RESEARCH Open Access
An in e ac ion map o ci cula ing
me aboli es, immune gene ne wo ks, and
hei gene ic egula ion
A ika P. Na h
1,2
, Sco C. Ri chie
2,3
, Sean G. Bya s
3,4
, Liam G. Fea nley
3,4
, Aki S. Ha ulinna
5,6
, Anni Joensuu
5
,
An i J. Kangas
7
, Pasi Soininen
7,8
, Annika Wenne s öm
5
, Lili Milani
9
, And es Me spalu
9
, Sa u Männis ö
5
,
Pe e Wü z
7,10
, Johannes Ke unen
5,7,8,11
, Emma Rai oha ju
12
, Mika Kähönen
13
, Ma kus Juonala
14,15
,
Aa no Palo ie
6,16,17,18
, Mika Ala-Ko pela
7,8,11,19,20
, Samuli Ripa i
6,21
, Te ho Leh imäki
12
, Gad Ab aham
2,3,4
,
Olli Rai aka i
22,23
, Veikko Salomaa
5
, Ma kus Pe ola
5,6,9
and Michael Inouye
1,2,3,4*
Abs ac
Backg ound: Immunome abolism plays a cen al ole in many ca diome abolic diseases. Howe e , a obus map o
immune- ela ed gene ne wo ks in ci cula ing human cells, hei in e ac ions wi h me aboli es, and hei gene ic con ol
is s ill lacking. He e, we in eg a e blood ansc ip omic, me abolomic, and genomic p o iles om wo popula ion-based
coho s ( o al N = 2168), including a subse o indi iduals wi h ma ched mul i-omic da a a 7-yea ollow-up.
Resul s: We iden i y opologically eplicable gene ne wo ks en iched o di e seimmune unc ions including cy o oxici y,
i al esponse, B cell, pla ele , neu ophil, and mas cell/basophil ac i i y. These immune gene modules show complex
pa e ns o associa ion wi h 158 ci cula ing me aboli es, including lipop o ein subclasses, lipids, a y acids, amino acids,
small molecules, and CRP. Genome-wide scans o module exp ession quan i a i e ai loci (mQTLs) e eal i e modules
wi h mQTLs ha ha e bo h cis and ans e ec s. The s onges mQTL is in ARHGEF3 ( s1354034) and a ec s a module
en iched o pla ele unc ion, independen o pla ele coun s. Modules o mas cell/basophil and neu ophil unc ion
show empo ally s able me aboli e associa ions o e 7-yea ollow-up, p o iding e idence ha hese modules and hei
cons i uen gene p oduc s may play cen al oles in me abolic in lamma ion. Fu he mo e, he s onges mQTL in
ARHGEF3 also displays clea empo al s abili y, suppo ing widesp ead ans e ec s a his locus.
Conclusions: This s udy p o ides a de ailed map o na u al a ia ion a he blood immunome abolic in e ace and i s
gene ic basis, and may acili a e subsequen s udies o explain in e -indi idual a ia ion in ca diome abolic disease.
Backg ound
O e he pas decade inc easing e idence has implica ed
in lamma ion as a p obable causal ac o in me abolic and
ca dio ascula diseases. Consequen ly, esea ch has begun
o ocus on he in e play be ween immuni y and me abol-
ism, o immunome abolism. While i is in ol ed in di e se
pa hophysiologies, immunome abolism is pa icula ly
ele an o diseases o immense global heal h bu den, such
as ype 2 diabe es (T2D) and a he oscle osis.
Fo T2D, immune o e ac i a ion in adipose issue has
been implica ed as a key d i e [1, 2]. S udies ha e
shown ha mac ophage in il a ion and subsequen
o e exp ession o p oin lamma o y cy okines, such as
TNF-α, in adipose issues is associa ed wi h insulin e-
sis ance [1, 2]. Mo eo e , e idence o me abolic in lam-
ma ion has been shown in o he issues whe e, in blood,
ele a ed glucose and ee a y acid le els po en ia e IL-
1β-media ed des uc ion o panc ea ic ß cells and subse-
quen T2D p og ession [3–5]. While ci cula ing me abo-
li es a e known o be associa ed wi h ca dio ascula
disease [6], in lamma ion is an inc easingly ecognized
ac o in pa hogenesis. In a he oscle osis, lipid-induced
in lamma o y esponse mechanisms ha e also been
* Co espondence: minouye@bake .edu.au
1
Depa men o Mic obiology and Immunology, The Uni e si y o Melbou ne,
Pa k ille 3010, Vic o ia, Aus alia
2
Sys ems Genomics Lab, Bake Hea and Diabe es Ins i u e, Melbou ne,
Vic o ia, Aus alia
Full lis o au ho in o ma ion is a ailable a he end o he a icle
© The Au ho (s). 2017 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
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he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e
(h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed.
Na h e al. Genome Biology (2017) 18:146
DOI 10.1186/s13059-017-1279-y
implica ed in p og ession o myoca dial in a c ion [7]. In
a he ogenic lesions, oxidized phospholipids a e known
o lead o a new mac ophage pheno ype [8], and choles-
e ol loading in mac ophages p omo es p oin lamma o y
cy okine sec e ion [9].
Pe haps su p isingly, ew la ge-scale s udies ha e sys-
ema ically assessed in e ac ions be ween he human im-
mune sys em and me aboli es. Recen s udies ha e
in es iga ed ma ched blood ansc ip omic and me abo-
lomic p o iles o unde s and hei in e play [10–16].
Howe e , hese s udies had modes sample sizes and
hus ha e no had he powe o ocus on he di e se
ange o immune p ocesses ha in e ac wi h ci cula ing
me aboli es. Fu he mo e, e en ewe ha e assessed e -
ec s o exp ession quan i a i e ai loci (eQTLs) on
immune gene ne wo ks. A obus in eg a ed map o
immunome abolic ela ionships and hei gene ic egula-
ion would p o ide a ounda ion o in es iga ing he di -
e en ial ca diome abolic disease suscep ibili y amongs
indi iduals while also iden i ying key a ge in e ac ions
o mechanis ic in i o and in i o ollow-up.
In his s udy, we p esen an in eg a ed immunome a-
bolic map using ma ched blood me abolomic and an-
sc ip omic p o iles om 2168 indi iduals om wo
popula ion-based coho s. We pe o m gene coexp es-
sion ne wo k disco e y and c oss-coho eplica ion o
iden i y obus gene modules which encode immune-
ela ed unc ions. Using a high- h oughpu quan i a i e
NMR me abolomics pla o m ha can sepa a e lipids
and lipop o ein sub- ac ions as well as quan i y a panel
o pola me aboli es, we iden i y signi ican in e ac ions
be ween immune gene modules and ci cula ing me abol-
i e measu es. Genome-wide scans o QTLs a ec ing im-
mune gene modules iden i y many cis and ans loci
a ec ing module exp ession. Finally, we es he long-
e m s abili y o gene modules, hei in e ac ions wi h
me aboli e measu es, and gene ic con ol using a 7-yea
ollow-up sampling o 333 indi iduals.
Resul s and discussion
Summa y o coho s and da a
We analyzed genome-wide geno ype, whole blood an-
sc ip omic, and se um me abolomics da a om wo
popula ion-based coho s (“Me hods”and Fig. 1). In
DILGOM07, 240 males and 278 emales aged 25–74
yea s we e ec ui ed ( o al N = 518). Da a we e a ailable
o a subse o 333 pa icipan s om DILGOM07 who
we e ollowed up a e 7 yea s (DILGOM14). In YFS,
ele an da a we e a ailable o 755 males and 895 e-
males aged 34–49 yea s ( o al N = 1650).
DILGOM and YFS geno yping was pe o med using
Illumina Human 610 and 670 a ays, espec i ely, wi h
subsequen geno ype impu a ion pe o med using IM-
PUTE2 [17] and he 1000 Genomes Phase I e sion 3
e e ence panel. Fo bo h coho s, whole blood an-
sc ip ome p o iling was pe o med using Illumina HT-12
a ays and se um me abolomics p o iling was ca ied
ou using he same se um NMR me abolomics pla o m
(B ainshake L d) [18]. Indi iduals on lipid-lowe ing
medica ion and p egnan women we e excluded om
he me abolome analyses (“Me hods”). O he 159 me-
aboli e measu es analyzed, 148 we e di ec ly quan i ied
and 11 de i ed (Addi ional ile 1: Table S1). A e il e -
ing, ma ched ansc ip ome and me abolome da a we e
a ailable o 440 indi iduals in DILGOM07 and 216 o
hese indi iduals (DILGOM14) who we e p o iled a 7-
yea ollow-up. In YFS, 1575 indi iduals we e a ailable
wi h simila da a (see “Me hods” o de ails).
Robus immune gene coexp ession ne wo ks om blood
We i s iden i ied ne wo ks o igh ly coexp essing
genes in DILGOM07 and hen used a pe mu a ion ap-
p oach, Ne Rep [19], o s a is ically es eplica ion pa -
e ns o densi y and connec i i y o hese ne wo ks in
YFS. Fo module de ec ion, we applied weigh ed gene
coexp ession ne wo k analysis (WGCNA) o all 35,422
p obes in he DILGOM07 da a, iden i ying a o al o 40
modules o coexp essed genes (“Me hods”). Fo each
module, we used Ne Rep o calcula e se en p ese a ion
s a is ics in he YFS, gene a e empi ical null dis ibu ions
o each o hese es s a is ics, and calcula e hei co e-
sponding P alues [19, 20]. A module was conside ed
s ongly p ese ed i he P alue was <0.001 o all se en
p ese a ion s a is ics (Bon e oni co ec ion o 40
modules). O he 40 DILGOM07 modules, 20 we e
s ongly p ese ed in YFS (Addi ional ile 2: Table S2).
Fo each o he 20 eplica ed modules, we de ined co e
gene p obes, hose which a e mos igh ly coexp essed
YFS DILGOM07 DILGOM14
S udy Popula ions
Molecula Da a
Ma ched T ansc ip ome-
Me abolome
Ma ched T ansc ip ome-
Geno ypes
N=333
N=258
N=294
Me abolome T ansc ip ome
Blood Samples
Geno ypes
Disco e Gene Modules
in DILGOM07
Ne wo k analysis
Replica e Gene Modules
in YFS and DILGOM14
SNPs
N=518
N=440
N=515
N=1650
N=1575
N=1400
Pe mu a ion
s a is ics
GO en ichmen
analysis
Immune-linked gene ne wo ks
Immune-me aboli e
associa ion
Gene ic d i e s o
immune modules
Analysis S a egy
T ansc ip ome
Fig. 1 The s udy design. GO Gene On ology, SNP single nucleo ide
polymo phism
Na h e al. Genome Biology (2017) 18:146 Page 2 o 15
and hus obus o clus e ing pa ame e s, using a
pe mu a ion es o module membe ship (“Me hods”;
Addi ional ile 3: Table S3).
To iden i y modules o pu a i e immune unc ion, we
ca ied ou Gene On ology (GO) biological p ocess en-
ichmen analysis using GO illa o he co e genes o
each eplica ed module [21]. Signi ican GO e ms ( alse
disco e y a e (FDR) <0.05) we e hen summa ized in o
ep esen a i e e ms based on seman ic simila i y using
REVIGO [22] (Addi ional ile 4: Figu e S1). A module
was conside ed immune- ela ed i i was signi ican ly
en iched o GO e ms “immune sys em p ocesses”
(GO:0002376) and/o “ egula ion o immune sys em
p ocesses”(GO:0002682) in he REVIGO ou pu . Six ou
o 20 modules we e en iched o a leas one o hese
e ms (Addi ional ile 5: Table S4). We also iden i ied
wo addi ional modules which we e no en iched o any
GO e ms bu ha e been p e iously linked o immune
unc ions ela ed o mas cell and basophil unc ion [13]
and pla ele agg ega ion ac i i y [23]. The eigh modules
encoded di e se immune unc ions, including cy o oxic,
i al esponse, B cell, pla ele , neu ophil, mas cell/baso-
phil, and gene al immune- ela ed unc ions. Each
immune module’s gene con en and pu a i e biological
unc ion is summa ized in Table 1.
Immune module associa ion analysis o eQTLs and
me aboli e measu es
Fo each gene module, we pe o med a genome-wide
scan o iden i y module QTLs (mQTLs) ha egula e ex-
p ession. In DILGOM07 and YFS, he module eigengene
was eg essed on each SNP, hen mQTL es s a is ics
we e combined in a me a-analysis (“Me hods”). Signi i-
can mQTLs we e u he examined a indi idual gene
exp ession le els. A genome-wide signi icance le el (P
alue <5 × 10
−8
) was used o iden i y mQTLs and signi i-
can ans e ec s on indi idual gene exp ession (Fig. 2
and Table 2). Leukocy e and pla ele coun s we e a ail-
able o YFS and we e used o es he obus ness o
module associa ions wi h mQTLs and me aboli e mea-
su es. Six modules showed s a is ically signi ican associ-
a ion wi h pla ele o leukocy e coun s (P alue <0.05)
(Addi ional ile 6: Table S5); howe e , adjus men o
leukocy e coun s did no a ec mQTL no module-
me aboli e measu e associa ions, wi h he excep ion o
he pla ele module (PM) and cy o oxic cell-like module
Table 1 Immune module gene con en and pu a i e biological unc ion based on GO e ms ( op h ee shown) and li e a u e
Module Size GO e ms Li e a u e-based immune- ela ed unc ion o genes
Cy o oxic cell-like
module (CCLM)
130
(115)
Immune sys em p ocess
De ense esponse
Immune esponse
Cy o oxic e ec o s (GZMA,GZMB,GZMM,CTSW,PRF1 [66]); su ace ecep o s
(IL2RB,SLAMF6,CD8A,CD8B,CD2,CD247,KLRD1,KLRG1 [66–68]); T and NK cell
di e en ia ion (ID2 and EOMES [69,70]), ac i a ion (ZAP70 and CBLB [71,72]),
and ec ui men (CX3CR1,CCL5,CCL4L2 [73])
Vi al esponse
module (VRM)
95
(88)
Response o i us
Type I in e e on signaling pa hway
Response o bio ic s imulus
Type I in e e on-induced an i i al ac i i y (IFITM1,IFIT1,IFIT2,IFIT3,IFIT5,IFI44,
IFI44L,IFI6,MX1,ISG15,ISG20,HERC5 [74,75]); i al RNA deg ada ion (OAS1,
OAS2,OAS3,OASL,DDX60 [30]); ype 1 in e e on-signaling pa hway (IRF9,
STAT1,STAT2 [76,77])
B-cell ac i i y module
(BCM)
54
(49)
Immune sys em p ocess
Immune esponse
B cell ac i a ion
B cell su ace ma ke s (CD79A,CD79B,CD22 [33,78]); B cell ac i a ion (BANK1,
BTLA,CD40,TNFRSF13B,TNFRSF13C [79]), de elopmen (POU2AF1,BCL11A,
RASGRP3 [80]), mig a ion (CXCR5,CCR6 [80,81]), and hei egula ion (CD83,
FCER2,FCRL5 [82]); an igen p esen a ion (HLA-DOA,HLA-DOB [83])
Pla ele module
(PM)
a
114
(106)
Coagula ion
Blood coagula ion
Cell ac i a ion
Pla ele ecep o signaling, ac i a ion, and coagula ion (GP6,GP9,ITGA2B,
ITGB3,ITGB5,MGLL,MPL,MMRN1,PTK2,VCL,THBS1,F13A1,VWF,[84]);
egula ing pla ele ac i i y (SEPT5,TSPAN9 [85,86])
Neu ophil module
(NM)
a
26
(26)
Killing o cells o o he o ganism
Cell killing
Response o ungus
An i-mic obial, - ungal, and - i al ac i i y (DEFA1,DEFA1B,DEFA3,DEFA4,
ELANE,BPI,RNASE2,RNASE3 [87–90]); neu ophil-media ed ac i i y (AZU1,
LCN2,MPO,CEACAM6,CEACAM8,OLFM4 [90,91]) and i s egula ion (LCN2,
CAMP,OLR1 [49,92,93])
Lipid-leukocy e
module (LLM)
a
13
(13)
Mas cell and basophil unc ion
b
Mas cell and basophil ela ed immune esponse and alle gic in lamma ion
(FCER1A,HDC,GATA2,SLC45A3,CPA3,MS4A3 [13,94,95])
Gene al immune
module A (GIMA)
509
(482)
Immune sys em p ocess
De ense esponse
Regula ion o esponse o s imulus
These modules con ain genes in ol ed in a b oad ange o immune
p ocesses and hei egula ion such as signaling; cell dea h; de ense esponse
o s ess, in lamma ion, and ex e nal s imuli; leukocy e ac i a ion, mig a ion,
and adhesion
Gene al immune
module B (GIMB)
74
(69)
Immune esponse-ac i a ing signal
ansduc ion
Posi i e egula ion o immune esponse
Ac i a ion o immune esponse
Size e e s o he numbe o co e genes in each module and he subse o hese co e genes wi h GO e m anno a ions a e lis ed in pa en heses. Func ions we e
assigned o each o hese modules based on GO en ichmen s and li e a u e-based sea ches o genes in he modules.
a
Modules p e iously epo ed o ha e immune ela ed unc ion
b
The LLM was no signi ican ly en iched o any GO e m
Na h e al. Genome Biology (2017) 18:146 Page 3 o 15
(CCLM) discussed below (Addi ional ile 7: Table S6).
Since we did no ha e cell coun s a ailable o DIL-
GOM07, all he associa ions be ween immune modules
and me aboli e measu es discussed below, unless o he -
wise no ed, ha e no been adjus ed o cell coun s.
Cy o oxic cell-like module
CCLM was associa ed wi h 24 me aboli e measu es,
mainly consis ing o a y acids, in e media e densi y
lipop o eins, and C- eac i e p o ein (CRP; Fig. 3;
Addi ional ile 8: Table S7). O hese, he a e age deg ee
Fig. 2 Module and exp ession QTL analysis. aManha an plo o me a-analyzed P alues om he DILGOM/YFS module QTL analysis. The lead
SNP and i s closes genes a e no ed. Each signi ican mQTL locus is colo ed by module. The ho izon al dashed line ep esen s genome-wide (me a-
P alue <5 × 10
−8
) signi icance. b–dCi cula plo s summa izing he indi idual gene associa ions (me a-P alue <5 × 10
−8
) o he lead module
QTLs in he VRM, PM, and NM. Lead SNPs and cis genes a e labeled ou side he ing. PM pla ele module, VRM i al esponse module, CCLM
cy o oxic cell-like module, NM neu ophil module, BCM B-cell ac i i y module
Table 2 QTLs o immune gene modules
Module Top SNP Ch Hg19 pos.
(Mb)
Allele
(mino /majo )
MAF
(a g)
P alue
DILGOM07
(e ec size)
P alue
YFS
(e ec size)
Me a
P alue
VRM s182710579 4 19768086 G/T 0.012 2.01 × 10
–4
(0.05) 8.10 × 10
–6
(0.02) 9.23 × 10
–9
s151234502 7 148950168 T/C 0.012 2.59 × 10
–1
(0.01) 5.31 × 10
–9
(0.03) 2.46 × 10
–8
s147742798 11 70947761 T/C 0.016 1.51 × 10
–3
(0.04) 1.66 × 10
–6
(0.02) 9.43 × 10
–9
BCM s2523489 6 31348878 T/C 0.186 1.42 × 10
–1
(0.005) 5.29 × 10
–8
(0.006) 6.27 × 10
–8
PM s1354034 3 56849749 T/C 0.284 7.11 × 10
–14
(-0.02) 1.51 × 10
–16
(-0.008) 7.35 × 10
–28
s28367734 6 3128657 A/G 0.108 5.40 × 10
–4
(0.02) 2.02 × 10
–5
(0.006) 5.44 × 10
–8
NM s2485364 6 159512260 C/T 0.466 1.78 × 10
–3
(0.009) 6.05 × 10
–7
(0.004) 3.93 × 10
–9
s13297295 9 131659724 C/T 0.085 4.26 × 10
–2
(0.009) 8.39 × 10
–11
(0.01) 3.93 × 10
–11
s140929198 20 38555870 A/G 0.031 2.98 × 10
–2
(0.03) 8.47 × 10
–9
(0.01) 1.41 × 10
–9
GIMA s2185366 8 131342722 T/C 0.421 2.0 × 10
–2
(0.007) 1.52 × 10
–6
(0.004) 1.05 × 10
–7
Modules: VRM i al esponse module, BCM B-cell ac i i y module, PM pla ele module, NM neu ophil module, GIMA gene al immune module A. MAF mino allele equency
Na h e al. Genome Biology (2017) 18:146 Page 4 o 15
o unsa u a ion in a y acids was he mos signi ican
associa ion (me a-P alue = 7.23 × 10
–7
). The immuno-
modula o y e ec s o polyunsa u a ed a y acids a e
well cha ac e ized; o example, omega-3 a y acids
ha e been shown o induce cy o oxici y in in i o can-
ce cell lines as well as animal models o umo inci-
dence and g ow h [24, 25]. Adjus men o he
associa ions be ween CCLM and me aboli e measu es
o leukocy e coun s esul ed in he gain o 38 add-
i ional associa ions and loss o ou (c ea inine, a io o
polyunsa u a ed a y acids o o al a y acids, e y low
densi y lipop o ein (VLDL) pa icle size, and CRP)
exis ing associa ions (Addi ional ile 7: Table S6). Va y-
ing p opo ions o leukocy e coun s can be co ela ed
wi h ansc ip ion-le el a ia ion in human blood [26]
bu no ac o con ound he la e 's associa ion wi h
pheno ypes. I his is he case, hen adjus ing o
leukocy e coun in he linea eg ession analysis can e-
duce noise and hus boos s a is ical powe o de ec an
associa ion, which may explain he addi ional associa-
ions no ed wi h he CCLM module. CCLM had no sig-
ni ican mQTLs.
IDL_P
L_LDL_P
VLDL_D
XL_HDL_P
XXL_VLDL_P
ACACE
ACE
ALA
ALB
APOA1
APOB
APOB/APOA1
IDL_L
L_LDL_L
LDL_D
XL_HDL_L
XXL_VLDL_L
BOHBUT
HDL_D
IDL_PL
L_LDL_PL
XL_HDL_PL
XXL_VLDL_PL
CIT
CREA
CRP
IDL_C
L_LDL_C
XL_HDL_C
XXL_VLDL_C
DHA
DHA/FA
IDL_CE
L_LDL_CE
XL_HDL_CE
XXL_VLDL_CE
EST_C
IDL_FC
L_LDL_FC
XL_HDL_FC
XXL_VLDL_FC
FAW3
FAW3/FA
FAW6
FAW6/FA
FREE_C
IDL_TG
L_LDL_TG
XL_HDL_TG
XXL_VLDL_TG
GLC
GLN
GLOL
GLY
GlycA
L_HDL_P
M_LDL_P
XL_VLDL_P
HDL_C
HDL_TG
HDL2_C
HDL3_C
HIS
L_HDL_L
M_LDL_L
XL_VLDL_L
ILE
L_HDL_PL
M_LDL_PL
L
P_LDLV_LX
L_HDL_C
M_LDL_C
C_LDLV_LX
L_HDL_CE
M_LDL_CE
XL_VLDL_CE
LA
LA/FA
LAC
LDL_C
LDL_TG
LEU
L_HDL_FC
M_LDL_FC
XL_VLDL_FC
MUFA
MUFA/FA
L_HDL_TG
M_LDL_TG
XL_VLDL_TG
L_VLDL_P
M_HDL_P
S_LDL_P
L_VLDL_L
M_HDL_L
S_LDL_L
PC
PHE
PUFA
PUFA/FA
PYR
L_VLDL_PL
M_HDL_PL
S_LDL_PL
L_VLDL_C
M_HDL_C
S_LDL_C
REMNANT_C
L_VLDL_CE
M_HDL_CE
S_LDL_CE
SERUM_C
SERUM_TG
SFA
SFA/FA
SM
L_VLDL_FC
M_HDL_FC
S_LDL_FC
TG_PG
TOT_CHO
TOT_FA
TOT_PG
TYR
L_VLDL_TG
M_HDL_TG
S_LDL_TG
UNSAT
M_VLDL_P
S_HDL_P
VAL
VLDL_C
VLDL_TG
M_VLDL_L
S_HDL_L
M_VLDL_PL
S_HDL_PL
M_VLDL_C
S_HDL_C
M_VLDL_CE
S_HDL_CE
M_VLDL_FC
S_HDL_FC
M_VLDL_TG
S_HDL_TG
S_VLDL_P
S_VLDL_L
S_VLDL_PL
S_VLDL_C
S_VLDL_CE
S_VLDL_FC
S_VLDL_TG
XS_VLDL_P
XS_VLDL_L
XS_VLDL_PL
XS_VLDL_C
XS_VLDL_CE
XS_VLDL_FC
XS_VLDL_TG
FAW6/FAW3
LLM (124)
NM (122)
GIMA (98)
GIMB (82)
PM (56)
CCLM (24)
BCM (14)
VRM (8)
FDR adjus ed P- alue
< 1.25 x 10
-04
1.25 x 10
-03
6.25 x 10
-03
> 6.25x10
-03
Signi icance
h eshold
1.25 x 10
-04
Lipop o ein Subclasses
HDL
IDL
LDL
VLDL
Lipop o ein
pa icle size
Apolipop o eins
Choles e ol
Glyce ides
& Phospholipids
Fa y Acids
Small
Me aboli es
Amino
Acids
In lamma o y
Ma ke s
Fig. 3 Me aboli e measu e associa ions wi h immune gene modules. Ci cula hea map o associa ions be ween indi idual me aboli e measu e
and he module eigengene o each module (colo ed by FDR-adjus ed P alues). Concen ic ci cles ep esen modules, wi h numbe s in pa en heses
deno ing o al numbe o me aboli e measu es associa ed wi h ha module a FDR-adjus ed P alue <6.25 × 10
–3
.NM neu ophil module, LLM lipid
leukocy e module, GIMA/GIMB gene al immune module A/B, PM pla ele module, CCLM cy o oxic cell-like module, BCM B-cell ac i i y module, VRM i al
esponse module. See Addi ional ile 1: Table S1 o ull me aboli e desc ip ions
Na h e al. Genome Biology (2017) 18:146 Page 5 o 15

Vi al esponse module
Th ee genome-wide signi ican mQTLs we e iden i ied
o he i al esponse module (VRM; Fig. 2a; Table 2).
The s onges mQTL, s182710579 (me a-P alue =
9.22 × 10
–9
), is wi hin a known lincRNA locus (RP11-
608O21.1) (Addi ional ile 4: Figu e S2a). Rs182710579
was a ans eQTL o h ee genes in he VRM (Fig. 2b;
Addi ional ile 9: Table S8). The s onges associa ion
was seen wi h CCL2 (me a-P alue = 6.78 × 10
–12
), a p o-
in lamma o y chemokine in ol ed in leukocy e ec ui -
men du ing i al in ec ion [27, 28]. Also, adipocy e-
de i ed CCL2 is known o play an impo an ole in
obesi y-associa ed adipose issue in lamma ion and
insulin esis ance [29]. The nex s onges mQTL,
s151234502, esides wi hin in on 4 o he ela i ely un-
s udied ZNF212, pa o a zinc inge gene clus e a
7q36 (Addi ional ile 4: Figu e S2b). Rs151234502 modu-
la ed exp ession o 11 VRM genes in ans (Fig. 2b;
Addi ional ile 9: Table S8). The s onges associa ion
was wi h OAS2 (me a-P alue = 8.98 × 10
–10
), an
in e e on-induced gene encoding an enzyme p omo ing
RNase L-media ed clea age o i al and cellula RNA
[30]. The hi d mQTL, s147742798, was an in e genic
SNP loca ed be ween SHANK2 and DHCR7 a 11q13.4
(Addi ional ile 4: Figu e S2c). Rs147742798 was a ans
eQTL o wo genes in he VRM, BST2 and PARP9
(Fig. 2b; Addi ional ile 9: Table S8). BST2 encodes a
ans-memb ane p o ein wi h in e e on-inducible an i-
i al unc ion [31]. S udies ha e p e iously shown induc-
ion o a y acid biosyn hesis by a ange o i uses [32].
VRM was associa ed wi h eigh me aboli e measu es, in-
cluding amino acids (alanine, phenylalanine), a y acids
(omega-6 a y acids, polyunsa u a ed a y acids, sa u-
a ed a y acids, and o al a y acids), and choles e ol
es e s in medium VLDL (Fig. 3; Addi ional ile 8: Table
S7). Consis en wi h i s pu a i e ole in i al esponse,
VRM was s ongly associa ed wi h CRP (me a P alue =
2.38 × 10
–10
).
B-cell ac i i y module
The B-cell ac i i y module (BCM) was associa ed wi h
14 me aboli e measu es including CRP, his idine, lac a e,
apolipop o iens, and mainly he medium high-densi y
lipop o ein (HDL) subclass o lipop o eins (Fig. 3;
Addi ional ile 8: Table S7). The s onges associa ion
was seen wi h CRP (me a-P alue = 2.65 × 10
–8
). His i-
dine was he second s onges associa ion. This is in e -
es ing gi en ha his idine is a subs a e o his amine,
and bo h his amine elease and B-cell ac i i y a e cen al
pa s o an alle gic eac ion. While no mQTLs o BCM
eached genome-wide signi icance, he e was some e i-
dence in he YFS o he MHC class I locus (Fig. 2a and
Table 2). The op signal was loca ed be ween HLA-B/C
and MICA ( s2523489, me a-P alue = 6.27 × 10
–8
;
Addi ional ile 4: Figu e S3). The HLA class I egion is well
known o be associa ed wi h au oimmune diseases, whe e
he ole o B cells is well ecognized. Rs2523489 was a
ans eQTL o CD79B (me a-P alue = 1.16 × 10
–9
), a sub-
uni o he an igen-binding B-cell ecep o complex [33].
Pla ele module
PM had he s onges mQTL o any gene module, an in-
onic SNP o he ARHGEF3 gene a 3p14.3 ( s1354034;
me a-P alue = 7.35 × 10
–28
, Fig. 2a; Table 2; Addi ional
ile 4: Figu e S4a). ARHGEF3 encodes a Rho guanine nu-
cleo ide exchange ac o , a ca alys o Rho GTPase con-
e sion om inac i e GDP-bound o ac i e GTP-bound
o m. Rs1354034 was an eQTL o he majo i y o genes
in he PM, all o which we e in ans. An in e genic
SNP, s2836773 (me a-P alue = 5.4 × 10
–8
), a he HLA
locus was also iden i ied as an mQTL o PM (Add-
i ional ile 4: Figu e S4b). The ARHGEF3 mQTL
( s1354034) exhibi ed a s ong ans- egula o y e ec
and was associa ed wi h 61 PM genes (65 unique p obes)
(Fig. 2c; Addi ional ile 9: Table S8). The op ans eQTL
was ITGB3 (me a-P alue = 5.09 × 10
–42
), a gene encod-
ing he β
3
subuni o he he e odime ic in eg in ecep o
(in eg in α
IIb
β
3
). This in eg in ecep o is mos highly
exp essed on ac i a ed pla ele s and plays a key ole in
media ing pla ele adhesion and agg ega ion upon bind-
ing o ib inogen and Willeb and ac o [34, 35]. Ou
da a a e consis en wi h p e ious obse a ions o he di-
e se ans eQTL e ec s o s1354034 [23], including
he pu a i e splice-QTL e ec s o s1354034 on TPM4,
a signi ican eGene in he PM.
ARHGEF3 i sel is o in ense in e es o pla ele biol-
ogy. I has p e iously been shown ha silencing o ARH-
GEF3 in zeb a ish p e en s h ombocy e o ma ion [36].
To es whe he ARHGEF3 exp ession had an e ec on
PM genes, we eg essed ou ARHGEF3 le els and e- an
he eQTL analysis. Adjus ing o ARHGEF3 did no a -
enua e he ans-associa ions o s1354034, sugges ing
ei he independence o downs eam unc ion o ARH-
GEF3 and s1354034 o pos - ansc ip ional modi ica-
ion o ARHGEF3. P e ious GWAS s udies ha e shown
s1354034 is associa ed wi h pla ele coun and mean
pla ele olume [36]; howe e , pe haps due o powe , we
ound no signi ican ela ionship be ween pla ele coun s
and s1354034 in YFS. While pla ele coun s we e posi-
i ely associa ed wi h he PM (β= 0.29; P alue = 8.23 ×
10
–30
; Addi ional ile 6: Table S5), he associa ion be-
ween s1354034 and he PM was s ill highly signi ican
when condi ioning on pla ele coun s (β=−0.33; P alue
= 1.40 × 10
–17
).
PM displayed di e se me abolic in e ac ions and was
associa ed wi h 55 me aboli e measu es, la gely comp is-
ing o lipop o ein subclasses and a y acids, as well as
CRP (Fig. 3; Addi ional ile 8: Table S7). Choles e ol
Na h e al. Genome Biology (2017) 18:146 Page 6 o 15
es e s in small HDL pa icles we e mos s ongly associ-
a ed wi h he PM (me a-P alue = 9.45 × 10
–20
). HDL has
been shown o exhibi an i h ombo ic p ope ies by
modula ing pla ele ac i a ion and agg ega ion and he
coagula ion pa hway [37]. Also, a ious LDL subclasses
o lipop o eins we e associa ed wi h he PM, which is
consis en wi h ou unde s anding ha LDL in luences
pla ele ac i i y. Fo example, LDL has been shown o
in luence pla ele ac i i y ei he by enhancing pla ele e-
sponsi eness o agg ega ing s imuli o by inducing ag-
g ega ion [38, 39]. Mo eo e , LDL-speci ic binding si es
on pla ele s ha e also been epo ed [40, 41]. As no ed
abo e, he PM was associa ed wi h pla ele coun s, and
adjus men o pla ele coun s in he YFS esul ed in a -
enua ion o app oxima ely hal o he weakes associa-
ions be ween PM and me aboli e measu es; howe e ,
he s onges we e main ained (Addi ional ile 7: Table
S6). Associa ion wi h VLDL pa icle size and h ee
o he s we e gained ollowing he adjus men (Addi ional
ile 7: Table S6).
Neu ophil module
Th ee loci we e iden i ied as mQTLs o he neu ophil
module (NM; Fig. 2a and Table 2). The op mQTL was
in onic o LRRC8A a 9q34.11 ( s13297295; me a-P
alue = 3.93 × 10
–11
; Addi ional ile 4: Figu e S5a).
LRRC8A encodes a ans-memb ane p o ein shown o
play a ole in B- and T-cell de elopmen and T cell unc-
ion [42, 43]. Two addi ional in e genic mQTLs we e lo-
ca ed a he TAGAP locus a 6q25.3 ( s2485364; me a-P
alue = 3.93 × 10
–9
) and a 20q12 ( s140929198; me a-P
alue = 1.41 × 10
–9
) (Addi ional ile 4: Figu e S5b, c).
Rs13297295 was a s ong ans egula o o NM and was
an eQTL o eigh NM genes ( en unique p obes), in
pa icula he majo alpha de ensins (DEFA1-DEFA4),
he genes o highes cen ali y in he module (Fig. 2d;
Addi ional ile 9: Table S8). Rs13297295 was a cis-eQTL
o ano he co e NM gene, LCN2 (pe mu ed me a-P
alue = 1 × 10
–4
) (Fig. 2d; Addi ional ile 9: Table S8).
LCN2 is exp essed in neu ophils and inducible by TLR
ac i a ion, ac ing as an an imic obial agen ia seques-
a ion o bac e ial side opho es o p e en i on up ake
[44–46]. LCN2’s ole in acu e phase esponse appea s o
be ela ed o ca dio ascula diseases, such as hea ail-
u e [47]. A he TAGAP locus, s2485364 was a ans-
eQTL o eigh NM genes ( en p obes) and was also a
s ong d i e o LCN2 (me a-P alue = 9.11 × 10
–17
)
(Fig. 2d and Addi ional ile 9: Table S8). Consis en wi h
ou indings, neu ophils om LCN2-de icien mice ha e
been shown o ha e impai ed chemo axis and phagocy ic
capabili y and inc eased suscep ibili y o bac e ial and
yeas in ec ions compa ed o wild ype [48, 49]. This
sugges s a possible unc ional ole o TAGAP a ian s in
egula ing neu ophil mig a ion h ough LCN2.
NM was associa ed wi h 121 ci cula ing me aboli e
measu es (~76% o all me aboli e measu es analyzed) as
well as CRP (Fig. 3; Addi ional ile 8: Table S7). The
s onges is he p e iously epo ed associa ion wi h in-
lamma o y bioma ke GlycA (me a-P alue = 2.68 × 10
–
25
) [10]; howe e , NM’s associa ion wi h a ious lipop o-
ein subclasses, pa icle sizes o lipop o eins, a y acids,
choles e ol, apolipop o eins, glyce ides and phospho-
lipids, amino acids, and o he small molecules indica es
i has a po en ially majo ole in linking neu ophil unc-
ion o me abolism.
Lipid-leukocy e module
Toge he wi h NM, he lipid-leukocy e module (LLM)
showed ex ensi e me abolic associa ions. O e all, 123
me aboli e measu es and CRP we e associa ed wi h
LLM, wi h he s onges being he a io o iglyce ides
o phosphoglyce ides (me a-P alue = 5.16 × 10
–138
; Fig. 3;
Addi ional ile 8: Table S7). Wi h he inclusion o he
YFS, hese indings s ongly eplica e p e ious associa-
ions be ween LLM and me aboli e measu es [14] as well
as de ec ing addi ional associa ions. We also con i m he
p e ious s ong nega i e associa ion be ween CRP and
LLM (me a-P alue = 8.16 × 10
–20
). Consis en wi h p e-
ious s udies, no mQTLs we e de ec ed o LLM.
Gene al immune modules A and B
No mQTLs we e associa ed wi h gene al immune mod-
ules A and B (GIMA and GIMB); howe e , hese modules
we e associa ed wi h 97 and 82 me aboli e measu es, e-
spec i ely (Fig. 3; Addi ional ile 8: Table S7). Choles e ol
es e s in small HDL and he mean diame e o VLDL
pa icles exhibi ed he s onges associa ions wi h GIMA
(me a-P alue = 1.56 × 10
–30
) and GIMB (me a-P alue =
1.83 × 10
–15
), espec i ely. The GIMA was also associa ed
wi h omega-3 a y acid le els (me a-P alue=4.1×10
–8
)
and CRP (me a-P alue = 5.7 × 10
–5
) while GIMB was no ,
pe haps due o he sub ly di e ence pa hway en ichmen s
o each module (Table 1). O he me aboli e measu es as-
socia ed wi h hese wo modules include mainly he VLDL
and HDL subclass o lipop o eins and a y acids; due o
hei la ge size and he e ogeneous composi ion, howe e ,
in e p e a ion o me abolic ela ionships o GIMA and
GIMB is limi ed.
Long- e m s abili y o in e ac ions be ween me aboli e
measu es, immune gene modules, and mQTLs
The 216 indi iduals in bo h he DILGOM 2007 and
2014 ollow-up allowed in es iga ion o he long- e m
s abili y o immunome abolic and mQTL ela ionships.
Ac oss his se en-yea pe iod, he eigh immune gene
coexp ession ne wo ks we e s ongly p ese ed (all
p ese a ion s a is ics’pe mu a ion P alues <0.001;
Addi ional ile 10: Table S9). The me aboli e–
Na h e al. Genome Biology (2017) 18:146 Page 7 o 15
me aboli e co ela ion s uc u e was also la gely con-
sis en be ween DIGOM07 and DILGOM14 (Addi ional
ile 4: Figu e S6).
Nex , we examined how me aboli e in e ac ions wi h
immune gene modules changed o e he 7-yea ime
pe iod (“Me hods”). The LLM–me aboli e measu e asso-
cia ions we e he mos consis en o e ime wi h 90 and
79 me aboli e measu es eaching signi icance in DIL-
GOM07 and DILGOM14, espec i ely, o which 74 we e
signi ican a bo h ime poin s (Fig. 4a; Addi ional ile
11: Table S10). The di ec ion and e ec size o LLM–
me aboli e measu e associa ions we e la gely main ained
(Fig. 4b). Fo he neu ophil module, he py u a e asso-
cia ion was signi ican ly main ained o e ime; howe e ,
he e was some e idence ha o he expec ed associa-
ions wi h NM we e s able o e ime, including GlycA
(Addi ional ile 11: Table S10). While no associa ions
wi h me aboli e measu es we e signi ican ly main ained
o he pla ele module, s1354034 was a empo ally
s able mQTL o PM (mQTL P alue = 4.87 × 10
–7
). No
o he mQTLs eached signi icance o empo al s abili y.
While we we e powe ed o opologically eplica e im-
mune modules be ween ime-poin s, powe o de ec
module–me aboli e associa ions and mQTLs was s ill
limi ed, wi h only he s onges associa ions eaching
signi icance. Fo he la e , he e ec sizes o module as-
socia ions we e gene ally consis en be ween he ime
poin s (Addi ional ile 4: Figu e S7), wi h he excep ion
o GIMB. Wi h he pa icula ly s ong consis ency o as-
socia ions o he LLM and NM, i may be ha smalle
modules, which cap u e mo e de ined ansc ip ional
p og ams, a e he mos empo ally s able in e ms o
hei pheno ype associa ions. Howe e , gi en he obus -
ness o hese associa ions be ween independen coho s,
we an icipa e ha , as long- e m omics ollow-up o
popula ion-based coho s inc ease in sample size, mo e
o hese disco e ed associa ions will become s a is ically
signi ican o e ime.
Conclusions
This s udy has u ilized o e 2000 indi iduals o map he
immuno-me abolic c oss alk ope a ing in ci cula ion.
We ha e iden i ied and cha ac e ized eigh obus im-
mune gene modules, hei gene ic con ol, and in e ac-
ions wi h di e se me aboli e measu es, including many
o clinical signi icance (e.g., iglyce ides, HDL, LDL,
b anched-chain amino acids). Also, se e al signi ican
me aboli e measu es iden i ied he e, pa icula ly
b anched chain amino acids and a y acids, ha e been
p e iously shown o be p edic i e o ca dio ascula
e en s and he de elopmen o T2D [6, 50]. Fu he -
mo e, ou indings a e consis en wi h and build upon
hose o p e ious s udies. In addi ion o i e newly iden-
i ied gene modules, hei mQTLs and me aboli e in e -
ac ions, we ha e eplica ed he p e iously cha ac e ized
LL module and con i m i s associa ion wi h lipop o ein
IDL_P
L_LDL_P
VLDL_D
XL_HDL_P
XXL_VLDL_P
ACACE
ACE
ALA
ALB
APOA1
APOB
APOB/APOA1
IDL_L
L_LDL_L
LDL_D
XL_HDL_L
XXL_VLDL_L
BOHBUT
HDL_D
IDL_PL
L_LDL_PL
XL_HDL_PL
XXL_VLDL_PL
CIT
CREA
CRP
IDL_C
L_LDL_C
XL_HDL_C
XXL_VLDL_C
DHA
DHA/FA
IDL_CE
L_LDL_CE
XL_HDL_CE
XXL_VLDL_CE
EST_C
IDL_FC
L_LDL_FC
XL_HDL_FC
XXL_VLDL_FC
FAW3
FAW3/FA
FAW6
FREE_C
IDL_TG
L_LDL_TG
XL_HDL_TG
XXL_VLDL_TG
GLC
GLN
GLOL
GLY
GP
L_HDL_P
M_LDL_P
XL_VLDL_P
HDL_C
HDL_TG
HDL2_C
HDL3_C
HIS
L_HDL_L
M_LDL_L
XL_VLDL_L
ILE
L_HDL_PL
M_LDL_PL
L_HDL_C
M_LDL_C
L_HDL_CE
M_LDL_CE
XL_VLDL_CE
LA
LA/FA
LAC
LDL_C
LDL_TG
LEU
L_HDL_FC
M_LDL_FC
XL_VLDL_FC
MUFA
MUFA/FA
L_HDL_TG
M_LDL_TG
XL_VLDL_TG
L_VLDL_P
M_HDL_P
S_LDL_P
L_VLDL_L
M_HDL_L
S_LDL_L
PC
PHE
PUFA
PUFA/FA
PYR
L_VLDL_PL
M_HDL_PL
S_LDL_PL
L_VLDL_C
M_HDL_C
S_LDL_C
REMNANT_C
L_VLDL_CE
M_HDL_CE
S_LDL_CE
SERUM_C
SERUM_TG
SFA
SFA/FA
SM
L_VLDL_FC
M_HDL_FC
S_LDL_FC
TG_PG
TOT_CHO
TOT_FA
TOT_PG
TYR
L_VLDL_TG
M_HDL_TG
S_LDL_TG
UNSAT
M_VLDL_P
S_HDL_P
VAL
VLDL_C
VLDL_TG
M_VLDL_L
S_HDL_L
M_VLDL_PL
S_HDL_PL
M_VLDL_C
S_HDL_C
M_VLDL_CE
S_HDL_CE
M_VLDL_FC
S_HDL_FC
M_VLDL_TG
S_HDL_TG
S_VLDL_P
S_VLDL_L
S_VLDL_PL
S_VLDL_C
S_VLDL_CE
S_VLDL_FC
S_VLDL_TG
XS_VLDL_P
XS_VLDL_L
XS_VLDL_PL
XS_VLDL_C
XS_VLDL_CE
XS_VLDL_FC
XS_VLDL_TG
Apolipop o eins
Choles e ol
Small
Me aboli es
Amino
Acids
In lamma o y
Ma ke s
Lipop o ein
pa icle size
Glyce ides
& Phospholipids
Fa y Acids
HDL
IDL
LDL
VLDL
FDR adjus ed P- alue
< 1.25 x 10
-04
1.25 x 10
-03
6.25 x 10
-03
> 6.25x10
-03
Signi icance
h eshold
1.25 x 10
-04
Lipop o ein Subclasses
Lipid Leukocy e Module (LLM)
DILGOM14 (N=79)
DILGOM07 (N=90)
XL_VLDL_PL
C_LDL
V
_LX
FAW6/FA
−0.50
−0.25
0
0.25
0.50
−0.50 −0.25 0 0.25 0.50
PUFA/FA
FAW6/FA
L_HDL_FC
L_HDL_FC
L_HDL_CE
XL_HDL_PL
UnSa
XL_HDL_FC
XL_HDL_CE
XL_HDL_C
HDL2_C L_HDL_PL
XL_HDL_P
TG_PG
S_VLDL_TG
M_HDL_TG
VLDL_TG
M_VLDL_TG
L_VLDL_PL
L_VLDL_TG
L_VLDL_C
HDL_TG
MUFA
S_LDL_TG
SFA
XXL_VLDL_PL
ApoB
Remnan _C
XS_VLDL_P
S_VLDL_C
M_VLDL_CE
Be a Es ima es DILGOM07
Be a Es ima es DILGOM14
Bo h DILGOM07 and DILGOM14
DILGOM07 only
DILGOM14 only
No signi ican
B
A
Signi ican me aboli e measu e
associa ions wi h LLM
LA/FA
XS_VLDL_L
L_VLDL_CE
Fig. 4 Tempo ally s able me aboli e measu e associa ions wi h he LLM. aCi cula hea map o associa ion be ween each me aboli e measu e
and he LLM. bCompa ison o he e ec size es ima es o me aboli e measu e associa ion wi h LLM in DILGOM07 and DILGOM14 shows ha he
o e all associa ion pa e ns a e consis en ac oss he wo ime-poin s. Colo s deno e me aboli es ha a e signi ican ly associa ed wi h he LLM in
DILGOM07 only (o ange), DILGOM14 only (blue), and ac oss bo h ime-poin s (g een). The g ey dashed line is he x = y line
Na h e al. Genome Biology (2017) 18:146 Page 8 o 15
subclasses, lipids, a y acids, and amino acids [13, 14].
Associa ions be ween he co e genes in he LL module
and isoleucine, leucine, and a ious lipids we e also
iden i ied independen ly in he KORA coho [12]. Im-
po an ly, we ha e shown he long- e m s abili y o LL
and neu ophil module coexp ession and in e ac ions
wi h me aboli e measu es, and we ha e g ea ly expanded
he numbe o known bioma ke s associa ed wi h he
NM om one (GlycA) o 123 [10]. Ou s udy has also
expanded he widesp ead ans eQTL e ec s a he
ARHGEF3 locus [23], shows hem o be s ongly main-
ained wi hin indi iduals o e ime, and u he iden i ies
ex ensi e in e ac ions wi h lipop o ein measu es ha
may be a consequence o hese ans e ec s.
Taken oge he , ou analyses illus a e he apidly
g owing body o e idence in ima ely linking he immu-
noin lamma o y esponse o he blood me abolome.
Wi h ine - esolu ion maps o hese in e ac ions, new
bioma ke s o ch onic and acu e in lamma o y s a es a e
likely o eme ge. Wi h in i o and in e en ional s udies,
modula ion o hese me aboli e–immune in e ac ions
h ough exis ing lipid-lowe ing medica ions, gu mic obe
e ec s, o die a y changes may p o ide new ways he
immune sys em i sel can be u ilized o lessen he bu -
den o ca diome abolic disease.
Me hods
S udy popula ions
This s udy used da a om wo popula ion-based co-
ho s, he Die a y, Li es yle, and Gene ic de e minan o
Obesi y and Me abolic synd ome (DILGOM; N = 518)
and he Ca dio ascula Risk in Young Finns S udy (YFS;
N = 1650), which ha e been desc ibed in de ail elsewhe e
[13, 51]. All subjec s en olled in hese s udies ga e w i -
en in o med consen .
The DILGOM s udy is a subsample o he FINRISK
2007 c oss-sec ional popula ion-based su ey, which e-
c ui ed a andom sample o 10,000 indi iduals be ween
25 and 74 yea s o age, s a i ied by sex and 10-yea age
g oups, om i e s udy a eas in Finland. All 6258 indi-
iduals who pa icipa ed in he FINRISK 2007 baseline
heal h examina ion we e in i ed o a end he DILGOM
s udy (N = 5024), 630 o whom unde wen a leas one
o he geno yping, ansc ip omics, o me abolomics
p o iling conside ed he e. In 2014, a ollow-up s udy was
conduc ed, o which 3735 indi iduals om he o iginal
s udy e-pa icipa ed. Samples collec ed in 2007 and
2014 a e e e ed o as DILGOM07 and DILGOM14,
espec i ely.
The YFS is a longi udinal p ospec i e coho s udy ha
s a ed in 1980, wi h ollow-up s udies ca ied ou e e y
3 yea s, o moni o ca dio ascula disease isk ac o s in
child en and adolescen s om i e majo egions o
Finland (Helsinki, Kuopio, Tu ku, Oulu, and Tampe e).
A o al o 3596 child en and adolescen s in age g oups
3, 6, 9, 12, 15, and 18 yea s pa icipa ed in he baseline
s udy; hese child en we e andomly selec ed om he
na ional public egis e and hei de ails a e desc ibed in
[51]. In his cu en s udy, da a collec ed om he 2011
ollow-up s udy (pa icipan s aged 34, 37, 40, 43, 46, and
49 yea s) we e analyzed.
Sample collec ion
Venous blood was collec ed ollowing an o e nigh as
in all h ee s udies. Samples we e cen i uged and he
esul ing plasma and se um samples we e aliquo ed in o
sepa a e ubes and s o ed a −70 °C o analyses. P o o-
cols o he blood sampling, physiological measu e-
men s, and clinical su ey ques ions we e simila ac oss
he YFS and DILGOM s udies and a e desc ibed ex en-
si ely in [13, 52].
Geno yping and impu a ion
Whole blood genomic DNA ob ained om bo h coho s
was geno yped using he Illumina 610-Quad SNP a ay
o DILGOM07 (N = 555) [13] and a cus om gene a ed
670 K Illumina BeadChip a ay o YFS (N = 2443) [53].
The 670 K a ay sha es 562,643 SNPs wi h he 610-quad
a ay. The 670 K a ay emo es poo ly pe o ming SNPs
om he 610-quad a ay and imp o es copy numbe
a ia ion co e age [53]. Geno ype calling was pe o med
wi h he Illuminus clus e ing algo i hm [54]. Quali y
con ol was as p e iously desc ibed in [13] and [53] o
DILGOM and YFS, espec i ely. Geno ypes we e im-
pu ed o he 1000 Genomes Phase 1 e sion 3 e e ence
panel using IMPUTE2 in bo h DILGOM and YFS [17].
Poo ly impu ed SNPs based on low call- a e (<0.90 o
DILGOM, <0.95 o YFS), low-in o ma ion sco e (<0.4),
mino allele equency <1%, and de ia ion om Ha dy–
Weinbe g equilib ium (P<5×10
–6
) we e hen emo ed.
A o al o 7,263,701 SNPs in DILGOM and 6,721,082 in
YFS passed quali y con ol, wi h 6,485,973 common be-
ween he wo. A o al o N = 518 samples in DILGOM
and N = 2443 samples in YFS indi iduals passed quali y
con ol il e s.
Me abolomics p o iling
Me aboli e concen a ions o DILGOM07 (N = 4816),
DILGOM14 (N = 1273), and YFS (N = 2046) we e quan i-
ied om se um samples u ilizing a high- h oughpu
NMR me abolomics pla o m (B ainshake L d, Helsinki,
Finland) [18, 55]. De ails o he expe imen al p o ocol, in-
cluding sample p epa a ion, NMR spec oscopy, and me-
aboli e iden i ica ion, ha e been p e iously desc ibed in
[13, 18]. A o al o 159 me aboli e measu es we e assessed,
o which 148 we e di ec ly measu ed and 11 we e de i ed
(Addi ional ile 1: Table S1). The 148 measu es include
he cons i uen s o 14 lipop o ein subclasses (98
Na h e al. Genome Biology (2017) 18:146 Page 9 o 15