bo h EO and AO exhibi ed
a deac i a ion ailu e when compa ed o HS in o e lapping egions in ol ing bila e al
s uc u es, such as he on al gy us (supe io , medial and in e io o bi al pa ), he ol-
ac o y a ea, he ec us and he an e io cingula e and he pa acingula e gy i, as well
as igh s uc u es, such as he supe io and middle empo al gy us, he pa ahippocam-
pal gy us, he hippocampus, he amygdala, he usi o m gy us and he cauda e nucleus
(Supplemen a y Figu e S4).
In . J. Mol. Sci. 2022,23, 7456 5 o 18
We de ec ed a signi ican diagnosis x HAP678 (GTT) in e ac ion o he EO s. HS
compa ison in he 2-back s. 1-back con as in one clus e loca ed a he supe io and
middle on al gy us, egions o he DLPFC (316 oxels, peak ac i a ion a MNI coo dina es
[
−
34,42,42], Zmax = 4.54, p= 0.0025, Figu e 1A). To u he in e p e his esul , mean
ac i i y sco es o he 1-back and 2-back con as s we e plo ed. As shown in Figu e 1B,
HS exhibi ed a clus e mean ac i i y o a ound ze o o he wo con as s, i espec i e o
hei haplo ypic p o ile. Subjec s wi h EO wi hou he isk haplo ype showed a pa e n
owa ds inc eased clus e ac i i y om 1-back o 2-back con as s, whe eas hose pa ien s
ca ying he isk haplo ype p esen ed a pa e n owa ds dec eased clus e ac i i y. We
did no obse e any signi ican in e ac ion on b ain ac i i y when we compa ed HS and
AO g oups.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 o 19
s uc u es, such as he on al gy us (supe io , medial and in e io o bi al pa ), he ol ac-
o y a ea, he ec us and he an e io cingula e and he pa acingula e gy i, as well as igh
s uc u es, such as he supe io and middle empo al gy us, he pa ahippocampal gy us,
he hippocampus, he amygdala, he usi o m gy us and he cauda e nucleus (Supplemen-
a y Figu e S4).
We de ec ed a signi ican diagnosis x HAP678 (GTT) in e ac ion o he EO s. HS
compa ison in he 2-back s. 1-back con as in one clus e loca ed a he supe io and
middle on al gy us, egions o he DLPFC (316 oxels, peak ac i a ion a MNI coo di-
na es [−34,42,42], Zmax = 4.54, p = 0.0025, Figu e 1A). To u he in e p e his esul , mean
ac i i y sco es o he 1-back and 2-back con as s we e plo ed. As shown in Figu e 1B,
HS exhibi ed a clus e mean ac i i y o a ound ze o o he wo con as s, i espec i e o
hei haplo ypic p o ile. Subjec s wi h EO wi hou he isk haplo ype showed a pa e n
owa ds inc eased clus e ac i i y om 1-back o 2-back con as s, whe eas hose pa ien s
ca ying he isk haplo ype p esen ed a pa e n owa ds dec eased clus e ac i i y. We did
no obse e any signi ican in e ac ion on b ain ac i i y when we compa ed HS and AO
g oups.
Figu e 1. (A) Axial iew o he b ain showing he signi ican clus e de i ed om he diagnosis x
NRN1 HAP678 GTT analysis in he 2-back s. 1-back con as . A sagi al iew wi h he ma ks o he
c oss slices is also included. The igh side o he image ep esen s he igh side o he b ain. The
MNI coo dina es a e gi en o he shown slices. Uni s o he ba co espond o he β alues o he
eg ession, s anda dised o Z sco es. (B) Ba plo s wi h he clus e mean ac i i y (es ima ed ma ginal
means and ±2 s anda d e o s (se)) o heal hy subjec s (HS; le , non-ca ie s: n = 27, ca ie s: n = 10)
and subjec s wi h ea ly-onse schizoph enia (EO; igh , non-ca ie s: n = 19, ca ie s: n = 20).
Figu e 1.
(
A
) Axial iew o he b ain showing he signi ican clus e de i ed om he diagnosis x
NRN1 HAP678 GTT analysis in he 2-back s. 1-back con as . A sagi al iew wi h he ma ks o he
c oss slices is also included. The igh side o he image ep esen s he igh side o he b ain. The
MNI coo dina es a e gi en o he shown slices. Uni s o he ba co espond o he
β
alues o he
eg ession, s anda dised o Z sco es. (
B
) Ba plo s wi h he clus e mean ac i i y (es ima ed ma ginal
means and
±
2 s anda d e o s (se)) o heal hy subjec s (HS; le , non-ca ie s: n = 27, ca ie s: n = 10)
and subjec s wi h ea ly-onse schizoph enia (EO; igh , non-ca ie s: n = 19, ca ie s: n = 20).
2.2.2. N-Back Beha iou al Response
Fi s , subjec s wi h EO exhibi ed a globally poo e pe o mance o he N-back ask
han HS in bo h di icul y le els (mean (SD) d’1: EO 3.07 (1.16) and HS 4.14 (0.68), F = 13.00,
p= 0.001; mean (SD) d’2: EO 2.06 (0.90) and HS 3.41 (0.88), F = 27.52, p< 0.001). While bo h
In . J. Mol. Sci. 2022,23, 7456 6 o 18
g oups showed di e en sco es a he wo le els o he ask, hei deg ee o dec ease in
pe o mance om he 1-back o 2-back was simila (F = 1.55, p= 0.22).
Second, AO and HS exhibi ed a simila pe o mance in he low memo y load condi ion,
bu hei pe o mance di e ged in he high memo y load condi ion (mean (SD) d’1: AO
3.81 (0.90) HS 4.14 (0.68), F = 0.33, p= 0.57; mean (SD) d’2: AO 2.48 (0.82) and HS 3.41 (0.88),
F = 15.27, p< 0.001). Then, as he pe o mance o he wo g oups was simila o he 1-back,
he deg ee o change om he 1-back o -back was mo e p onounced in subjec s wi h AO
han HS (F = 8.11, p= 0.01).
Thi d, EO pe o mance a he low memo y load condi ion was modula ed by NRN1
haplo ypic a iabili y. Subjec s ca ying he HAP678 GTT showed a poo e pe o mance
when compa ed o hose wi hou he isk haplo ype (mean (SD) d’1: non-ca ie s 3.67 (0.22)
and ca ie s 2.86 (0.19), F = 5.66, p= 0.02) (Figu e 2). No e ec o NRN1 haplo ypic a iabili y
on ask pe o mance was de ec ed in ei he HS o AO subjec s.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 o 19
2.2.2. N-Back Beha iou al Response
Fi s , subjec s wi h EO exhibi ed a globally poo e pe o mance o he N-back ask
han HS in bo h di icul y le els (mean (SD) d’1: EO 3.07 (1.16) and HS 4.14 (0.68), F =
13.00, p = 0.001; mean (SD) d’2: EO 2.06 (0.90) and HS 3.41 (0.88), F = 27.52, p < 0.001). While
bo h g oups showed di e en sco es a he wo le els o he ask, hei deg ee o dec ease
in pe o mance om he 1-back o 2-back was simila (F = 1.55, p = 0.22).
Second, AO and HS exhibi ed a simila pe o mance in he low memo y load condi-
ion, bu hei pe o mance di e ged in he high memo y load condi ion (mean (SD) d’1:
AO 3.81 (0.90) HS 4.14 (0.68), F = 0.33, p = 0.57; mean (SD) d’2: AO 2.48 (0.82) and HS 3.41
(0.88), F = 15.27, p < 0.001). Then, as he pe o mance o he wo g oups was simila o he
1-back, he deg ee o change om he 1-back o -back was mo e p onounced in subjec s
wi h AO han HS (F = 8.11, p = 0.01).
Thi d, EO pe o mance a he low memo y load condi ion was modula ed by NRN1
haplo ypic a iabili y. Subjec s ca ying he HAP678 GTT showed a poo e pe o mance
when compa ed o hose wi hou he isk haplo ype (mean (SD) d’1: non-ca ie s 3.67
(0.22) and ca ie s 2.86 (0.19), F = 5.66, p = 0.02) (Figu e 2). No e ec o NRN1 haplo ypic
a iabili y on ask pe o mance was de ec ed in ei he HS o AO subjec s.
Figu e 2. Ba plo s wi h mean pe o mance (es ima ed ma ginal means and ±2 s anda d e o s(se))
o heal hy subjec s (HS; le , non-ca ie s: n = 27, ca ie s: n = 10) and subjec s wi h ea ly-onse
schizoph enia (EO; igh , non-ca ie s: n = 19, ca ie s: n = 20) by NRN1 HAP678 GTT.
3. Discussion
In his s udy, we combined gene ic associa ion and neu oimaging app oaches o
deepen in o he ole o NRN1 in he age a onse o SZ. Rega ding he gene ic associa ion
app oach, ou s udy adds o he only wo p e ious s udies on he associa ion o he NRN1
gene wi h SZ and o he diso de s wi hin he spec um [35,36], bu i is he i s o be de-
eloped h ough amily-based and case–con ol designs in wo independen samples. Ou
esul s, de i ed om he wo samples, sugges ha he a iabili y a NRN1 may explain a
modes p opo ion o he isk o EO. Conce ning he neu oimaging app oach, ou s udy
ep esen s he i s o explo e WM neu al co ela es o NRN1. Ou indings indica e ha
NRN1 a ian s con e ing isk o SZ also ha e an e ec on he pe o mance o he N-back
ask, speci ically wi hin EO subjec s. Addi ionally, we epo b ain ac i i y di e ences be-
ween EO subjec s and HS loca ed a he DLPFC condi ional o he same gene ic a ian s.
Ou gene ic associa ion analyses iden i ied di e en SNPs and haplo ypes a NRN1
associa ed wi h EO in SSD. We de ec ed a signi ican unde - ansmission o he HAP678
GCT om pa en s o a ec ed o sp ing, speci ically in EO amilies. In pa allel, h ough a
case–con ol app oach, we iden i ied he e ec o SNP6, 7 and 8 on he isk o EO SSD
and he associa ion o a isk haplo ype encompassing hese same polymo phisms HAP678
Figu e 2.
Ba plo s wi h mean pe o mance (es ima ed ma ginal means and
±
2 s anda d e o s(se))
o heal hy subjec s (HS; le , non-ca ie s: n = 27, ca ie s: n = 10) and subjec s wi h ea ly-onse
schizoph enia (EO; igh , non-ca ie s: n = 19, ca ie s: n = 20) by NRN1 HAP678 GTT.
3. Discussion
In his s udy, we combined gene ic associa ion and neu oimaging app oaches o
deepen in o he ole o NRN1 in he age a onse o SZ. Rega ding he gene ic associa ion
app oach, ou s udy adds o he only wo p e ious s udies on he associa ion o he NRN1
gene wi h SZ and o he diso de s wi hin he spec um [
35
,
36
], bu i is he i s o be
de eloped h ough amily-based and case–con ol designs in wo independen samples.
Ou esul s, de i ed om he wo samples, sugges ha he a iabili y a NRN1 may explain
a modes p opo ion o he isk o EO. Conce ning he neu oimaging app oach, ou s udy
ep esen s he i s o explo e WM neu al co ela es o NRN1. Ou indings indica e ha
NRN1 a ian s con e ing isk o SZ also ha e an e ec on he pe o mance o he N-back
ask, speci ically wi hin EO subjec s. Addi ionally, we epo b ain ac i i y di e ences
be ween EO subjec s and HS loca ed a he DLPFC condi ional o he same gene ic a ian s.
Ou gene ic associa ion analyses iden i ied di e en SNPs and haplo ypes a NRN1
associa ed wi h EO in SSD. We de ec ed a signi ican unde - ansmission o he HAP678
GCT om pa en s o a ec ed o sp ing, speci ically in EO amilies. In pa allel, h ough a
case–con ol app oach, we iden i ied he e ec o SNP6, 7 and 8 on he isk o EO SSD
and he associa ion o a isk haplo ype encompassing hese same polymo phisms HAP678
GTT. On he con a y, we did no de ec any associa ion wi h AO SSD. The e o e, ou da a
con e ge in o he iew o polymo phisms a NRN1 (SNP6, SNP7, SNP8) as a ele an
gene ic a iabili y sou ce in modi ying he neu ode elopmen p ocesses ela ed o he
ea lie eme gence o hese diso de s. I is o no e ha hese SNPs ha e been p e iously
associa ed wi h he isk o SZ in he wo-p eceding wo ks [
35
,
36
]. Mo eo e , one o hese
s udies also iden i ied a ole o NRN1 in age a onse o SSD [
36
]. Howe e , ou esul s
In . J. Mol. Sci. 2022,23, 7456 7 o 18
should be in e p e ed in he con ex o he polygenic a chi ec u e o hese diso de s, as he
e ec o he SNPs and haplo ypes is small (see he co esponding ORs). S ill, his e idence
sugges s ha hose genes ha in luence b ain de elopmen , such as NRN1, may modi y
illness ai s, such as age a onse , and ul ima ely a ec he isk o hese diso de s.
Due o he ew s udies ocused on examining he associa ion o he NRN1 gene
wi h SZ, da a om whole-genome app oaches mus be aken in o conside a ion o he
u he in e p e a ion o ou esul s. Fi s , di e en gene ic linkage s udies mapping SZ
o a genomic loca ion poin ed owa ds he associa ion o ch omosome egion 6p24-25
and highligh ed NRN1 as a posi ional candida e gene [
50
–
53
]. Howe e , as a as we
know, NRN1 has no appea ed as a signi ican locus in he la es genome-wide associa ion
s udies [
3
]. These nega i e esul s could be explained due o he modi ie p ope ies o
NRN1, which means ha , as ou esul s sugges , NRN1 modula es SSD pheno ype h ough
i s impac on age a onse . Some linkage and whole-genome s udies ha speci ically
aimed o iden i y modi ie loci ela ed o he age a onse in SZ ha e highligh ed he
ch omosome egion 6p24 and some NRN1 neighbou ing in e genic a ian s wi h pu a i e
egula o y oles on i s exp ession [
21
,
54
]. Addi ionally, whole-genome app oaches ha e
also linked NRN1 and SZ h ough epigene ic mechanisms. In his espec , Pidsley e al.,
2014 [
55
] iden i ied, h ough a me hylomic app oach in human pos -mo em p e on al
co ex samples, a wide gene ic egion ha is hypome hyla ed in pa ien s compa ed o
con ols, spanning he body o NRN1. This esul sugges s ha NRN1 could be di e en ially
exp essed in he p e on al co ex o subjec s wi h SZ, a b ain egion epe i i ely desc ibed
o be al e ed in his diso de [56].
To explo e he neu obiological ansla ion o he obse ed gene ic a ian s con e ing
a highe isk o EO SZ, we de eloped a neu oimaging gene ic s udy in a ma ched case–
con ol sub-se . Ou unc ional da a sugges ha he isk haplo ype (HAP678 GTT) ha is
associa ed wi h he ea lie eme gence o he diso de is also associa ed wi h DLPFC ac i i y
changes wi hin his g oup o pa ien s. Conc e ely, h ough he analysis o di e ences
be ween he wo le els o he N-back ask (2-back s. 1-back con as ), we obse ed ha
EO subjec s no ca ying he isk haplo ype changed DLPFC ac i i y owa ds ac i a ion
in esponse o he ask’s inc easing di icul y. A he same ime, hose ca ying he isk
haplo ype we e p one o dec eased ac i i y. The p e ious ew s udies explo ing whole-b ain
ac i i y di e ences be ween subjec s wi h EO and HS ha e epo ed inconsis en indings
ega ding he implica ed egions. Some epo ed educed ac i a ion o he le VLPFC and
ex as ia e isual co ex [
43
], while o he s desc ibed VLPFC hype ac i a ion [
47
]. O he
wo ks epo ed he educed engagemen o he DLPFC, he ACC, on al ope culum and
in e io and pos e io pa ie al and cauda e [
44
–
46
], con a y o o he in es iga ions ha
sugges ed inc eased ac i a ions in he ACC, medial empo al lobe s uc u es, he insula
and bila e al la e al empo al lobes [
48
]. In his espec , ou esul s shed ligh on hose
con o e sial indings, as hey p o ide e idence ha gene ic ac o s, in his case, he NRN1
gene, could be unde lying hese di e ences in DLPFC ac i i y.
To in e p e ou unc ional esul s in he DLPFC, i is impo an o in eg a e b ain
ac i i y indings wi h N-back beha iou al da a. On he one hand, he sus ained ac i a ion
o he p e on al ci cui s is conside ed a key mechanism o execu ing high-memo y-load
asks [
57
]. On he o he hand, i is also known ha he deg ee o change in DLPFC
ac i i y is ela ed o he cogni i e e o needed o pe o m he ask. In o he wo ds, i
he compu a ional cos is unlikely o esul in he accu a e pe o mance o he ask, he
p e on al esou ces ge disengaged [
58
]. In his sense, EO subjec s no ca ying he isk
haplo ype displayed a be e pe o mance a low memo y load and exhibi ed a highe
deg ee o DLPFC modula ion owa ds ac i a ion in esponse o a highe memo y load le el.
This esul sugges s ha EO subjec s wi hou he isk haplo ype may use g ea e p e on al
esou ces in esponse o ask di icul y inc ease han hose wi h he isk haplo ype, who
seem o each ac i a ion and pe o mance peaks a a lowe p ocessing load.
On he whole, ou unc ional and beha iou al esul s align wi h he p eceding e idence
linking NRN1 and cogni i e pe o mance in SZ [
35
,
36
] and execu i e unc ion in HS [
59
].
In . J. Mol. Sci. 2022,23, 7456 8 o 18
Fu he mo e, hey sugges ha hese p e on al ne wo ks o sus ained ac i a ion du ing
WM, in which NRN1 appea s o ha e a ele an ole, migh be especially sensi i e o he
ea lie onse o psychosis. The speci ic e ec s in EO o ms o SZ seem easonable since
se e al in es iga ions ha e desc ibed adolescence as a c ucial pe iod o he de elopmen
o he p e on al co ex [
60
] and he eo ganisa ion o he WM ne wo k [
61
,
62
]. This is
also suppo ed by se e al s udies showing he g ea e ec ui men o WM egions in
adul s han in child en [
63
]. In his iew, he ea lie onse o he diso de migh s ongly
impac he neu al ajec o ies associa ed wi h WM de elopmen , po en ially leading o
WM-cha ac e is ic impai men s in EO compa ed o AO [
64
]. In e es ingly, ega ding he
speci ic ole o DLPFC ac i i y and NRN1 as po en ial ma ke s o EO, a ecen s udy using
an inno a i e ansc ip omic app oach de ined wo molecula ly dis inc subg oups o
subjec s wi h SZ [
65
]. The i s p esen ed a DLPFC ansc ip ome e y simila o ha
o HS, while he second exhibi ed a s ikingly di e en DLPFC ansc ip ome, wi h he
NRN1 gene included among he di e en ially exp essed genes. These da a sugges ha
undamen al biologic di e ences exis be ween subjec s diagnosed wi h SZ. Thus, ou
esul s on he modula ion e ec o NRN1 haplo ypic a iabili y on b ain unc ion and
pe o mance con ibu e o b idging he gap be ween he ole o NRN1 in synap ic plas ici y
p ocesses and he pa hophysiological mechanisms unde lying SZ.
Towa ds a u he unde s anding o such mechanisms, conside ing he pu a i e e ec s
o he analysed polymo phic si es on gene exp ession egula o y mechanisms ep esen s a
aluable esou ce o p o ide addi ional meaning and impo ance o ou associa ion da a.
Among he h ee a ian s encompassed by he HAP678 isk haplo ype (LD = 0.99 in bo h
samples), da a om he RegulomeDB and Haplo eg highligh he unc ional e ec s o SNP6
( s3763180). The e is e idence ha his a ian could modi y he his one enhance and
p omo e ma ks in he b ain, con ibu ing o he ch oma in s a e a his locus. Mo eo e ,
his a ian is p edic ed o al e mo i s ha o e lap he ecogni ion sequence o di e en
ansc ip ion ac o s, such as he alpha iso o m o he CCAAT-enhance binding p o eins
(C/EBP), PBX homeobox 3 (PBX3) and he Neu on-Res ic i e Silencing Fac o (NRSF).
In e es ingly, he change o a T o a G in ha posi ion is linked o inc eased NRSF a ini y,
implica ed in he p og amming o s ess-sensi i e neu ons by neona al expe ience h ough
epigene ic mechanisms, p omo ing esilience o s ess- ela ed emo ional diso de s [
66
].
The o he wo a ian s included in he HAP678 p esen ed a lowe unc ionali y sco e;
s ill, da a show hei pu a i e modula o y e ec s on he a ini y o some ansc ip ion
ac o s. Fo ins ance, he SNP7 ( s10484320) is sugges ed o modi y he binding o he
TATA-binding p o ein (TBP) and PU.1. The TBP has been associa ed wi h he isk o SZ,
age a onse and p e on al unc ion [
67
]. Addi ionally, highe le els o he ansc ip ion
ac o PU.1, equi ed o he de elopmen o he immune sys em, ha e been de ec ed in
pos -mo em b ain samples om indi iduals diagnosed wi h SZ compa ed o HS [
68
].
Addi ionally, acco dingly o B ainiac da a, when he e ec o hese h ee SNPs s a i ies he
exp ession o NRN1 ansc ip s, geno ype-based di e ences eme ge in he hippocampus,
and a end e ec is de ec ed in he co ex. These lines o e idence sugges pu a i e
molecula mechanisms by which he SNPs included in he HAP678 may a ec he complex
pheno ype o SZ. Ne e heless, u he unc ional da a on hese SNPs a e needed o ully
cha ac e ise hei impac on he unde lying mechanisms ha connec NRN1 and age a
onse o psychosis.
Finally, ou s udy should be in e p e ed in he con ex o some limi a ions. Fi s ,
ega ding ou gene ic associa ion app oach, he samples could be conside ed o be ela i ely
small. Howe e , acco ding o he s a is ical powe o ou analyses and a e mul iple
es ing co ec ion p ocedu es, we concu en ly iden i ied, in wo independen samples,
he impac o NRN1 gene ic a ian s on he isk o he ea lie onse o SZ. Second, all
he a ian s included in he p esen s udy a e polymo phic; howe e , i is known ha a
ce ain p opo ion o he a iance in gene ic liabili y o SZ is also accoun ed o by a e
a ian s [
69
,
70
]. The e o e, di e en app oaches analysing he combined ole o common
and low- equency a ian s along NRN1 gene on SZ would be o po en ial in e es . Thi d,
In . J. Mol. Sci. 2022,23, 7456 9 o 18
while he p esen s udy has no di ec ly analysed he unc ional consequences o he NRN1
a ian s associa ed wi h EO, ou esul s and he a ailable unc ional da a sugges he
need o cell-based s udies in eg a ing gene ic a iabili y in o ma ion. Fou h, in he
case o neu oimaging app oaches, al hough we compa ed EO subjec s and HS, pa ien s
we e scanned in hei adul hood, yea s a e he onse o he illness. The e o e, illness
du a ion and ela ed clinical a iables could ha e a ec ed he esul s. Based on his, we
checked he possible impac o illness du a ion o medica ion on he mean ac i i y and
he d’ sco es h ough eg essions. While we canno comple ely ule ou he e ec o hese
a iables he lack o signi icance sugges s ha ou esul s a e no modula ed by hem.
Addi ionally, i should be unde lined ha ac i a ion di e ences a p e on al egions ha e
been obse ed in una ec ed i s ela i es o SZ pa ien s [
39
], indi iduals a clinical high
isk o psychosis [
71
] and indi iduals wi h ea men -naï e i s episode psychosis [
72
],
sugges ing ha his pa e n may ep esen an in insic ea u e o SZ a he han a medica ion
e ec . Las ly, he absence o ep esen a ion o di e se e hnic g oups and he low p opo ion
o emales wi hin ou EO g oup hampe s he ex apola ion o ou esul s and demands he
need o new s udies in la ge samples wi h equal ep esen a ion o hose popula ions.
4. Ma e ials and Me hods
4.1. Sample
This s udy included 798 indi iduals (Table 2). Two independen samples we e used o
de elop sepa a e gene ic associa ion analyses: (i) Sample 1 comp ised 151 ios (wi h an
o sp ing diagnosed wi h schizoph enia spec um diso de s (SSD) plus 302 heal hy pa en s),
(ii) Sample 2 consis ed o 225 independen pa ien s diagnosed wi h SSD and 120 HS. Also,
om Sample 2, a sub-se o cases wi h SZ (39 EO and 39 AO) and HS (39) (ma ched
by sex, age and es ima ed IQ) was selec ed o de elop a neu oimaging gene ic analysis
(Sample 3)
. Pa icipan s we e d awn om admissions o bo h Child and Adolescen and
Adul Psychia ic Uni s. All HS we e ec ui ed om non-medical s a wo king in he
hospi al, hei ela i es and acquain ances, plus independen sou ces in he communi y.
All pa ien s we e e alua ed by expe ienced psychia is s and me he DSM-IV-TR
c i e ia o SSD, including schizoph enia, schizoph eni o m diso de , schizoa ec i e diso -
de and psychosis diso de no o he wise speci ied (Table 2). Pa ien s up o 17 yea s old
we e diagnosed ollowing Kiddie Schedule o A ec i e Diso de s and Schizoph enia (KS-
DAS, [73]), while he Comp ehensi e Assessmen o Symp oms and His o y (CASH, [74])
o he S uc u ed Clinical In e iew o DSM Diso de s (SCID, [
75
]) was used o adul
pa ien s. Age a onse o he i s episode was de e mined using hese clinical schedules
and/o he Symp om Onse in Schizoph enia in en o y (SOS, [
76
]). Following p e ious
s udies [
12
], subjec s wi h SSD we e classi ied as ei he EO when he i s episode occu ed
be o e o a 18 yea s, o as adul -onse AO when p esen ed a age 19 o olde .
The gene al exclusion c i e ia included an age abo e 65 yea s, majo medical illnesses
ha could a ec b ain unc ions, subs ance-induced psycho ic diso de , neu ological con-
di ions and ha ing had a leas one pa en no om Eu opean ances y. Mo eo e , all he
ela i es and HS unde wen a clinical in e iew on pe sonal and/o amilial psychia ic
his o y using Family In e iew o Gene ic S udies (FIGS) [
77
] and hose who epo ed a
pe sonal his o y o men al illness o ea men wi h psycho opic medica ion we e excluded.
Fo he subjec s included in he neu oimaging s udy, he exclusion c i e ia also in-
cluded an es ima ed IQ unde 70, le manual dominance, and a his o y o head auma
wi h loss o consciousness. The e alua ion o pa ien s comp ised he Posi i e and Neg-
a i e Symp oms Scale (PANSS), while he es ima ed IQ o bo h pa ien s and con ols,
was assessed using he Wo d Accen ua ion Tes [
78
], which equi es he p onuncia ion o
30 low- equency Spanish wo ds whose accen s we e emo ed.
All pa icipan s p o ided w i en consen a e being in o med abou he s udy p oce-
du es and implica ions. In he case o pa ien s below he age o 18, w i en consen was
also ob ained om hei pa en s. The s udy was pe o med ollowing he guidelines o he
In . J. Mol. Sci. 2022,23, 7456 16 o 18
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