ARTICLE
En e o i us-associa ed changes in blood ansc ip omic p o iles
o child en wi h gene ic suscep ibili y o ype 1 diabe es
Niina Lie zen
1
&Le T. T. An
1
&Ma ia K. Jaakkola
1,2
&Henna Kallionpää
1
&
Sami Oika inen
3,4
&Juha Mykkänen
5,6
&Mikael Knip
7,8,9,10
&Rii a Veijola
11,12
&
Jo ma Ilonen
13,14
&Jo ma Toppa i
5,15
&Heikki Hyö y
3,4
&Rii a Lahesmaa
1
&
Lau a L. Elo
1
Recei ed: 8 June 2017 /Accep ed: 24 Augus 2017 /Published online: 8 No embe 2017
#The Au ho (s) 2017. This a icle is an open access publica ion
Abs ac
Aims/hypo hesis En e o i us in ec ions ha e been associa ed
wi h he de elopmen o ype 1 diabe es in mul iple s udies,
bu li le is known abou en e o i us-induced esponses in
child en a isk o de eloping ype 1 diabe es. Ou aim was
o use genome-wide ansc ip omics da a o cha ac e ise
en e o i us-associa ed changes in whole-blood samples om
child en wi h gene ic suscep ibili y o ype 1 diabe es.
Me hods Longi udinal whole-blood samples (356 samples in
o al) collec ed om 28 pai s o child en a inc eased isk o
de eloping ype 1 diabe es we e sc eened o he p esence o
en e o i us RNA. Se en o hese samples we e de ec ed as
en e o i us-posi i e, each o hem collec ed om a di e en
child, and ansc ip omics da a om hese child en we e
analysed o unde s and he indi idual-le el esponses associ-
a ed wi h en e o i us in ec ions. T ansc ip clus e s wi h
peaking o d opping exp ession a he ime o en e o i us
posi i i y we e selec ed as he en e o i us-associa ed signals.
Resul s S ong signs o ac i a ion o an in e e on esponse
we e de ec ed in ou child en a en e o i us posi i i y, while
Niina Lie zen and Le T. T. An con ibu ed equally o he s udy.
Rii a Lahesmaa and Lau a L. Elo a e join senio au ho s.
Elec onic supplemen a y ma e ial The online e sion o his a icle
(h ps://doi.o g/10.1007/s00125-017-4460-7) con ains pee - e iewed bu
unedi ed supplemen a y ma e ial, which is a ailable o au ho ised use s.
*Niina Lie zen
niina.lie zen@u u. i
*Lau a L. Elo
lau a.elo@u u. i
1
Tu ku Cen e o Bio echnology, Uni e si y o Tu ku and Åbo
Akademi Uni e si y, Tykis öka u 6, FI-20520 Tu ku, Finland
2
Depa men o Ma hema ics and S a is ics, Uni e si y o Tu ku,
Tu ku, Finland
3
Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e,
Tampe e, Finland
4
Fimlab Labo a o ies, Pi kanmaa Hospi al Dis ic , Tampe e, Finland
5
Depa men o Pedia ics, Tu ku Uni e si y Hospi al, Tu ku, Finland
6
Resea ch Cen e o Applied and P e en i e Ca dio ascula
Medicine, Uni e si y o Tu ku, Tu ku, Finland
7
Child en’s Hospi al, Uni e si y o Helsinki and Helsinki Uni e si y
Hospi al, Helsinki, Finland
8
Resea ch P og ams Uni , Diabe es and Obesi y, Uni e si y o
Helsinki, Helsinki, Finland
9
Folkhälsan Resea ch Cen e , Helsinki, Finland
10
Tampe e Cen e o Child Heal h Resea ch, Tampe e Uni e si y
Hospi al, Tampe e, Finland
11
Depa men o Pedia ics, PEDEGO Resea ch Uni , Uni e si y o
Oulu, Oulu, Finland
12
Depa men o Child en and Adolescen s, Medical Resea ch Cen e ,
Oulu Uni e si y Hospi al, Oulu, Finland
13
Immunogene ics Labo a o y, Ins i u e o Biomedicine, Uni e si y o
Tu ku, Tu ku, Finland
14
Depa men o Clinical Mic obiology, Tu ku Uni e si y Hospi al,
Tu ku, Finland
15
Depa men o Physiology, Ins i u e o Biomedicine, Uni e si y o
Tu ku, Tu ku, Finland
Diabe ologia (2018) 61:381–388
DOI 10.1007/s00125-017-4460-7
ansc ip omic changes in he o he h ee child en indica ed
ac i a ion o adap i e immune esponses. Addi ionally, a la ge
p opo ion o he en e o i us-associa ed changes we e speci ic
o indi iduals. An en e o i us-induced signa u e was buil
using 339 genes peaking a en e o i us posi i i y in ou o
he child en, and 77 o hese genes we e also up egula ed in
human pe iphe al blood mononuclea cells in ec ed in i o
wi h di e en en e o i uses. These genes sepa a ed he ou
en e o i us-posi i e samples clea ly om he emaining 352
blood samples analysed.
Conclusions/in e p e a ion We ha e, o he i s ime, iden i-
ied en e o i us-associa ed ansc ip omic p o iles in whole-
blood samples om child en wi h gene ic suscep ibili y o
ype 1 diabe es. Ou esul s p o ide a s a ing poin o unde -
s anding he indi idual esponses o en e o i us in ec ions in
blood and hei po en ial connec ion o he de elopmen o
ype 1 diabe es.
Da a a ailabili y The da ase s analysed du ing he cu en
s udy a e included in his published a icle and i s supple-
men a y in o ma ion iles (www.b k. i/ esea ch/
compu a ional-biomedicine/1234-2) o a e a ailable om
he Gene Exp ession Omnibus (GEO) eposi o y (accession
GSE30211).
Keywo ds Clinical immunology .En e o i us .Human .
Mic oa ay .P edic ion and p e en ion o ype 1 diabe es
Abb e ia ions
DAVID Da abase o Anno a ionVisualiza ion and
In eg a ed Disco e y
DIPP Type 1 Diabe es P edic ion and P e en ion
FDR False-disco e y a e
IPA Ingenui y Pa hway Analysis
PBMC Pe iphe al blood mononuclea cell
RMA Robus mul ia ay a e age
UPC Uni e sal exP ession Codes
In oduc ion
En e o i uses a e among he mos common i uses causing
in ec ions in humans. They a e single-s anded RNA i uses
ha eplica e ypically in he in es ine, bu can occasionally
sp ead also o blood and ce ain in e nal o gans. Al hough
en e o i us in ec ions a e mos ly asymp oma ic o cause only
mild symp oms, hey can also cause se e e illnesses such as
meningi is, myoca di is and hand- oo -and-mou h disease.
Se e al s udies ha e associa ed i al in ec ions, especially
human en e o i us in ec ions, wi h he de elopmen o ype 1
diabe es [1–4]. En e o i uses ha e a clea opism o panc e-
a ic be a cells [5], and low-g ade en e o i us in ec ion has
been de ec ed in panc ea ic isle s o li ing indi iduals wi h
ecen ly diagnosed ype 1 diabe es [6]. P ospec i e s udies
ha e also ound signs o en e o i us in ec ions mo e common-
ly in child en who la e de elop ype 1 diabe es au oan ibodies
o clinical ype 1 diabe es han in con ol child en [1,2,7].
The p esence o en e o i uses is no , howe e , hough o di-
ec ly esul in an inc eased isk o ype 1 diabe es. The ou -
comes o in ec ion likely depend on complex ela ionships
be ween he hos and he i us: o example, he gene ic back-
g ound and indi idual p ope ies o he hos [8–11], he iming
o in ec ions [12] and he ype o en e o i us in ading he hos
[13,14]. Cu en ly, only limi ed da a a e a ailable ega ding
in i o en e o i us esponses in child en a isk o de eloping
ype 1 diabe es, and mos s udies s ill ely on in i o in ec ion
models. The e o e, be e unde s anding o he indi idual-
le el esponses o en e o i us in ec ion is equi ed o gain
insigh s in o he a iable ou comes o hese in ec ions.
In his s udy, we pe o med, o he i s ime, genome-wide
ansc ip omic analysis o en e o i us-associa ed changes in
child en wi h gene ic suscep ibili y o ype 1 diabe es. We
analysed mic oa ay da a om 44 longi udinally collec ed
whole-blood samples [15] om se en child en who we e
en e o i us-posi i e in one o he ollow-up samples. Ou
aim was o unde s and he indi idual-le el ansc ip omic
changes associa ed wi h en e o i us in ec ions and o cha ac-
e ise he common ea u es o en e o i us esponses in young
child en.
Me hods
S udy pa icipan s and sample selec ion The mic oa ay
da a used in his s udy a e pa o he da ase published by
Kallionpää e al [15] (GEO accession GSE30211) co e ing
356 PAXGene whole-blood RNA samples measu ed using
he A yme ix Human Genome U219 A ay (A yme ix,
San a Cla a, CA, USA). The samples we e collec ed om
28 pai s o child en pa icipa ing in he Finnish Type 1
Diabe es P edic ion and P e en ion (DIPP) s udy in Tu ku,
Finland. All child en in he DIPP s udy ca y HLA-
con e ed gene ic isk o ype 1 diabe es, and hey ha e been
obse ed om bi h a egula in e als [16]. All child en had
w i en pa en al consen and he E hics Commi ee o Tu ku
Uni e si y Hospi al had g an ed app o al o he DIPP s udy.
The s udy was ca ied ou in acco dance wi h he p inciples o
he Decla a ion o Helsinki.
The p esence o en e o i us RNA was s udied using quan i-
a i e RT-PCR as desc ibed p e iously [17] om he same 356
RNA samples used o he mic oa ay analyses. The RT-PCR
was ca ied ou in h ee pa allel eac ions. I all h ee eac ions
ga e a posi i e esul , he sample was classi ied as s ongly
en e o i us-posi i e; i only one o he eac ions was posi i e,
he sample was classi ied as weakly en e o i us-posi i e. In o al,
se en samples we e de ec ed as en e o i us-posi i e, each
382 Diabe ologia (2018) 61:381–388
collec ed om a di e en child. Mic oa ay da a o all 44 sam-
ples om hese en e o i us-posi i e child en we e selec ed o
u he analyses (elec onic supplemen a y ma e ial [ESM]
Table 1).
Mic oa ay da a p ocessing and clus e ing The mic oa ay
da a we e p e-p ocessed using he obus mul ia ay a e age
(RMA) me hod implemen ed in he Bioconduc o package
a y e sion 1.44.0 (h p://bioconduc o .o g/packages/ elease/
bioc/h ml/a y.h ml), and log
2
- ans o med. The Uni e sal
exP ession Codes (UPC) me hod o he Bioconduc o pack-
age SCAN.UPC e sion 2.12.1 (h ps://bioconduc o .o g/
packages/ elease/bioc/h ml/SCAN.UPC.h ml)[18] was used
o il e ou p obe se s wi h low exp ession (UPC < 0.5 in all
he samples).
Fo each p obe se , he RMA-no malised exp ession alues
we e ans o med in o zsco es based on hei child-speci ic mean
and s anda d de ia ion o e he i us-nega i e samples. The
zsco e p o iles we e clus e ed sepa a ely o each child using
he k-means algo i hm, wi h Pea son co ela ion and k = 10. Fo
each child, he clus e s wi h he highes and lowes cen oid
alues a he ime o en e o i us posi i i y we e iden i ied
( e e ed o as peaking and d opping clus e s, espec i ely).
Func ional da a analysis Func ional classi ica ion o he da a
was pe o med using he Da abase o Anno a ion, Visualiza ion
and In eg a ed Disco e y (DAVID; h ps://da id.nci c .go /;
accessed No embe o Decembe 2016) [19] and Ingenui y
Pa hway Analysis (IPA; Qiagen Bioin o ma ics, Aa hus,
Denma k, www.qiagenbioin o ma ics.com/p oduc s/ingenui y-
pa hway-analysis/; accessed No embe o Decembe 2016)
ools. Gene on ology classes wi h DAVID alse-disco e y a e
(FDR) < 0.05 and IPA pa hways wi h p alue < 0.001 we e
conside ed signi ican ly en iched. The In e e ome 2.01 da abase
(www.in e e ome.o g; accessed No embe o Decembe 2016)
[20] was used o s udy he p esence o human in e e on-
egula ed genes.
Mic oa ay da a om human PBMCs in ec ed in i o
The ansc ip omics da a om child en a isk o ype 1 diabe-
es we e compa ed wi h mic oa ay da a (HumanHT-12 V3.0
BeadChip, Illumina, San Diego, CA, USA) om pe iphe al
blood mononuclea cells (PBMCs) in ec ed wi h en e o i us
in i o [14], including h ee eplica e samples o PBMCs in-
ec ed wi h ATCC s ain o echo i us 9 o wild- ype Coxsackie
B1 i us s ains CDC10802 and CDC10796 o 48 h, and
unin ec ed con ol PBMCs (see Da ase 1 published on
h ps://www.b k. i/1234-2/). The da a we e p e-p ocessed using
he a iance-s abilising no malisa ion o he Bioconduc o lumi
package e sion 2.18.0 (h ps://bioconduc o .o g/packages/
elease/bioc/h ml/lumi.h ml)[21]. The UPC me hod was used
o il e ou p obes wi h low exp ession un il eaching he
numbe o p obes equal o he da ase om he en e o i us-
posi i e child en. Di e en ial exp ession was de e mined using
he Bioconduc o ROTS [22] package e sion 1.1.1 (h ps://
bioconduc o .o g/packages/ elease/bioc/h ml/ROTS.h ml)and
cu -o alues FDR < 0.05 and old change > 1.5. To enable
compa ison be ween he Illumina and A yme ix pla o ms,
he p obes and p obe se s we e mapped o genes using IPA
(Qiagen; accessed No embe o Decembe 2016).
Resul s
En e o i us RNA was de ec ed in se en o 356 whole-blood
RNA samples, wi h i e s ongly en e o i us-posi i e and wo
weakly en e o i us-posi i e samples each aken om a di e -
en child.
To cha ac e ise en e o i us-associa ed changes in whole-
blood ansc ip ome, we s udied longi udinal gene exp ession
p o iles o hese en e o i us-posi i e child en by di iding all
p obe se s child-speci ically in o en clus e s (ESM Fig. 1;see
also Da ase 2 published on h ps://www.b k. i/1234-2/). Fo
each child, he clus e s wi h he highes and lowes cen oid
alue a he ime o en e o i us posi i i y we e selec ed as he
en e o i us-associa ed signals (Fig. 1a–g).
Fo ou s ongly en e o i us-posi i e child en, he o e laps
be ween peaking and d opping clus e s we e highe (a e age
o e laps o 46% and 37%, espec i ely) han hose be ween he
o he child en (a e age o e laps o 8%) (Fig. 1h, i). In o al,
593 p obe se s mapping o 339 dis inc genes we e de ec ed in
he peaking clus e s o all ou child en. This se was de ined as
he en e o i us-induced signa u e. Howe e , app oxima ely
20% o he p obe se s in each o hese peaking clus e s we e
child-speci ic, indica ing he p esence o indi idual di e ences
in en e o i us esponses. The o he h ee child en had lowe
o e laps wi h each o he and wi h all o he child en.
Genes in ol ed in an i i al immune esponses and espe-
cially in e e on signalling we e signi ican ly en iched in bo h
he peaking clus e s o he ou s ongly en e o i us-posi i e
child en and he en e o i us-induced signa u e (Fig. 2a; see
also Da ase 2 published on h ps://www.b k. i/1234-2/). Also,
child-speci ic exp ession p o iles o wo in e e on signalling
genes, STAT2 and MX1, showed clea peaks in he ou chil-
d en a en e o i us posi i i y (Fig. 2b–h). Fu he mo e, 74%
o he signa u e genes we e egula ed by in e e ons based on
he In e e ome da abase. The B cell ecep o signalling pa h-
way was among he mos signi ican ly en iched pa hways in
he peaking clus e s o he o he h ee child en, po en ially
e lec ing he ac i a ion o adap i e immune esponses.
Based on he d opping clus e s, en e o i us in ec ion was also
associa ed wi h down egula ion o genes in ol ed in mRNA
p ocessing, ansc ip ion o ansla ion in six o he se en chil-
d en (see Da ase 3 published on h ps://www.b k. i/1234-2/).
En e o i us RNA is de ec able in blood o only a ew days
du ing he acu e phase o in ec ion. To es ima e he iming o
Diabe ologia (2018) 61:381–388 383
in ec ion ela i e o sample collec ion, we compa ed ou peaking
and d opping clus e s wi h whole-blood ansc ip ional changes
du ing acu e and eco e y phases a e in luenza i us in ec ion,
as epo edbyZhaie al[23] (see Da ase 2 published on h ps://
www.b k. i/1234-2/). O he 25 op genes up egula ed du ing he
acu e phase o in luenza in ec ion [23], 23 o e lapped ou
en e o i us-induced signa u e. Also, all se en na u al kille
(NK) cell ac i a ion signa u e genes associa ed wi h he acu e
phase o in luenza in ec ion [23] peaked in mo e han one o he
en e o i us-posi i e child en. O e laps wi h he op up- and
down egula ed genes speci ic o he eco e y phase a e in lu-
enza i us in ec ion [23] we e low o all he child en.
We also compa ed ou esul s wi h en e o i us-induced
esponses in human PBMCs in ec ed in i o wi h h ee di e -
en en e o i uses [14]. The genes up egula ed in he in i o
in ec ions we e en iched wi h hose associa ed wi h he de ence
esponse o i us and he ype I in e e on signalling pa hway,
simila ly o he genes in ou en e o i us-induced signa u e. O
he genes up egula ed by any o he en e o i uses, 70% we e
p esen in a leas one o he peaking clus e s. O e all, 77 genes
p esen in he en e o i us-induced signa u e we e up egula ed
in all h ee in i o in ec ions (ESM Fig. 2a). O hese genes, 73
we e in e e on- egula ed based on he In e e ome da abase.
Al hough only app oxima ely 50% o he genes down egula ed
in he in i o in ec ions we e p esen in any o he d opping
clus e s o en e o i us-posi i e child en, genes associa ed wi h
ansla ion we e en iched in bo h da ase s.
As en e o i uses a e known o in ec panc ea ic isle s [5]
and ha e been ound in panc eases o indi iduals wi h ype 1
diabe es mo e o en han in non-diabe ic con ol g oups [24,
25], we also compa ed ou esul s wi h en e o i us-induced
esponses in human panc ea ic isle s in ec ed in i o wi h en-
e o i uses [10,26]. App oxima ely hal o he en e o i us-
induced genes in human panc ea ic isle s [10,26]we ealso
p esen in ou en e o i us-induced blood ansc ip omic signa-
u e in child en a isk o de eloping ype 1 diabe es (ESM
Fig. 2b), while he o e laps wi h he genes p esen in he
peaking clus e s o he o he h ee ype 1 diabe es isk child en
we e low (ESM Fig. 2b). In o al, he e we e 64 en e o i us-
induced genes common o he in i o in ec ed panc ea ic isle s
2314
2219
3425
2325
2508
1318
2572
2515
3844
4590
2360
1532
1258
2417
S ong 1
S ong 2
S ong 3
S ong 4
S ong 5
Weak 1
Weak 2
S ong 1
S ong 2
S ong 3
S ong 4
S ong 5
Weak 1
Weak 2
Clus e
cen oid alue
Clus e
cen oid alue
Clus e
cen oid alue
Clus e
cen oid alue
Clus e
cen oid alue
Clus e
cen oid alue
Clus e
cen oid alue
1
2
0
-1
-2
1
2
0
-1
-2
1
2
0
-1
-2
Time om EV+ (mon hs) Time om EV+ (mon hs)
Time om EV+ (mon hs) Time om EV+ (mon hs) Time om EV+ (mon hs)
Time om EV+ (mon hs) Time om EV+ (mon hs)
a
1
2
0
-1
-2
d
1
2
0
-1
-2
e
5
1
2
0
-1
-2
●
●
●
●
1
2
0
-1
-2
g
hi
S ong 1
S ong 2
S ong 3
S ong 4
S ong 5
Weak 1
Weak 2
S ong 1
S ong 2
S ong 3
S ong 4
S ong 5
Weak 1
Weak 2
b
0 10203040 0 5 10 15 20 25 30
0 5 10 15 20 25 0 10 15 -20 -15 -10 -5 0
-5 0 5 10 -10 0 10 20
c
Fig. 1 (a–g) A e age exp ession p o iles o clus e s peaking o d opping
a en e o i us posi i i y o he se en en e o i us-posi i e child en:
S ong 1 (a), S ong 2 (b), S ong 3 (c), S ong 4 (d), S ong 5 (e),
Weak 1 ( )andWeak2(g). Red, peaking clus e s; blue, d opping clus e s.
EV+, en e o i us-posi i e. (h,i) O e lapping p obe se s be ween he
peaking and d opping clus e s. The black a eas indica e he p opo ion
o o e lapping p obe se s ela i e o he child/clus e no ed a he op o
he column. The boxes highligh ed in he ou lined ame show he
peaking (h) and d opping (i) clus e s o he ou s ongly en e o i us-
posi i e child en wi h he mos simila en e o i us-associa ed changes.
The o al numbe s o p obe se s in each clus e a e p esen ed in he
diagonal. The i e child en wi h s ongly en e o i us-posi i e blood
samples a e deno ed as S ong 1–S ong 5. The wo child en wi h weakly
en e o i us-posi i e blood samples a e deno ed as Weak 1 and Weak 2
384 Diabe ologia (2018) 61:381–388
[10,26] and ou en e o i us-induced blood ansc ip omic sig-
na u e, all o which we e associa ed wi h an i i al in e e on
esponses, including IFIH1,IRF7,MX1,STAT1 and STAT2.
Finally, we es ed whe he he 339 en e o i us-induced sig-
na u e genes o he 77 genes also up egula ed in all h ee
in i o in ec ions o human PBMCs could di e en ia e be-
ween he en e o i us-posi i e and en e o i us-nega i e blood
samples in he ull ansc ip omics da ase by Kallionpää e al
[15]. The ou en e o i us-posi i e samples wi h clea signs o
in e e on esponse ac i a ion we e clea ly sepa a ed om all
o he samples using ei he o he gene se s, while he o he h ee
en e o i us-posi i e samples we e no sepa a ed om he
en e o i us-nega i e samples (Fig. 3a, ESM Fig. 3a). To ensu e
ha he obse ed sepa a ion was no due o he use o he same
ou en e o i us-posi i e child en in de ining he signa u e, a
simila analysis was pe o med using he genes up egula ed in
he in i o in ec ed PBMCs by any o he h ee en e o i uses.
He e also, he ou s ongly en e o i us-posi i e samples we e
6
8
10
12
−5
6
8
10
12
−10
6
8
10
12
6
8
10
12
6
8
10
12
6
8
10
12
−20 −10
0 10203040 0 5 10 01020 0 5 10 20 30
0 5 10 15 20 25 0 5 10 15 0
6
8
10
12
15 25
Time om EV+ (mon hs)
Log2 exp ession
Time om EV+
(
mon hs
)
Log2 exp ession
Time om EV+ (mon hs)
Log2 exp ession
Time om EV+ (mon hs)
Log2 exp ession
Time om EV+ (mon hs)
Log2 exp ession
Time om EV+
(
mon hs
)
Log2 exp ession
Time om EV+
(
mon hs
)
Log2 exp ession
bcde
gh
a
Nucleus
Cy oplasm
IFN IFN
R
NF-KB
BCL-2
BAX
BAK
IRF1
TAP1
IFITM3
IFITM1
IFITM2
IFITM1
IFITM2 IFIT3 IFI35
G1P3
IRF9
DRIP150 IRF9
PSMB8
MX1
G1P2
IFI35
IFIT1
OAS1
ISRE
Glucoco icoid
IRF9
PSMB8
p65
JAK1
TC-PTP
TC-PTP
STAT1
STAT1
STAT2
SOCS1
P
STAT1
PSTAT1
P
STAT2
P
P
STAT1
PIAS1
P
STAT1
PSTAT2
P
STAT1
P
PP
TC-PTP
JAK2 JAK1 JAK1
JAK2 TYK2
R
IFNA
R1
/
IFNA
R2
GAS
γ
IFN
γ
IFN
γ
IFN
γ
IFN
γ
αβ
RR
α
α
β
β
STAT1
Fig. 2 (a) Genes in peaking clus e s mapping o he in e e on signalling
pa hway based on he IPA ool. Red, genes p esen in a leas ou peaking
clus e s o he s ongly en e o i us-posi i e child en; pink, genes p esen
in a leas one peaking clus e . (b–h) Child-speci ic exp ession p o iles o
wo genes o he in e e on signalling pa hway, MX1 (g ey) and STAT2
(black). IFNγis also known as IFNG;IFNα/βis also known as IFNA1/
B1;TC-PTP is also known as PTPN2;NF-κB p65 is also known as
RELA;BCL-2 is also known as BCL2;BAK is also known as BAK1;
DRIP150 is also known as MED14;G1P2 is also known as ISG15;
G1P3 is also known as IFI6. EV+, en e o i us-posi i e; GAS, IFNG-
ac i a ed sequence; ISRE, in e e on-s imula ed egula o y elemen . The
i e child en wi h s ongly en e o i us-posi i e blood samples a e deno -
ed as S ong 1–S ong 5 (b– ). The wo child en wi h weakly en e o i us-
posi i e blood samples a e deno ed as Weak 1 and Weak 2 (g,h)
Diabe ologia (2018) 61:381–388 385

clea ly sepa a ed om he en e o i us-nega i e samples (ESM
Fig. 3b).
Up egula ion o in e e on- egula ed genes du ing en e o-
i us in ec ion is one o he conspicuous ea u es in his s udy,
and ac i a ion o in e e on signalling has been obse ed in
he blood o child en who ha e de eloped diabe es- ela ed
au oan ibodies o clinical ype 1 diabe es be o e he i s de-
ec ion o au oan ibodies [15,27]. Howe e , he exp ession o
he 339 en e o i us-induced signa u e genes (ESM Figs 3a, c
and 4) o he 77 genes also up egula ed wi h in i o en e o-
i us in ec ion (Fig. 3a, b) did no show ma ked di e ences
be ween au oan ibody-nega i e and au oan ibody-posi i e
child en be o e o a e se ocon e sion based on he longi u-
dinal blood ansc ip omics da a om child en a isk o ype
1 diabe es epo ed by Kallionpää e al [15] o Fe ei a e al
[27]. Mo eo e , ewe han 50% o he genes up egula ed in
child en who ha e de eloped diabe es- ela ed au oan ibodies
o clinical ype 1 diabe es in he wo a o emen ioned s udies
[15,27] o e lapped wi h he en e o i us-induced signa u e
(Fig. 3c, ESM Fig. 3c).
Discussion
In he cu en s udy, we ha e iden i ied en e o i us-associa ed
ansc ip omic p o iles in whole-blood samples om se en chil-
d en wi h gene ic suscep ibili y o ype 1 diabe es and
cha ac e ised hei indi idual esponses o en e o i us in ec ions.
In e e on esponse is a cen al pa o he inna e an i i al
immune esponse, and se e al en e o i us s ains induce a
p o ound in e e on esponse in human blood cells [14,28].
We de ec ed clea signs o in e e on esponse ac i a ion in
ou s ongly en e o i us-posi i e child en. En e o i us-
associa ed changes in hese child en esembled p e iously e-
po ed di e ences occu ing du ing he acu e phase o i us
in ec ion [23], indica ing ha hese samples we e collec ed
du ing he acu e phase o in ec ion cha ac e ised by high i us
load. In wo child en wi h only weakly en e o i us-posi i e
blood samples and one child wi h a s ongly en e o i us-
posi i e blood sample, no s ong signs o in e e on esponse
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
Aab−
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
Pos Se o
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
P eSe o
19
Ve i ied DIPP en e o i us
up egula ed genes
Kallionpää e al
up egula ed genes
Fe ei a e al IFN
g
enes
Sum o z sco es
150
100
50
0
-50
a
b
c
Aab−Pos Se oP eSe o
Sum o z sco es
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
1500
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●●
●
●
●●
●
●
●
●
●●
●
●
●
●●
●
●
●
●
●●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●●
●
●●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●●
●
●
●●
●
●●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●●
●
●
●
●
●
●
●
●●
●
●
●●
●
●
●
●
1000
500
0
3143
19
22
33
151

Fig. 3 Exp ession o he 77 genes p esen in ou en e o i us-induced
signa u e and up egula ed in all h ee in i o en e o i us in ec ions in
mic oa ay da a published by: (a) Kallionpää e al [15]; and (b) Fe ei a
e al [27]. Sums o child-speci ic zsco es o e he 77 genes we e
calcula ed o each o he 356 whole blood samples by Kallionpää e al
[15] (GEO: GSE30211) and he 454 PBMC samples by Fe ei a e al [27]
(A ay Exp ess: E-MTAB-1724), as desc ibed in Me hods, using he
published p e-p ocessed da ase s and sample in o ma ion based on
pe sonal communica ions wi h Fe ei a e al. All p obes (a)o he
highes -in ensi y exons mapping o genes (b) o e lapping wi h he 77
genes we e summed. (a) Black, s ongly en e o i us-posi i e blood
samples; whi e, weakly en e o i us-posi i e blood samples; g ey,
en e o i us-nega i e blood samples. (a,b) P eSe o, samples collec ed
om be o e se ocon e sion om child en wi h au oan ibody posi i i y
o ype 1 diabe es (in a,n=22;inb,n= 65); Pos Se o, samples collec ed
a e se ocon e sion om child en wi h au oan ibody posi i i y o ype 1
diabe es (in a,n=169;bn=84),Aab
−
, samples collec ed om
au oan ibody-nega i e child en (in a,n=165;inb,n=305).(c)Venn
diag am showing he o e laps be ween he 77 genes p esen in he
en e o i us-induced signa u e and up egula ed in all h ee in i o
en e o i us in ec ions; genes up egula ed be o e o a e se ocon e sion
in au oan ibody-posi i e child en based on he esul s by Kallionpää e al
[15] and he 225 in e e on-inducible genes de ec ed by Fe ei a e al [27]
386 Diabe ologia (2018) 61:381–388
we e de ec ed, bu changes implying he ac i a ion o adap i e
immune esponses we e obse ed. En e o i us-associa ed
down egula ion o ansc ip ion, ansla ion o mRNA
p ocessing-associa ed genes was obse ed in six child en, al-
hough he indi idual p obes and genes mapping o hese ca -
ego ies a ied be ween indi iduals. Up egula ion o in e e on
esponse genes and down egula ion o ansla ion-associa ed
genes we e also de ec ed in human PBMCs in ec ed in i o
wi h h ee di e en en e o i uses. Finally, up egula ion o
genes associa ed wi h in e e on esponses was he common
ea u e be ween en e o i us-induced blood ansc ip omic
changes in ou child en a isk o de eloping ype 1 diabe es
and in i o en e o i us-in ec ed human panc ea ic isle s [10,
26], c ea ing a link be ween he i us–hos in e play in blood
and in he panc eas.
We buil an en e o i us-induced signa u e co e ing 339
genes p esen in he peaking clus e s o he ou child en wi h
clea indica ions o in e e on esponse ac i a ion, and a mo e
selec i e signa u e o 77 genes addi ionally up egula ed in
human PBMCs in ec ed in i o wi h h ee di e en en e o i-
uses. Bo h signa u es sepa a ed he ou s ongly en e o i us-
posi i e samples om he o he samples in he ull mic oa ay
da ase published by Kallionpää e al [15].
The en e o i us-associa ed signa u e showed only mode -
a e o e lap wi h he up egula ed genes in Kallionpää e al [15]
and Fe ei a e al [27], and could no di e en ia e be ween
child en who de eloped ype 1 diabe es au oan ibodies o
clinical ype 1 diabe es and au oan ibody-nega i e child en
in hose s udies (ESM Fig. 3). Al hough ac i a ion o in e e -
on signalling has been shown o p ecede he de elopmen o
au oimmuni y in child en a isk o ype 1 diabe es, ou esul s
indica e di e ences be ween en e o i us-associa ed and ype
1 diabe es-associa ed in e e on signals.
The ou child en wi h clea signs o in e e on esponse
ac i a ion included wo pe sis en ly au oan ibody-nega i e
child en, one child who la e became posi i e o mul iple ype
1 diabe es au oan ibodies and one au oan ibody-posi i e child
who la e de eloped clinical ype 1 diabe es. Wi h he limi ed
numbe o child en a ailable o he cu en s udy, and he
signi ican amoun o he e ogenei y in en e o i us-associa ed
changes be ween he child en, i is no possible o d aw con-
clusions ega ding connec ions be ween en e o i us in ec ions
and ype 1 diabe es.
The e a e se e al ac o s ha can explain he obse ed
he e ogenei y in he en e o i us-associa ed esponses. Fi s ,
ea lie in i o s udies ha e shown ha he magni ude o
in e e on esponse induc ion in PBMCs a ies signi ican ly
be ween di e en en e o i uses [14]. Second, he apid kine -
ics o an i i al immune esponses can be a sou ce o signi i-
can he e ogenei y when cha ac e ising en e o i us-associa ed
blood ansc ip omic changes in ollow-up s udies wi h long
sampling in e als, al hough en e o i us RNA can be de ec ed
in blood o only a ew days du ing he acu e phase o
in ec ion. Thi d, hos esponses o acu e in ec ions caused by
di e en i uses can be simila , and some imes he di e -
gences be ween i uses a e be e explained by he di e en
magni udes o he e ec han by he ac ual genes esponding
o in ec ion [23,29]. Al hough ou en e o i us-induced signa-
u e has a high o e lap wi h in i o en e o i us-induced
changes in human PBMCs and panc ea ic isle s, we canno
conclude ha hese changes a e uniquely obse ed a e in ec-
ion wi h en e o i uses. Finally, al hough en e o i us in ec-
ions a e o en asymp oma ic, clinical symp oms we e epo -
ed o i e o he se en child en less han a week be o e he
collec ion o he en e o i us-posi i e blood samples. Th ee
child en we e su e ing om e e a ound he ime o
en e o i us-posi i e sample collec ion, including one child
also su e ing om conjunc i i is, and common cold-like
symp oms we e epo ed o wo child en. In e es ingly, he
h ee child en wi h e e a ound he ime o en e o i us-
posi i e sample collec ion we e s ongly en e o i us-posi i e
based on quan i a i e RT-PCR and had clea signs o in e e -
on esponse ac i a ion associa ed wi h he en e o i us-
posi i e blood sample.
Despi e he limi a ions o he cu en s udy, i p o ides a
s a ing poin o unde s anding he indi idual esponses o
en e o i us in ec ions in i o, and how hese esponses a e
e lec ed in he mRNA exp ession p o iles in whole blood.
Fu he longi udinal s udies wi h la ge coho s, sho e sam-
pling in e als and be e knowledge o he ac ual i us s ains
in ec ing he indi iduals will p o ide deepe insigh s in o he
associa ions be ween en e o i us in ec ions and ype 1
diabe es.
Acknowledgemen s The au ho s a e g a e ul o he DIPP amilies o
hei pa icipa ion and he s a o he DIPP s udy o wo king wi h he
amilies and ob aining he samples o he s udy. We hank O. Simell, he
hono a y p incipal in es iga o o he DIPP s udy, o his wo k. We also
hank R.C. Fe ei a (Uni e si y o Camb idge, Camb idge, UK) and
co-au ho s o sha ing addi ional me ada a o hei samples (E-MTAB-
1724 [27]) wi h us.
Da a a ailabili y The da ase s analysed du ing he cu en s udy a e
included in his published a icle and i s supplemen a y in o ma ion iles
(www.b k. i/ esea ch/compu a ional-biomedicine/1234-2)o a e
a ailable om he GEO eposi o y (accession GSE30211).
Funding This wo k was inancially suppo ed by he Eu opean
Resea ch Council (ERC) (decision numbe 677943), JDRF (g an s 17-
2013-533 and 2-2013-32), he Academy o Finland (Cen e o Excellence
in Molecula Sys ems Immunology and Physiology Resea ch, 2012–
2017, decision numbe 250114 and g an s 287423, 288671, 292482,
292335, 294337, 296801 and 304995), he Eu opean Union’s Ho izon
2020 esea ch and inno a ion p og amme (decision numbe 675395),
Tekes, he Finnish Funding Agency o Inno a ion (1877/31/2016), he
Sig id Jusélius Founda ion, he Y jö Johansson Founda ion, he Finnish
Diabe es Resea ch Founda ion, he Reino Lah ika i Founda ion, he
Eu opean Commission (Pe sis en Vi us In ec ion in Diabe es Ne wo k
Diabe ologia (2018) 61:381–388 387
[PEVNET] F ame P og amme 7, con ac numbe 261441) and he Paulo
Founda ion.
Duali y o in e es The au ho s decla e ha he e is no duali y o in e -
es associa ed wi h his manusc ip .
Con ibu ion s a emen LTTA and LLE planned he da a analyses.
LTTA was esponsible o analysing he da a and pa icipa ed in w i ing
he manusc ip and p epa ing he igu es. NL pa icipa ed in planning he
da a analyses, in e p e ed he esul s and pa icipa ed in w i ing he man-
usc ip and p epa ing he igu es. HK pa icipa ed in planning he da a
analyses and in e p e ing he esul s. MKJ pa icipa ed in analysing he
da a and p epa ing he igu es. JM, JT, JI, MK and RV p o ided and
in e p e ed he clinical in o ma ion o he s udy child en. HH and SO
we e esponsible o he i us analysis wi hin he s udy, p o ided he
in i o in ec ion da a and con ibu ed o he ini ia ion and design o he
s udy. RL and LLE ini ia ed and designed he s udy, supe ised he s udy
and pa icipa ed in in e p e a ion o he esul s and w i ing he manusc ip .
All au ho s edi ed/ e ised and app o ed he inal e sion o he manu-
sc ip . LLE is he gua an o o his wo k.
Open Access This a icle is dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion 4.0 In e na ional License (h p://
c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use,
dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app o-
p ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he
C ea i e Commons license, and indica e i changes we e made.
Re e ences
1. Yeung WC, Rawlinson WD, C aig ME (2011) En e o i us in ec-
ion and ype 1 diabe es melli us: sys ema ic e iew and me a-
analysis o obse a ional molecula s udies. BMJ 342:d35
2. Lai inen OH, Honkanen H, Pakkanen O e al (2014) Coxsackie i us
B1 is associa ed wi h induc ion o be a-cell au oimmuni y ha po -
ends ype 1 diabe es. Diabe es 63:446–455
3. Beye lein A, Donnachie E, Je gens S, Ziegle AG (2016) In ec ions in
ea ly li e and de elopmen o ype 1 diabe es. JAMA 315:1899–1901
4. Hyo y H (2016) Vi uses in ype 1 diabe es. Pedia Diabe es
17(Suppl 22):56–64
5. Ylipaas o P, Klingel K, Lindbe g AM e al (2004) En e o i us in-
ec ion in human panc ea ic isle cells, isle opism in i o and
ecep o in ol emen in cul u ed isle be a cells. Diabe ologia 47:
225–239
6. K og old L, Edwin B, Buanes T e al (2015) De ec ion o a low-
g ade en e o i al in ec ion in he isle s o Lange hans o li ing pa-
ien s newly diagnosed wi h ype 1 diabe es. Diabe es 64:1682–1687
7. Honkanen H, Oika inen S, Nu minen N e al (2017) De ec ion o
en e o i uses in s ools p ecedes isle au oimmuni y by se e al
mon hs: Possible e idence o slowly ope a ing mechanisms in
i us-induced au oimmuni y. Diabe ologia 60:424–431
8. San in I, Eizi ik DL (2013) Candida e genes o ype 1 diabe es
modula e panc ea ic isle in lamma ion and be a-cell apop osis.
Diabe es Obes Me ab 15(Suppl 3):71–81
9. Ash on MP, Eugs e A, Wal he D e al (2016) Incomple e immune
esponse o coxsackie B i uses associa es wi h ea ly au oimmuni y
agains insulin. Sci Rep 6:32899
10. Domsgen E, Lind K, Kong L e al (2016) An IFIH1 gene polymo -
phism associa ed wi h isk o au oimmuni y egula es canonical
an i i al de ence pa hways in coxsackie i us in ec ed human pan-
c ea ic isle s. Sci Rep 6:39378
11. Schul e BM, Gielen PR, Ke s-Rebel ED e al (2016) En e o i us
exposu e uniquely disc imina es ype 1 diabe es pa ien s wi h a
homozygous om a he e ozygous melanoma di e en ia ion-
associa ed p o ein 5/in e e on induced wi h helicase C domain 1
A946T geno ype. Vi al Immunol 29:389–397
12. Oika inen S, Ma iskainen M, Tau iainen S e al (2011) En e o i us
RNA in blood is linked o he de elopmen o ype 1 diabe es.
Diabe es 60:276–279
13. Oika inen S, Tau iainen S, Hobe D e al (2014) Vi us an ibody
su ey in di e en eu opean popula ions indica es isk associa ion
be ween coxsackie i us B1 and ype 1 diabe es. Diabe es 63:655–
662
14. Hamalainen S, Nu minen N, Ahl o s H e al (2014) Coxsackie i us
B1 e eals s ain speci ic di e ences in plasmacy oid dend i ic cell
media ed immunogenici y. J Med Vi ol 86:1412–1420
15. Kallionpää H, Elo LL, Laajala E e al (2014) Inna e immune ac i i y
is de ec ed p io o se ocon e sion in child en wi h HLA-con e ed
ype 1 diabe es suscep ibili y. Diabe es 63:2402–2414
16. Kupila A, Muona P, Simell T e al (2001) Feasibili y o gene ic and
immunological p edic ion o ype I diabe es in a popula ion-based
bi h coho . Diabe ologia 44:290–297
17. Honkanen H, Oika inen S, Pakkanen O e al (2013) Human en e o-
i us 71 s ains in he backg ound popula ion and in hospi al pa-
ien s in inland. J Clin Vi ol 56:348–353
18. Piccolo SR, Wi he s MR, F ancis OE, Bild AH, Johnson WE
(2013) Mul ipla o m single-sample es ima es o ansc ip ional ac-
i a ion. P oc Na l Acad Sci U S A 110:17778–17783
19. Huang da W, She man BT, Lempicki RA (2009) Sys ema ic and
in eg a i e analysis o la ge gene lis s using DAVID bioin o ma ics
esou ces. Na P o oc 4:44–57
20. Rusino a I, Fo s e S, Yu S e al (2013) In e e ome 2.0: An up-
da ed da abase o anno a ed in e e on- egula ed genes. Nucleic
Acids Res 41:D1040–D1046
21. Du P, Kibbe WA, Lin SM (2008) Lumi: a pipeline o p ocessing
illumina mic oa ay. Bioin o ma ics 24:1547–1548
22. Suomi T, Seyednas ollah F, Jaakkola MK, Faux T, Elo LL (2017)
ROTS: an R package o ep oducibili y-op imized s a is ical es -
ing. PLoS Compu Biol 13:e1005562
23. Zhai Y, F anco LM, A ma RL e al (2015) Hos ansc ip ional
esponse o in luenza and o he acu e espi a o y i al in ec-
ions—a p ospec i e coho s udy. PLoS Pa hog 11:e1004869
24. Richa dson SJ, Willcox A, Bone AJ, Foulis AK, Mo gan NG
(2009) The p e alence o en e o i al capsid p o ein p1 immuno-
s aining in panc ea ic isle s in human ype 1 diabe es. Diabe ologia
52:1143–1151
25. Richa dson SJ, Lee e P, Bone AJ, Foulis AK, Mo gan NG (2013)
Exp ession o he en e o i al capsid p o ein VP1 in he isle cells o
pa ien s wi h ype 1 diabe es is associa ed wi h induc ion o p o ein
kinase R and down egula ion o mcl-1. Diabe ologia 56:185–193
26. Ylipaas o P, Ku lu B, Rasilainen S e al (2005) Global p o iling o
coxsackie i us- and cy okine-induced gene exp ession in human
panc ea ic isle s. Diabe ologia 48:1510–1522
27. Fe ei a RC, Guo H, Coulson RM e al (2014) A ype I in e e on
ansc ip ional signa u e p ecedes au oimmuni y in child en gene -
ically a isk o ype 1 diabe es. Diabe es 63:2538–2550
28. Alidjinou EK, Sane F, Engelmann I, Hobe D (2013) Se um-
dependen enhancemen o coxsackie i us B4-induced p oduc ion
o IFNalpha, IL-6 and TNFalpha by pe iphe al blood mononuclea
cells. J Mol Biol 425:5020–5031
29. Hu X, Yu J, C osby SD, S o ch GA (2013) Gene exp ession p o iles
in eb ile child en wi h de ined i al and bac e ial in ec ion. P oc
Na l Acad Sci U S A 110:12792–12797
388 Diabe ologia (2018) 61:381–388