He nangomez‑Lade ase al.
Biology o Sex Di e ences (2023) 14:86
h ps://doi.o g/10.1186/s13293‑023‑00572‑1
RESEARCH
Sex bias inceliac disease: XWAS
andmonocy e eQTLs inwomen iden i y
TMEM187 asa unc ional candida e gene
Alba He nangomez‑Lade as1,2, A iadna Cille os‑Po e 1,2, Sil ia Ma ínez Velasco2,3, Se gi Ma í1,2,
Ma ía Lega da2,3, Bá ba a Paola González‑Ga cía1,2, Ca los Tu au2,3, I aia Ga cía‑San is eban1,2, Iñaki I as o za2,3,
No a Fe nandez‑Jimenez1,2* and Jose Ramon Bilbao1,2,4*
Abs ac
Backg ound Celiac disease (CeD) is an immune‑media ed diso de ha de elops in gene ically p edisposed indi‑
iduals upon glu en consump ion. HLA isk alleles explain 40% o he gene ic componen o CeD, so he e ha e been
con inuing e o s o unco e non‑HLA loci ha can explain he emaining he i abili y. As in mos au oimmune diso ‑
de s, he p e alence o CeD is signi ican ly highe in women. He e, we in es iga ed he possible in ol emen o he X
ch omosome on he sex bias o CeD.
Me hods We pe o med a X ch omosome‑wide associa ion s udy (XWAS) and a gene‑based associa ion s udy
in women om he CeD Immunochip (7062 cases, 5446 con ols). We also cons uc ed a da abase o X ch omo‑
some cis‑exp ession quan i a i e ai loci (eQTLs) in monocy es om uns imula ed (n = 226) and lipopolysaccha ide
(LPS)‑s imula ed (n = 130) emale dono s and pe o med a Summa y‑da a‑based MR (SMR) analysis o in eg a e XWAS
and eQTL in o ma ion. We in e oga ed he exp ession o he po en ially causal gene (TMEM187) in pe iphe al blood
mononuclea cells (PBMCs) om celiac pa ien s a onse , on a glu en‑ ee die , po en ial celiac pa ien s and non‑celiac
con ols.
Resul s The XWAS and gene‑based analyses iden i ied 13 SNPs and 25 genes, espec i ely, 22 o which had no been
p e iously associa ed wi h CeD. The X ch omosome cis‑eQTL analysis ound 18 genes wi h a leas one cis‑eQTL
in naï e emale monocy es and 8 genes in LPS‑s imula ed emale monocy es, 2 o which we e common o bo h si u‑
a ions and 6 we e unique o LPS s imula ion. SMR iden i ied a po en ially causal associa ion o TMEM187 exp ession
in naï e monocy es wi h CeD in women, egula ed by CeD‑associa ed, eQTL‑SNPs s7350355 and s5945386. The CeD‑
isk alleles we e co ela ed wi h lowe TMEM187 exp ession. These esul s we e eplica ed using eQTLs om LPS‑s im‑
ula ed monocy es. We obse ed highe le els o TMEM187 exp ession in PBMCs om emale CeD pa ien s a onse
compa ed o emale non‑celiac con ols, bu no in male CeD indi iduals.
Conclusion Using X ch omosome geno ypes and gene exp ession da a om emale monocy es, SMR has iden i ied
TMEM187 as a po en ially causal candida e in CeD. Fu he s udies a e needed o unde s and he implica ion o he X
ch omosome in he highe p e alence o CeD in women.
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Biology o Sex Di e ences
*Co espondence:
No a Fe nandez‑Jimenez
[email p o ec ed]
Jose Ramon Bilbao
[email p o ec ed]
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Page 2 o 10
He nangomez‑Lade ase al. Biology o Sex Di e ences (2023) 14:86
Backg ound
Celiac disease (CeD) is an immune-media ed en e opa hy
ha de elops in gene ically p edisposed indi iduals as
a eac ion o glu en inges ion [1]. The global p e alence
o CeD is 1.4% acco ding o se ological diagnosis, and
0.7% based on biopsy con i ma ion [2]. As in o he au o-
immune diseases, he p e alence o CeD is signi ican ly
highe in women [3]. Almos all CeD pa ien s ca y he
Human Leucocy e An igen (HLA) alleles ha encode
he HLA-DQ2 and/o HLA-DQ8 molecules. Howe e ,
HLA is necessa y, bu no su icien o de elop he dis-
ease, and only explains 40% o he o e all gene ic isk [4,
5]. GWAS and ine-mapping s udies like he Immunochip
p ojec ha e iden i ied mo e han 40 non-HLA loci asso-
cia ed wi h CeD [6–8]. Ne e heless, mos o he SNPs
loca ed in hese loci ei he map o non-coding egions o
he genome, a away om genes, o a e in s ong link-
age disequilib ium (LD) wi h o he associa ed a ian s,
making i di icul o iden i y he genes ha a e unc ion-
ally in ol ed in he disease [9]. Al oge he , HLA and non-
HLA a ian s iden i ied so a explain a ound 50% o CeD
he i abili y [10]. The missing gene ic he i abili y hypo h-
esis sugges s ha addi ional, uniden i ied gene ic and
en i onmen al ac o s a e in ol ed in he de elopmen o
CeD. In his sense, he X ch omosome has been his o i-
cally igno ed in mos GWAS, o has been analyzed as i
i we e ano he au osome, wi hou accoun ing o male
hemizygosi y and emale X ch omosome inac i a ion
(XCI), wi h only e y ew s udies ha ake hese consid-
e a ions in o accoun [11–13].
Se e al s udies ha e shown a ela ionship be ween he
isk o di e en au oimmune diseases, including sys emic
lupus e y hema osus (SLE), Sjög en synd ome, ype 1
diabe es melli us and CeD, and X ch omosome ane-
uploidies like Kline el e , T iple X o Tu ne synd omes
[14–17]. Addi ionally, many o he app oxima ely 1100
genes on he X ch omosome a e hough o be ela ed o
he immune unc ion [18, 19]. These indings sugges a
ole o he X ch omosome in he biased sex-p e alence
o hese condi ions. In he case o CeD, mos isk loci a e
loca ed on he au osomes bu X ch omosome genes ha e
also been iden i ied, including TLR7 and TLR8, HCFC1,
TMEM187 and IRAK1 [6, 7].
Monocy es a e a undamen al pa o he inna e
immune de ense agains mic oo ganisms [20]. Di e -
en s udies ha e also ela ed his ype o immune cells o
CeD, and gliadin pep ides s imula e hei p oduc ion o
IL-8 and TNF-α, especially in celiac pa ien s [21, 22]. I
Highligh s
• The XWAS and gene‑based associa ion s udy iden i ied 13 gene ic a ian s and 25 genes signi ican ly associa ed
wi h CeD in women, bu no in men.
• eQTL analyses in naï e and LPS‑s imula ed emale monocy es iden i ied 16 unique genes wi h a leas one
cis‑eQTL in he naï e condi ion, 6 in he LPS condi ion and 2 genes, namely ZNF185 and TMEM187, common
o bo h condi ions.
• SMR iden i ied h ee SNPs ( s7350355, s5945386, s80208125) associa ed wi h gene TMEM187. The CeD isk
alleles we e nega i ely co ela ed wi h TMEM187 exp ession.
• PBMCs om ac i e emale CeD pa ien s showed signi ican ly highe TMEM187 exp ession han con ols,
bu no di e ences we e obse ed in men.
Keywo ds Celiac disease, XWAS, Mendelian andomiza ion, TMEM187, Monocy es, eQTLs
Plain language summa y
Celiac disease (CeD) is an immune‑ ela ed condi ion igge ed by glu en consump ion in gene ically suscep ible
indi iduals. Women p esen highe p e alence o CeD han men, bu he biological explana ion o such di e ence
has no been elucida ed. In his s udy, we in es iga ed whe he speci ic gene ic a ia ions on he X ch omosome
we e associa ed wi h CeD in each sex. Su p isingly, we ound 13 gene ic a ian s and 25 genes signi ican ly linked
o CeD in women, bu no in men. Addi ionally, we iden i ied gene ic a ian s on he X ch omosome associa ed
wi h gene exp ession o monocy es, a ype o immune cells ha is ac i a ed in CeD a e glu en in ake. In eg a ing
hese da a wi h ou p e ious indings, we ound ha lowe exp ession o a gene e med TMEM187 migh be associ‑
a ed wi h a po en ial inc ease in CeD isk in women. Finally, alida ion expe imen s con i med highe TMEM187 le els
in blood cells om emale CeD pa ien s compa ed o non‑celiac women, while no such di e ence was seen in males.
In summa y, ou s udy sugges s ha he X‑ch omosome gene TMEM187 may play a key ole in CeD de elopmen ,
p o iding insigh s in o he highe p e alence o CeD in emales.
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He nangomez‑Lade ase al. Biology o Sex Di e ences (2023) 14:86
has been sugges ed ha he esponse igge ed by glia-
din in monocy es is simila o ha induced by lipopoly-
saccha ide (LPS) h ough ecep o s such as TLR4 [23].
Fai ax e al. analyzed how gene ic a ian s shape gene
exp ession in monocy es, unde di e en in i o s im-
uli, including LPS, and showed ha mo e han hal o
he monocy e eQTLs a e speci ic o he en i onmen al
s imulus, bu he X ch omosome was no included in he
analysis [24].
Mendelian andomiza ion (MR), and mo e speci ically
he Summa y-da a-based MR (SMR), in eg a es GWAS
and eQTL summa y s a is ics in o de o de ec he unc-
ional in ol emen o genes unde he GWAS peaks [25].
Pa icula ly, associa ion hi s a e ansla ed in o po en-
ially causal ela ionships be ween exp ession le els o
candida e genes and complex ai s, in ele an issues,
cell ypes and con ex . Again, p e ious analyses in CeD
ha ha e made use o his me hod o combine GWAS
and eQTL summa y s a is ics ha e omi ed he X ch o-
mosome [26, 27].
In his s udy, we hypo hesized ha he X ch omosome
could ha bo addi ional suscep ibili y loci ha could
help explain bo h he missing he i abili y and he highe
p e alence in women. The e o e, we aimed o iden-
i y genes on he X ch omosome ha migh pa icipa e
in he pa hogenesis and also con ibu e o he sex bias
in CeD, h ough hei speci ic ansc ip ional p o ile in
monocy es. Fo ha pu pose, we pe o med an X-ch o-
mosome associa ion s udy (XWAS) in women om he
Immunochip p ojec , and cons uc ed a da abase o X
ch omosome cis-eQTLs in emale monocy es. Finally, we
combined he wo da ase s using SMR in o de o ind
monocy e-speci ic unc ional candida es on he X ch o-
mosome in ol ed in CeD.
Ma e ials andme hods
Immunochip da a andX ch omosome associa ion analyses
The CeD Immunochip da ase was il e ed o include
only X ch omosome a ian s wi h geno yping a e > 95%,
mino allele equency (MAF) > 1% and P- alue om
Ha dy Weinbe g equilib ium (PHWE) es > 1 × 10–6
using PLINK1.9 [28]. We emo ed indi iduals wi h call
a e < 97% and he e ozygosi y de ia ing mo e han 4
s anda d de ia ions om he mean (> 4SD). Geno ypes
we e impu ed a he Michigan Impu a ion Se e [29]
using HRC 1.1 2016 (GRCh37/hg19) as a e e ence
panel, using only he Eu opean popula ion, Minimac4
as he impu a ion so wa e and phasing wi h Eagle 2.4.
Impu ed SNPs wi h an R2 impu a ion accu acy abo e 0.8,
MAF > 1% and PHWE > 1 × 10–6 we e kep . A e applying
hese il e s, 12,508 emale samples (7062 cases and 5446
con ols) and 9474 males (3712 cases and 5762 con ols)
and 1611 SNPs we e e ained. We calcula ed he i s en
p incipal componen s o he geno ypes using PLINK1.9
o con ol o po en ial popula ion s a i ica ion in down-
s eam analyses. The op associa ed SNP was eplica ed
in he Dubois e al. s udy GWAS da ase [6].
The CeD XWAS was pe o med sepa a ely o men and
women using he newml me hod implemen ed in SNPT-
EST ( e sion 2.5.6), assuming a comple e inac i a ion o
one ch omosome in emales and equal e ec size in bo h
sexes [30]. Speci ically, his me hod uses a logis ic eg es-
sion model encoding geno ypes in males as 0/1 and in
emales as 0 / ½ / 1. The analysis was pe o med assuming
an addi i e gene ic model and he i s en p incipal com-
ponen s o he geno ype da a we e included as co a ia es.
The P- alue h eshold o s a is ical signi icance was se
a P < 8.68 × 10–5 a e Bon e oni co ec ion acco ding o
he numbe o independen es s, as de e mined wi h he
SimpleM me hod [31]. Resul s we e plo ed on a Man-
ha an plo gene a ed wi h he qqman R package [32].
A gene-based associa ion analysis was ca ied ou
in women using he Fas BAT me hod a ailable in he
Genome-wide Complex T ai Analysis (GCTA) so -
wa e package [33, 34]. This me hod in eg a es GWAS
summa y s a is ics and LD in o ma ion o calcula e he
P- alue o a se o a ian s wi hin a p ese dis ance om
a gene. The analysis was pe o med using he de aul
se ings sugges ed by GCTA-Fas BAT: gene egions
ex ended 50kb away om bo h he 3′ and 5′ UTRs o
he genes, and SNPs in s ong LD ( 2 ≥ 0.9) we e p uned.
We conduc ed he analysis o 2393 genes and 1611 SNPs
and he P- alue signi icance h eshold was se a P < 0.05
a e False Disco e y Ra e (FDR) co ec ion. A egional
associa ion plo combining XWAS and gene-based asso-
cia ion esul s was gene a ed wi h an open-sou ce R
sc ip (h ps:// gi hub. com/ Geeke ics/ Locus Zooms).
Monocy e cis‑eQTLs
A ca alog o naï e and LPS-s imula ed monocy e cis-
eQTLs o he X ch omosome was cons uc ed in women
using SNP geno ype and exp ession da a om a gen-
e al popula ion s udy by Fai ax e al. [24]. X ch omo-
some a ian s and indi iduals we e il e ed and impu ed
as desc ibed abo e. Addi ionally, only hose SNPs ha
we e homozygous o he mino allele in a leas 5 sam-
ples we e e ained. A e he QC, 233 emale samples and
165,648 a ian s emained o subsequen analyses.
Monocy e exp ession da a ha had al eady unde gone
da a no maliza ion, ans o ma ion wi h Va iance S a-
bilizing T ans o ma ion (VST), ba ch e ec co ec ion
and emo al o ou lie s, we e subjec ed o addi ional
QC s eps using he IluminaHuman 4.db [35] o emo e
hose p obes ha ma ched mo e han one locus, hose
on he au osomes, Y ch omosome o wi hou ch omo-
some epo ed, hose con aining SNPs wi h MAF > 0.1,
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He nangomez‑Lade ase al. Biology o Sex Di e ences (2023) 14:86
and hose desc ibed as Bad o No ma ch. This esul ed
in 1347 p obes mapping o 1250 s able gene IDs in he X
ch omosome.
We combined geno ype (165,648 SNPs) and gene
exp ession (1250 genes) in o ma ion o 226 samples om
he naï e monocy e emale da ase using he QTL ools
so wa e [36]. The associa ions be ween SNP geno ypes
and gene exp ession le els we e es ed wi h simple linea
eg essions assuming no mal dis ibu ion o he da a, and
he i s 10 p incipal componen s o he geno ype we e
included as co a ia es. Only SNPs wi hin a 1-Mb window
om he ansc ip ion s a si e (TSS) o a gene we e ana-
lyzed. PeQTL alues we e calcula ed wi h he nominal pass
op ion and a PeQTL < 5 × 10–8 h eshold was se o iden-
i y signi ican esul s. The same analysis was pe o med
wi h exp ession da a om LPS-s imula ed monocy es in
women (n = 130).
SMR analyses
SMR was pe o med combining he summa y s a is ics
om he XWAS and he eQTLs o naï e and LPS-s im-
ula ed monocy es using he SMR so wa e [25]. B ie ly,
SMR uses cis-eQTLs as ins umen al a iables, gene
exp ession as he exposu e, and CeD as he ou come, o
in e genes pleio opically o causally associa ed wi h
CeD. SMR esul s we e subjec ed o he he e ogenei y in
dependen ins umen s (HEIDI) es o de ec he p es-
ence o LD. In his es , a signi ican P- alue sugges s ha
he associa ion de ec ed could be he esul o wo gene ic
a ian s in s ong LD, whe eas a non-signi ican P- alue
indica es ha a single a ian is associa ed wi h bo h gene
exp ession and he disease. We used he ollowing de aul
pa ame e s sugges ed by SMR: he cis-window was se a
2Mb, he h eshold PeQTL o he SMR analysis was se
a 5 × 10–8, he h eshold PeQTL o he HEIDI es was
se a 1.57 × 10–3 and SNPs wi h a LD 2 > 0.9 o 2 < 0.05
wi h he op associa ed eQTL we e p uned. SMR esul s
wi h an FDR q- alue < 0.05 and PHEIDI > 0.05, we e consid-
e ed as pleio opic o causal associa ions. We plo ed he
TMEM187 egion (2000kb) on he X ch omosome using
he SMRLocusPlo sc ip a ailable in he SMR websi e.
Exp ession analysis inpedia ic CeD pa ien s
TMEM187 exp ession was quan i ied in pe iphe al
blood mononuclea cells (PBMCs) om CeD child en
a onse (20 emales, 8 males), pa ien s on glu en- ee
die (GFD) (6 emales, 9 males), po en ial CeD pa ien s
(5 emales, 6 males) and non-celiac con ols (17 emales,
10 males). CeD was diagnosed a he Pedia ic Gas oen-
e ology Uni o C uces Uni e si y Hospi al. The s udy
was app o ed by he Clinical Resea ch Boa d o C uces
Uni e si y Hospi al. Samples (2.5 ml in EDTA-con aining
ubes) we e collec ed a e in o med consen had been
ob ained om hei pa en s o gua dians and ans e ed
o he Basque Biobank o Resea ch. PBMCs we e iso-
la ed using he MACSp ep™ PBMC Isola ion ki (Mil e-
nyi Bio ec SL, Mad id, Spain; ca . no. 130–115-169),
RNA was pu i ied using he NucleoSpin® RNA mini ki
(Mache ey–Nagel, Dü en, Ge many; ca . no. 740955.250
4,392,653) and s o ed a –80°C un il use.
The exp ession o TMEM187 was quan i ied by RT-
qPCR using he TaqMan RNA- o-C 1-S ep Ki (The mo
Fishe Scien i ic Inc., Wal ham, MA, USA; ca . no.
4392653) and a comme cially a ailable TaqMan Gene
Exp ession assay (The mo Fishe Scien i ic Inc., ca . no.
Hs01920894_s1) on a Bio-Rad CFX Real Time PCR sys-
em (Bio Rad Scien i ic, He cules, CA, USA). The house-
keeping gene RPLPO was simul aneously measu ed and
used as an endogenous con ol. Rela i e exp ession in
each sample was calcula ed using he 2−ΔΔC me hod and
he e o e, he exp ession o each sample was no malized
o bo h he exp ession o RPLPO and he a e age o he
con ols. Di e ences in gene exp ession le els we e ana-
lyzed wi h Mann–Whi ney U- es using G aphPad P ism
.8.0.1. Finally, he exp ession o TMEM187 in di e -
en immune cell ypes (B, NK, T cells, and monocy es)
was analyzed and sex-s a i ied exp ession plo s we e
cons uc ed using he de aul se ings o he Da abase
o Immune Cell Exp ession eQTL Epigenomics (DICE,
h ps:// dice- da ab ase. o g/) online b owse [37].
Resul s
XWAS andgene‑le el associa ion analyses inCeD
To de e mine whe he he sex bias in CeD p e alence is
ela ed o he X ch omosome, we pe o med he XWAS
o CeD in women and men sepa a ely (Addi ional ile1).
We iden i ied a single associa ion peak on Xq28 including
13 signi ican SNPs (PXWAS < 8.68 × 10–5), wi h s78237385
(PXWAS = 2.30 × 10–5, OR = 1.20 ± 0.10) as he op SNP
(Addi ional ile2). The male XWAS did no de ec any
signi ican associa ion (Addi ional ile3). The op SNP
was also sugges i ely associa ed wi h CeD in emales
om he Dubois e al. s udy (P- alue = 1.86 × 10–4) bu
was no signi ican (P- alue = 0.19) in men om he same
s udy (Addi ional ile4).
We also pe o med a gene-based associa ion analy-
sis in women, ha akes in o accoun he agg ega ed
e ec o se s o SNPs. We in oduced 2392 genes and
1611 SNPs in he analysis and de ined each gene egion
as ± 50 kb om bo h 3′ and 5′ UTRs. As a esul , 276
associa ion es s we e pe o med and 25 candida e
genes in he X ch omosome we e iden i ied o be asso-
cia ed wi h CeD in women (FDR q- alue < 0.05) (Addi-
ional ile5). Ou o he 25 genes, 22 a e no el genes
associa ed wi h CeD, al hough ARHGAP4, RENBP,
NAA10, AVPR2 o MECP2 had been p e iously
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He nangomez‑Lade ase al. Biology o Sex Di e ences (2023) 14:86
iden i ied in o he au oimmune diseases [38–40].
XWAS and gene-le el analysis esul s a e summa ized
in Fig.1.
Cis‑eQTL analysis o heX ch omosome inmonocy es
o women
In o de o ob ain an addi ional laye o in o ma ion o
in e p e he XWAS signal, we calcula ed he X ch o-
mosome eQTLs in monocy es om women. We ana-
lyzed 226 samples wi h in o ma i e geno ype and gene
exp ession da a om naï e monocy es, and pe o med
mul iple linea eg essions be ween 165,648 SNPs and
1250 p obes co esponding o 819 genes. Applying a
h eshold PeQTL alue o 5 × 10–8, we iden i ied 1097
cis-eQTLs in ol ing 1054 SNPs, 19 p obes and 18 inde-
penden genes (Addi ional ile6). The analysis in 130
LPS-s imula ed emale monocy e samples e ealed 150
cis-eQTLs, co esponding o 94 SNPs, 9 p obes and
8 genes (Addi ional ile 7). Two genes (ZNF185 and
TMEM187) we e common o bo h si ua ions, 16 we e
unique o naï e emale monocy es, and 6 o emale
monocy es a e LPS s imula ion.
XWAS andcis‑eQTL SMR andgene exp ession analyses
The SMR and HEIDI analyses o he summa y s a is-
ics o he emale CeD XWAS and he naï e monocy e
cis-eQTL da ase iden i ied wo SNPs ( s7350355 and
s5945386) ha we e associa ed wi h wo exp ession
p obes (ILMN_2198185 and ILMN_3242211, espec-
i ely) co esponding o he same gene, TMEM187,
wi h a q- alue < 0.05 and PHEIDI > 0.05. The mino alleles
s7350355*G and s5945386*G a e bo h he CeD isk
alleles and we e nega i ely co ela ed wi h TMEM187
exp ession (Fig. 2; Addi ional ile 8). The SMR analy-
sis was eplica ed wi h cis-eQTLs om LPS-s imu-
la ed emale monocy es and e ealed a single SNP
( s80208125) ha was associa ed wi h he same wo
TMEM187 p obes (Addi ional ile9; Addi ional ile10).
We in es iga ed he exp ession o TMEM187 in
PBMCs om emale CeD pa ien s a diagnosis, on
GFD, po en ial CeD pa ien s and non-celiac emale
con ols. TMEM187 showed a signi ican ly highe
exp ession in ac i e CeD pa ien s compa ed o con ols
(P- alue = 0.0417) and no di e ences we e obse ed in
po en ial CeD and GFD- ea ed indi iduals (Fig.3A).
We also s udied TMEM187 exp ession in men bu no
Fig. 1 Locus zoom plo 200 kb ups eam and downs eam o he XWAS op SNP. The XWAS op SNP, s78237385 is ep esen ed wi h a pu ple
diamond. In he op panel, he colo schema ep esen s he LD be ween he op SNP and he SNPs included in he egion. In he bo om panel,
he colo schema ep esen s he P‑ alue om he gene‑based associa ion analysis
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He nangomez‑Lade ase al. Biology o Sex Di e ences (2023) 14:86
signi ican di e ences we e ound (Fig. 3B). On he
o he hand, he exp ession o TMEM187 a ied among
di e en immune cell ypes (Addi ional ile11).
Discussion
The p e alence o CeD is signi ican ly highe in women,
as in he case o o he au oimmune diseases [3]. A
highe p e alence o immune-media ed diso de s has
Fig. 2 SMR locus plo o he esul s o he SMR analysis be ween he CeD XWAS and he naï e emale monocy e eQTLs. In he op panel, g ey do s
ep esen − log10(P‑ alues) o he emale XWAS SNPs. Diamonds ep esen − log10(P‑ alues) o p obes om he SMR analysis and illed diamonds
show hose ha pass he HEIDI es . In he middle panel, he ed c osses ep esen − log10(P‑ alues) o gene p obes in he eQTL analysis. In
he bo om panel, he loca ion o he p obes on he X ch omosome is shown
Fig. 3 Resul s o he exp ession analysis. A Exp ession o TMEM187 in PBMCs o emale samples. B Exp ession o TMEM187 in PBMCs o male
samples. Bo h emale and male samples we e classi ied in o ou g oups: non‑celiac con ols, celiac pa ien s a onse , po en ial celiac pa ien s
and celiac pa ien s on GFD ep esen ed by whi e, ligh g ey, da k g ey and black ci cles, espec i ely
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He nangomez‑Lade ase al. Biology o Sex Di e ences (2023) 14:86
also been obse ed in indi iduals wi h synd omes
ela ed o X ch omosome aneuploidies [14, 17], sug-
ges ing an implica ion o he X ch omosome in he
isk o au oimmune diseases, p obably also in he isk
o CeD. In he cu en s udy, we ha e ocused on gene
exp ession in emale monocy es in o de o iden i y
genes on he X ch omosome ha a e in ol ed in CeD
and could explain he sex bias in he disease p e alence.
Fo ha pu pose, we conduc ed a sex-speci ic XWAS in
women om he Immunochip da ase and iden i ied 25
genes associa ed wi h CeD in women, o which 22 ha e
no been p e iously epo ed. Rema kably, 24 ou o he
25 genes ha we e signi ican in he analysis a e loca ed
on ch omosome egion Xq28, which has been p e i-
ously associa ed wi h di e en au oimmune condi ions
such as SLE [39], heuma oid a h i is [40], sys emic
scle osis [38] o CeD [7].
On he o he hand, i has been sugges ed ha gliadin
igge s an inna e esponse in monocy es simila o ha
p oduced by LPS [23]. We pe o med an X ch omo-
some cis-eQTL analysis in emale naï e and LPS-s imu-
la ed monocy es and iden i ied 6 eQTL-genes unique o
LPS-s imula ed emale monocy es, some o which ha e
been p e iously associa ed wi h au oimmune diso de s,
including PLXNA3 [41].
We hen in eg a ed he XWAS wi h cis-eQTL da a
om emale naï e and LPS-s imula ed monocy es using
an MR app oach. To ou knowledge, his is he i s
SMR analysis o he X ch omosome in CeD. We iden i-
ied wo SNPs ( s7350355 and s5945386) ha egula e
he exp ession o TMEM187 in naï e monocy es and a
di e en SNP ( s80208125) associa ed wi h TMEM187
in LPS-s imula ed monocy es, sugges ing ha he
gene ic backg ound may be mo e impo an han LPS
s imula ion in he egula ion o TMEM187 exp ession.
s7350355 is a missense a ian loca ed wi hin exon 2 o
TMEM187, s5945386 is an in e genic a ian loca ed
21 kb downs eam o TMEM187, and s80208125 is
loca ed on he 5’ UTR o TMEM187. All hese a ian s
exhibi s ong LD (> 0.87) in he B i ish popula ion in
England and Sco land, om he 1000 Genomes P ojec
[42]. In all h ee cases, he CeD isk alleles ( s7350355*G,
s5945386*G and s80208125*G) we e he mino alleles
(MAF a ound 0.2) and we e nega i ely co ela ed wi h
TMEM187 exp ession. TMEM187 encodes a mul ipass
ansmemb ane p o ein o unknown unc ion [43], and i
has been p oposed as a pu a i e candida e gene in CeD
oge he wi h IRAK1 and HCFC1, loca ed in he same
locus [7]. As a as we know, he only s udy ha obse ed
TMEM187 dys egula ion in CeD is he one ca ied ou by
Pascual e al. in duodenal biopsies [44]. The TMEM187
locus has also been associa ed wi h o he au oimmune
diso de s such as SLE o heuma oid a h i is [39, 45],
suppo ing he hypo hesis o a sha ed gene ic back-
g ound in au oimmune diso de s.
In ou s udy, we in e oga ed he exp ession o
TMEM187 in PBMCs om celiac pa ien s a onse , celiac
pa ien s on GFD, po en ial celiac pa ien s and non-celiac
con ols. PBMCs a e a mix u e o immune cells ha
con ain monocy es (10–20%), oge he wi h lympho-
cy es (70–90%) and dend i ic cells (1–2%), among o he s
[46]. We obse ed a highe exp ession o TMEM187 in
PBMCs om emale pedia ic pa ien s a disease onse
compa ed o non-celiac child en. These esul s a e con-
sis en wi h a s udy published in 2016 by Pascual e al.
ha showed an up egula ion o TMEM187 exp ession
in biopsies o celiac adul s [44]. The up egula ion o
TMEM187 in emale CeD pa ien s was no eplica ed in
male PBMCs, sugges ing possible ole o TMEM187 in
he sex bias o CeD ha ne e heless needs o be con-
i med wi h addi ional in es iga ions.
The o e exp ession epo ed o TMEM187 in emale
CeD pa ien s a onse is appa en ly con adic o y o he
ac ha CeD isk alleles co ela e wi h lowe exp ession.
This obse ed di e gence could be due o di e en ea-
sons: i s , he CeD isk eQTLs could ha e an e ec a he
p o ein le el, aking in o accoun ha s7350355 is a mis-
sense a ian ha could al e he unc ion o TMEM187,
ega dless o mRNA quan i y. On he o he hand, we a e
unable o de ini i ely asce ain which o he eQTLs is he
causal SNP, gi en he s ong LD. Ano he eason could
be ha PBMCs con ain a ela i ely modes p opo ion o
monocy es, and he SNP could ha e di e en e ec s on
he gene exp ession in o he cell ypes, he e o e explain-
ing he appa en con adic ion. The highly a iable
exp ession o TMEM187 in di e en immune cell ypes
wa an s u he esea ch on i s ole in he immune sys-
em. In addi ion, we ha e o bea in mind ha he p esen
exp ession analyses ha e been ca ied ou in a pedia ic
coho o diagnosed celiac child en, while emale dono s
in he monocy e exp ession s udy we e non-celiac adul s.
I has been well epo ed ha disease and immunogenic
insul can some imes su pass he geno ypic e ec , and
lead o his kind o appa en ly con adic o y si ua ions
[47]. Finally, Pascual e al. obse ed di e ences in he
gene exp ession p o ile o suscep ibili y genes in CeD
be ween child en and adul s, including TMEM187 [44].
Las bu no leas , i is wo h men ioning ha he main
objec i e in he p esen wo k was no o iden i y SNPs
wi h a unc ional in ol emen in CeD, no o asce ain
he mechanism by which hey exe hei unc ion, bu
o highligh po en ially causal genes ha pa icipa e in
he pa hogenesis o he disease h ough hei exp ession
in monocy es. We ca ied ou bo h he XWAS and he
eQTL calcula ions wi h he aim o ob aining ins umen s
o pe o m downs eam analyses such as SMR, being
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He nangomez‑Lade ase al. Biology o Sex Di e ences (2023) 14:86
awa e ha in ou esul s SNPs will lose hei ele ance
and will be eplaced by unc ional candida e genes.
One limi a ion o his s udy is ha bo h he lack o
associa ion as well as he absence o signi ican di e en-
ial exp ession be ween CeD cases and con ols obse ed
in men could be due o he smalle sample size o he
male coho . This is pa ly a consequence o he highe
incidence o CeD in women and an impo an ac o o
conside . Howe e , we ha e s udied he associa ion o
he op SNP o ou emale XWAS ( s78237385) in an
independen da ase [6] and he P- alues in men and
women a e 0.19 and 1.86 × 10–4, espec i ely. Addi ion-
ally, he SNPs wi h signi ican esul s in he SMR analysis
( s5945386, s7350355 and s80208125) ha e P- alues in
women o 8.29 × 10–5, 4.66 × 10–3 and 3.34 × 10–3, espec-
i ely, while in men, hey show P- alues o 0.50, 0.49 and
0.57, espec i ely. We conside hese P- alues unlikely o
become signi ican e en wi h a highe numbe o male
samples, and his lack o a signi ican associa ion in men
could imply a possible di e gen mechanism o pa hogen-
esis be ween sexes, ha could explain he inc eased p e -
alence o CeD, and o he au oimmune diseases obse ed
in women [48].
Pe spec i es andsigni icance
This is he i s SMR app oach in he X ch omosome in
CeD. We ha e iden i ied TMEM187 as a candida e gene
in CeD in monocy es and alida ed i s di e en ial exp es-
sion in PBMCs om emale CeD pa ien s a onse . The
ac ha bo h he gene ic associa ion and he di e en ial
gene exp ession a e no ound in male pa ien s sugges s a
ole o TMEM187 in he sex bias obse ed in CeD. SMR
appea s as a use ul app oach o iden i y po en ially causal
genes unde associa ion peaks, including he X ch omo-
some. Fu he s udies a e needed o iden i y he unc ion
o TMEM187 and unde s and i s beha io in di e en
cell ypes and disease s a us, and o cla i y i s ole in CeD
pa hogenesis and he sex bias.
Supplemen a y In o ma ion
The online e sion con ains supplemen a y ma e ial a ailable a h ps:// doi.
o g/ 10. 1186/ s13293‑ 023‑ 00572‑1.
Addi ional ile1. Sex‑speci ic summa y s a is ics om Immunochip
XWAS in women and men. BP: posi ion o he a ian ; OA: o he allele; EA:
e ec allele; F: equency o he e ec allele; B: be a alue, SE: s anda d
e o o he be a, P: P‑ alue.
Addi ional ile2. Manha an plo o he CeD XWAS in women. The op
SNP s78237385 (P‑ alue = 2.30 × 10−5) is shown as a ed ci cle. The blue
line ep esen s he signi ican h eshold acco ding o he Bon e oni co ‑
ec ion o he numbe o independen es s (P‑ alue = 8.68 × 10−5).
Addi ional ile3. Manha an plo o he CeD XWAS o CeD in men. The
blue line ep esen s he signi ican h eshold acco ding o he Bon e oni
co ec ion o he numbe o independen es s (P‑ alue = 8.68 × 10−5).
Addi ional ile4. Nominal P‑ alues o he op Immunochip emale XWAS
SNP ( s78237385) in he di e en da ase s s a i ied by sex. EAF: equency
o he e ec allele in cases; EAF: equency o he e ec allele in con ols;
OR: odd a io.
Addi ional ile5. CeD candida e genes on he X ch omosome iden i ied
by gene‑based associa ion analysis a q‑ alue < 0.05. Top associa ed SNP:
he op associa ed XWAS SNP in he egion; Top PXWAS: P‑ alue o he
op associa ed XWAS SNP in he egion; P as BAT: gene‑based es P‑ alue;
q‑ alue: FDR adjus ed gene‑based es P‑ alue.
Addi ional ile6. cis‑eQTLs iden i ied on he X ch omosome by cis‑eQTL
analysis o naï e emale monocy es a nominal P‑ alue < 5 × 10–8. A o al
o 1,097 cis‑eQTLs, implica ing 1,054 SNPs, 19 p obes and 18 genes we e
iden i ied on he X ch omosome o naï e emale monocy es. n_ a _in_cis:
he o al numbe o a ian s es ed in cis; dis _gene_ a : he dis ance
be ween he gene and he es ed a ian ; a _posi ion: he posi ion o he
a ian ; nom_p al: he nominal P‑ alue o he associa ion be ween he
a ian and he gene; _squa ed: he co ela ion coe icien o he linea
eg ession; slope: he slope (be a) o he linea eg ession; slope_se: he
s anda d e o o he be a.
Addi ional ile7. cis‑eQTLs iden i ied in he X ch omosome by cis‑eQTL
analysis o LPS‑s imula ed emale monocy es a nominal P‑ alue < 5 × 10–8.
A o al o 150 cis‑eQTLs, implica ing 94 SNPs, 9 p obes and 8 genes we e
iden i ied on he X ch omosome o LPS‑s imula ed emale monocy es.
n_ a _in_cis: he o al numbe o a ian s es ed in cis; dis _gene_ a :
he dis ance be ween he gene and he es ed a ian ; a _posi ion: he
posi ion o he a ian ; nom_p al: he nominal P‑ alue o he associa ion
be ween he a ian and he gene; _squa ed: he co ela ion coe icien
o he linea eg ession; slope: he slope (be a) o he linea eg ession;
slope_se: he s anda d e o o he be a.
Addi ional ile8. Summa y o he SMR analysis be ween he CeD XWAS
and he naï e emale monocy e eQTLs. A1: he e ec allele; A2: he o he
allele; b_XWAS: he e ec size om XWAS; p_XWAS: P‑ alue om he
XWAS; b_eQTL: he e ec size om eQTL analysis; p_eQTL: P‑ alue om
he eQTL analysis; b_SMR: he e ec size om he SMR analysis; p_SMR:
nominal P‑ alue om he SMR analysis; q‑ alue: FDR adjus ed P‑ alue.
Resul s in bold indica e s a is ical signi icance a e mul iple es ing
co ec ion.
Addi ional ile9. SMR locus plo o he esul s o he SMR analysis
be ween he CeD XWAS and he LPS‑s imula ed emale monocy e eQTLs.
In he op panel, g ey do s ep esen ‑log10(P‑ alues) o he emale XWAS
SNPs. Diamonds ep esen ‑log10(P‑ alues) o p obes om he SMR
analysis and illed diamonds show hose ha pass he HEIDI es . In he
middle panel, he ed c osses ep esen ‑log10(P‑ alues) o gene p obes in
he eQTL analysis. In he bo om panel, he loca ion o he p obes on he
X ch omosome is shown.
Addi ional ile10. Summa y o he SMR analysis be ween he CeD XWAS
and he LPS‑s imula ed emale monocy e eQTLs. A1: he e ec allele; A2:
he o he allele; b_XWAS: he e ec size om XWAS; p_XWAS: P‑ alue om
he XWAS; b_eQTL: he e ec size om eQTL analysis; p_eQTL: P‑ alue
om he eQTL analysis; b_SMR: he e ec size om he SMR analysis;
p_SMR: nominal P‑ alue om he SMR analysis; q‑ alue: FDR adjus ed
P‑ alue. Resul s in bold indica e s a is ical signi icance a e mul iple es ‑
ing co ec ion.
Addi ional ile11. TMEM187 exp ession in di e en immune cells. Red
and blue boxes ep esen TMEM187 exp ession in he di e en immune
cells om emales and males, espec i ely.
Acknowledgemen s
We acknowledge all he pa ien s and hei amilies, as well as he Basque
Biobank o cus ody and p epa a ion o he clinical samples.
Au ho con ibu ions
JRB and NF‑J designed and supe ised he esea ch; AH‑L pe o med he
compu a ional and expe imen al analyses; AH‑L, AC‑P and SM p epa ed he
code and o ma ed he da a o he compu a ional analyses, BPG and IG‑S
helped o design and pe o m he expe imen al analyses; SMV, ML, CT and
Page 9 o 10
He nangomez‑Lade ase al. Biology o Sex Di e ences (2023) 14:86
II diagnosed pa ien s and collec ed biological samples; JRB, NF‑J and AH‑L
w o e he i s d a o he manusc ip , and all au ho s con ibu ed o he
a icle and app o ed he inal e sion.
Funding
JRB is unded by Resea ch G an PID2019‑106382RB‑I00 om he MCIN/
AEI/h ps://doi.o g/10.13039/501100011033. AH‑L is a p edoc o al el‑
low suppo ed by g an PRE‑C‑2020‑0091 om he MCIN/AEI/h ps://doi.
o g/10.13039/501100011033and by ESF In es ing in you u u e. NFJ is unded
by esea ch g an s 2019/111085 om he Basque Depa men o Heal h, and
PI21/01491 om he Ins i u o de Salud Ca los III (ISCIII), co‑ unded by he
Eu opean Union.
A ailabili y o da a and ma e ials
The signi ican esul s o XWAS, gene‑based associa ion analysis, eQTL analysis,
and SMR analysis a e included a icle and i s addi ional iles. The comple e
summa y s a is ics gene a ed du ing he cu en s udy a e a ailable om he
co esponding au ho on easonable eques . Monocy e da a we e ob ained
om public eposi o ies, acco ding o a Da a T ans e Ag eemen be ween he
Uni e si y o he Basque Coun y (UPV/EHU) and he Uni e si y o Ox o d. The
geno yping da a we e downloaded om he Eu opean Genome‑phenome
A chi e (h ps:// www. ebi. ac. uk/ ega/ da as e s/; expe imen EGAS00000000109)
and he gene exp ession mic oa ay da a om he same indi iduals we e
downloaded om he EBI A ayExp ess da abase (h ps:// www. ebi. ac. uk/ a ay
exp e ss/ expe imen s/; expe imen numbe E‑MTAB‑2232). This s udy makes
use o he Immnochip da a gene a ed by he Wellcome T us Case–Con ol
Conso ium (WTCC da a se s EGAD00010000246, EGAD00010000248 and
EGAD00010000250). A ull lis o he in es iga o s who con ibu ed o he
gene a ion o he da a is a ailable om h p:// www. w ccc. o g. uk. Funding o
he p ojec was p o ided by he Wellcome T us unde awa ds 076113, 085475
and 090355.
Decla a ions
E hics app o al and consen o pa icipa e
The s udies in ol ing human pa icipan s we e e iewed and app o ed by
E hical Commi ee o Clinical Resea ch o C uces Uni e si y Hospi al. W i en
in o med consen o pa icipa e in his s udy was p o ided by he pa icipan s’
legal gua dian/nex o kin.
Consen o publica ion
No applicable.
Compe ing in e es s
The au ho s decla e ha he esea ch was conduc ed in he absence o any
comme cial o inancial ela ionships ha could be cons ued as a po en ial
con lic o in e es .
Au ho de ails
1 Depa men o Gene ics, Physical An h opology and Animal Physiology,
Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Basque Coun y, Spain.
2 Biobizkaia Heal h Resea ch Ins i u e, Ba akaldo, Basque Coun y, Spain.
3 Pedia ic Gas oen e ology Uni , C uces Uni e si y Hospi al, Ba akaldo, Basque
Coun y, Spain. 4 Spanish Biomedical Resea ch Cen e in Diabe es and Associ‑
a ed Me abolic Diso de s (CIBERDEM), Mad id, Spain.
Recei ed: 28 July 2023 Accep ed: 28 No embe 2023
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