Open Access
A ailable online h p://a h i is- esea ch.com/con en /11/3/R69
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Vol 11 No 3
Resea ch a icle
Replica ion o ecen ly iden i ied sys emic lupus e y hema osus
gene ic associa ions: a case–con ol s udy
Ma ian Sua ez-Ges al1, Manuel Calaza1, Emöke End e y2, Rudol Pullmann3, Josep O di-Ros4,
Gian Domenico Sebas iani5, Sa ka Ruzicko a6, Ma ia Jose San os7,8, Ch yssa Papas e iades9,
Mau izio Ma chini10, Fo ini N Skopouli11, Ana Sua ez12, F ancisco J Blanco13, Sand a D'Al onso14,
Ma c Bijl15, Pa icia Ca ei a16, To s en Wi e17, Se gio Miglia esi18, Juan J Gomez-Reino1,19,
An onio Gonzalez1 o he Eu opean Conso ium o SLE DNA Collec ions
1Labo a o io de In es igacion 10 and Rheuma ology Uni , Hospi al Clinico Uni e si a io de San iago, San iago de Compos ela 15706, Spain
2Paedia ics Depa men , Albe Szen -Gyö gyi Medical and Pha maceu ical Cen e, Uni e si y o Szeged, Szeged 6721, Hunga y
3Ins i u e o Clinical Biochemis y, Ma in Facul y Hospi al, Jessenius Medical Facul y, Kollá o a 2, 036 59 Ma in, Slo akia
4In e nal Medicine, Resea ch Labo a o y in Au oimmune Diseases, Hospi al Vall d'Heb on, 08035 Ba celona, Spain
5Ospedale S Camillo-Fo lanini, U O Complessa di Reuma ologia, 00151 Roma, I aly
6Molecula Biology and Immunogene ics Depa men , Ins i u e o Rheuma ology, 128 50 P ague 2, Czech Republic
7Rheuma ology Depa men , Hospi al Ga cia de O a, Almada, Po ugal
8Rheuma ology Resea ch Uni , Ins i u o Medicina Molecula , Faculdade de Medicina da Uni e sidade de Lisboa, Po ugal
9Depa men o His ocompa ibili y and Immunology, E angelismos Hospi al, 10676 A hens, G eece
10Clinical Immunology, Uni e si y o Milan and Fondazione IRCCS Ospedale Maggio e Policlinico, Mangiagalli e Regina Elena, 20122 Milan, I aly
11Pa hophysiology Depa men , A hens Uni e si y Medical School, A hens 115 27, G eece
12Depa men o Func ional Biology, Hospi al Uni e si a io Cen al de As u ias, Uni e sidad de O iedo, O iedo 33006, Spain
13INIBIC-CH Uni e si a io A Co uña, 15006 A Co uña, Spain
14Dep Medical Sciences and IRCAD, Eas e n Piedmon Uni e si y, 28100 No a a, I aly
15Depa men o Rheuma ology and Clinical Immunology, Uni e si y Medical Cen e G oningen, 9713 G oningen, The Ne he lands
16Rheuma ology Uni Hospi al 12 de Oc ub e, 28041 Mad id, Spain
17Di ision o Clinical Immunology, Depa men o In e nal Medicine o he Hanno e Medical School, D-30625 Hanno e , Ge many
18Rheuma ology Uni , Second Uni e si y o Naples, 81100 Naples, I aly
19Depa men o Medicine, Uni e si y o San iago de Compos ela, San iago de Compos ela, 15706, Spain
Co esponding au ho : An onio Gonzalez, anlugon@ho mail.com
Recei ed: 24 Ma 2009 Re isions eques ed: 29 Ap 2009 Re isions ecei ed: 8 May 2009 Accep ed: 14 May 2009 Published: 14 May 2009
A h i is Resea ch & The apy 2009, 11:R69 (doi:10.1186/a 2698)
This a icle is online a : h p://a h i is- esea ch.com/con en /11/3/R69
© 2009 Sua ez-Ges el e al.; licensee BioMed Cen al L d.
This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0),
which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Abs ac
In oduc ion We aimed o eplica e associa ion o newly
iden i ied sys emic lupus e y hema osus (SLE) loci.
Me hods We selec ed he mos associa ed SNP in 10 SLE loci.
These 10 SNPs we e analysed in 1,579 pa ien s wi h SLE and
1,726 con ols o Eu opean o igin by single-base ex ension.
Compa ison o allele equencies be ween cases and con ols
was done wi h he Man el–Haenszel app oach o accoun o
he e ogenei y be ween sample collec ions.
Resul s A p e iously con o e sial associa ion wi h a SNP in he
TYK2 gene was eplica ed (odds a io (OR) = 0.79, P = 2.5 ×
10-5), as well as associa ion wi h he X ch omosome MECP2
gene (OR = 1.26, P = 0.00085 in women), which had only been
epo ed in a single s udy, and associa ion wi h ou o he loci,
1q25.1 (OR = 0.81, P = 0.0001), PXK (OR = 1.19, P =
0.0038), BANK1 (OR = 0.83, P = 0.006) and KIAA1542 (OR
= 0.84, P = 0.001), which ha e been iden i ied in a genome-
wide associa ion s udy, bu no ound in any o he s udy. All
hese eplica ions showed he same disease-associa ed allele
as o iginally epo ed. No associa ion was ound wi h he LY9
SNP, which had been epo ed in a single s udy.
Conclusions Ou esul s con i m nine SLE loci. Fo six o hem,
TYK2, MECP2, 1q25.1, PXK, BANK1 and KIAA1542, his
BANK1: B-cell sca old p o ein wi h anky in epea s 1; BLK: B-lymphoid y osine kinase; GWA: genome-wide associa ion; IL: in e leukin; ITGAM:
in eg in alpha M; LY9: lymphocy e an igen 9; MECP2: me hyl CpG binding p o ein 2; OR: odds a io; PXK: PX domain con aining se ine/ h eonine
kinase; SLE: sys emic lupus e y hema osus; SLEGEN: In e na ional Conso ium o Sys emic Lupus E y hema osus Gene ics; SNP: single nucleo ide
polymo phism; STAT4: signal ansduce and ac i a o o ansc ip ion; TYK2: y osine kinase 2.
A h i is Resea ch & The apy Vol 11 No 3 Sua ez-Ges al e al.
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eplica ion is impo an . The o he h ee loci, ITGAM, STAT4 and
C8o 13-BLK, we e al eady clea ly con i med. Ou esul s also
sugges ha MECP2 associa ion has no in luence in he sex
bias o SLE, con a y o wha has been p oposed. In addi ion,
none o he o he associa ions seems impo an in his espec .
In oduc ion
Sys emic lupus e y hema osus (SLE) is a complex au oim-
mune disease o wide a iabili y in i s mani es a ions and clini-
cal e olu ion ha cha ac e is ically in ol es mul iple
au oan ibodies agains ubiqui ous nuclea an igens. I s
gene ic componen is e y signi ican , as shown by a sibling
ecu ence isk a io o 20 and a 10- old excess in SLE con-
co dance be ween monozygo ic wins o e dizygo ic wins
[1,2].
Linkage s udies ha e indica ed ha his gene ic componen is
due o mul iple low-pene ance common gene ic ac o s [1].
Only a ew ac o s had consis en ly been demons a ed un il
2008: he class II HLA alleles, low-a ini y ecep o s o he
cons an ac ion o IgG, and he PTPN22 and IRF5 genes.
This scena io has been d ama ically imp o ed by new echnol-
ogies and genome esou ces [2]. Fou genome-wide associa-
ion (GWA) s udies we e published in 2008 [3-6] ha ,
oge he wi h o he la ge-scale s udies, ha e g ea ly enla ged
he numbe o con incing SLE-associa ed loci. No all o he
newly desc ibed indings, howe e , ha e a ained he same
deg ee o con i ma ion [2]. Some o hem a e al eady de ini-
i ely con i med by eplica ion in di e en sample collec ions
by he same au ho s and also by independen au ho s in sep-
a a e s udies (Table 1). In his g oup a e he SLE associa ions
wi h he ITGAM [3,4,6,7], STAT4 [3,4,6,8-12] and C8o 13-
BLK egions [3,4,6]. O he indings a e e y solid bu hey s ill
equi e con i ma ion by independen s udies. In his g oup a e
he associa ed loci ha we e only epo ed in a single GWA
s udy bu no in he o he s udies, such as BANK1 [5], PXK
[3], KIAA1542 [3] and 1q25.1 [3], o hose ha we e epo ed
in a single la ge s udy bu no in any o he ou GWA s udies,
such as MECP2 [13] and LY9 [14]. Finally, he TYK2 associ-
a ion is mo e con o e sial because i was ound in a la ge
s udy wi h Scandina ian amilies [15], pa ially eplica ed in a
la ge s udy o UK amilies [16], and excluded in one o he
GWA s udies [3].
In he p esen pape , he e o e, we ha e analysed SLE associ-
a ion o each o hese loci in mo e han 1,500 SLE pa ien s
and 1,700 con ols – and all o hem excep LY9 ha e been
clea ly eplica ed. In addi ion, we ha e ound ha many o
hese loci a e also impo an o SLE in men whe e da a om
p e ious epo s is almos comple ely absen .
Ma e ials and me hods
Sample collec ion
We used DNA samples om SLE pa ien s and e hnically
ma ched heal hy con ols o 16 collec ions om nine Eu o-
pean coun ies (see Table S1 in Addi ional da a ile 1). Mos o
hese samples ha e al eady been desc ibed [17]. Two new
sample collec ions we e om As u ias, Spain and Almada,
Po ugal. Each ec ui ing cen e was asked o abou 100 SLE
pa ien s and 100 e hnically ma ched con ols. A o al o 1,579
cases and 1,726 con ols we e ob ained in his way. All SLE
pa ien s me he e ised Ame ican College o Rheuma ology
classi ica ion c i e ia [18]. Clinical cha ac e is ics o he
pa ien s a e p o ided in Table S2 in Addi ional da a ile 1.
Pa ien s and con ols ga e w i en in o med consen . Sample
collec ion was app o ed by he espec i e e hical commi ees.
Geno yping
We selec ed a SNP o each o he 10 associa ed loci ha we
in ended o eplica e (Table 1). The SNPs we e selec ed
because hey we e s ongly associa ed wi h SLE o because
hey we e desc ibed as p obable causal polymo phisms.
These 10 SNPs we e ampli ied in a single PCR wi h he Qia-
gen Mul iplex PCR ki (Qiagen, Cha swo h, CA, USA) wi h 20
ng genomic DNA and 0.2 μM o each p ime ( o p ime s and
p obes, see Table S3 in Addi ional da a ile 1). The PCR p od-
uc s we e pu i ied by diges ion wi h Exonuclease I (Epicen e,
Madison, WI, USA) and sh imp alkaline phospha ase (GE
Heal hca e, Ba celona, Spain). Pu i ied PCR p oduc s we e
geno yped by single-base ex ension wi h he SNaPsho Mul i-
plex Ki (Applied Biosys ems, Fos e Ci y, CA, USA) and spe-
ci ic p obes. A e a second pu i ica ion wi h sh imp alkaline
phospha ase (GE Heal hca e), samples we e analysed in he
Abi P ism 3130xl Gene ic Analyze (Applied Biosys ems) and
geno ypes assigned by he GeneMappe so wa e. All geno-
ype calls we e manually e iewed and con lic ing esul s we e
libe ally e-assayed o e-geno yped by sequencing wi h he
Big Dye Ready Reac ion Ki 3.1 (Applied Biosys ems).
Sequence eac ions ollowed he ki manu ac u e p o ocol
and we e also analysed in he Abi P ism 3130xl Gene ic Ana-
lyze .
S a is ical analysis
Some o he sample collec ions in ou s udy ha e al eady been
used o he analysis o speci ic associa ions included in his
p ojec . They ha e been excluded om he ele an analyses
o a oid da a duplica ion; his ci cums ance is de ailed in Table
S4 in Addi ional da a ile 1, whe e aw geno ype da a om
each sample collec ion a e epo ed. Ha dy-Weinbe g equilib-
ium es s in con ol samples we e pe o med wi h Haplo iew
wi h a h eshold o 0.05 unco ec ed o mul iple es s [19].
O he s a is ical analyses we e ca ied ou in a cus omized e -
sion o he S a is ica 7.0 p og am (S a So , Tulsa, OK, USA).
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Compa ison o cases and con ols was ca ied ou wi h he
Man el–Haenszel app oach because allele equency di e -
ences a e p obable be ween sample collec ions e en i spe-
ci ic e ec s on he pheno ype a e cons an . Spu ious alse
posi i e o alse nega i e esul s he e o e become likely i he
allele di e ences a e no accoun ed o . To a oid his, he Man-
el–Haenszel app oach combines e ec sizes aken as he
odds a io (OR) in each s a um allowing o he e ogenei y in
allele equencies. This app oach p o ides an accu a e com-
bined s a is ic i he he e ogenei y o e ec sizes, e alua ed
wi h he B eslow–Day es , is excluded. Signi ican he e oge-
nei y o e ec s is he e o e excluded by he B eslow–Day es
and allele equency he e ogenei y is accoun ed o wi h he
Man el–Haenszel app oach. These analyses we e also con-
duc ed a e s a i ying he samples by gende . Uni a ia e
logis ic eg ession models we e used o es he i o he da a
o addi i e, ecessi e and dominan gene ic models. S a is ical
powe was es ima ed wi h he Powe and sample size calcula-
ions so wa e [20].
Table 1
Newly sys emic lupus e y hema osus-associa ed loci ha we e examined wi h p e ious e idence o associa ion
Locus Ch . Loca ion SNP AllelesaOR (95% CI) Associa ed Sample size Popula ion Re e ence
ITGAM 16 Exon 3 s1143679 G/A 1.78 (1.6 o 2.0) Yes 3,818 Eu opean Ame ican [7]
1.55 (1.2 o 2.0) Yes 1,289 A ican Ame ican [7]
2.07 (1.3 o 3.4) Yes 271 Gullah [7]
O he SNPs Yes [3,4,6]
STAT4 2 In on 3 s7574865 G/T 1.5 (1.2 o 1.8) Yes 3,057 Eu opean [3]
1.50 (1.4 o 1.7) Yes 4,651 Eu opean Ame ican [4]
1.55 (1.3 o 1.8) Yes 2,287 Eu opean Ame ican [8]
1.61 (1.4 o 1.9) Yes 2,495 Japanese [9]
1.62 (1.2 o 2.2) Yes 565 Colombians [10]
1.56 (1.4 o 1.7) Yes 3,958 Eu opean [12]
O he SNPs Yes [6,11]
C8o 13-BLK 8 In e genic s13277113 G/A 1.39 (1.3 o 1.5) Yes 6,301 Eu opean Ame ican [4]
O he SNPs Yes [3,6]
TYK2 19 Exon 8 s2304256 C/A 0.625 (0.5 o 0.8) Yes 966 Swedish/Finnish [15]
105:127bNo 380cEu opean/Indo-
Pakis ani
[16]
O he SNPs No [3]
MECP2 X In on 2 s17435 A/T 1.58 (1.3 o 1.9) Yes 1,364 Ko ean [13]
1.29 (1.1 o 1.5) Yes 2,160 Eu opean [13]
1q25.1 1 In e genic s10798269 G/A 0.82 (0.8 o 0.9) Yes 6,728 Eu opean [3]
BANK1 4 In on 1 s17266594 T/C 0.74 (0.6 o 0.9) Yes 927 Scandina ian [5]
0.70 (0.5 o 0.9) Yes 576 Ge man [5]
0.63 (0.5 o 0.8) Yes 450 I alian [5]
0.78 (0.6 o 0.9) Yes 1,136 Spanish [5]
0.58 (0.4 o 0.8) Yes 620 A gen inian [5]
KIAA1542 11 In on 4 s4963128 G/A 0.78 (0.7 o 0.9) Yes 6,728 Eu opean [3]
PXK 3 In on 5 s6445975 T/G 1.25 (1.2 o 1.4) Yes 6,728 Eu opean [3]
LY9 1 Exon 8 s509749 A/G 377:403dYes 510cUK Caucasian [14]
237:251dNo 270cCanadian [14]
Loci o de ed as p esen ed in Table 2. The SNPs selec ed o eplica ion a e de ailed. Ch , ch omosome; CI, con idence in e al; OR, odds a io.
aMajo /mino alleles. bT ansmi ed:un ansmi ed. cNumbe o amilies. dObse ed:expec ed.
A h i is Resea ch & The apy Vol 11 No 3 Sua ez-Ges al e al.
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Resul s
A o al o 1,579 SLE pa ien s and 1,726 con ols om 16
Eu opean collec ions we e a ailable o s udy (Tables S1 and
S2 in Addi ional da a ile 1). The geno yping call a e was
99.9% and he geno ypes in con ols we e in Ha dy-Weinbe g
equilib ium. Indi idual collec ion da a o each SNP is shown
in Tables S4 and S5 in Addi ional da a ile 1. Combined anal-
ysis o he SNP e ec s ac oss ou sample collec ions was pe -
o med wi h he Man el–Haenszel app oach, which is a
me hod co ec ing o a iabili y in allele equencies be ween
collec ions p o ided ha he e ec sizes ( ha is, ORs) a e no
signi ican ly di e gen . This condi ion was ul illed because no
signi ican he e ogenei y in OR was de ec ed o any o he
SNPs (Table 2, inal column).
The combined da a showed signi ican di e ences be ween
SLE cases and con ols o eigh o he nine SNPs loca ed in
au osomal ch omosomes (Table 2). All o he signi ican di e -
ences be ween cases and con ols we e in he same di ec ion
as o iginally epo ed (Tables 1 and 2). We ound associa ion
o he ou SNPs ha ha e been epo ed in a single GWA and
no ye eplica ed by independen s udies: s10798269 in
1q25.1 (OR = 0.81, P = 0.00013), s6445975 in PXK (OR =
1.19, P = 0.0038), s17266594 in BANK1 (OR = 0.83, P =
0.0062) and s4963128 in KIAA1542 (OR = 0.84, P =
0.0011). The e was also signi ican associa ion o wo o he
h ee SNPs ha we e desc ibed in la ge s udies bu ha we e
no obse ed in any o he GWA s udies: s2304256 in TYK2
(OR = 0.79, P = 2.5 × 10-5) and s17435 in MECP2 (analysis
o his SNP was pe o med sepa a ely in women and men
because his gene is in ch omosome X; see below). Only
s509749 in LY9 was simila in cases and con ols. Ou s udy
had su icien powe (80%) o de ec associa ion a his SNP
wi h an e ec size equi alen o OR > 1.15 wi h P < 0.05 (o
OR > 1.23 o P < 0.001).
In addi ion o hese impo an esul s o eplica ion, we ound
associa ion wi h he h ee loci ha ha e al eady been epli-
ca ed in GWA s udies: s1143679 in ITGAM (OR = 1.70, P
= 1.1 × 10-16), s7574865 in STAT4 (OR = 1.62, P = 2.4 ×
10-12) and s13277113 in C8o 13-BLK (OR = 1.34, P = 5.1
× 10-7). The e ec sizes o hese h ee associa ion signals
( ha is, hei ORs) we e la ge han o all he o he signals,
pe haps explaining he mo e consis en eplica ion o hei
associa ion. Geno ype compa isons o he di e en SNPs
we e conco dan wi h an addi i e gene ic model and yielded
e y simila esul s o he allele equency analyses (da a no
shown).
Combined analysis was also conduc ed in women (Table 3).
This was pa icula ly necessa y o he MECP2 SNP s17435,
loca ed in he X ch omosome. This SNP showed a signi ican
di e ence be ween SLE women and con ol women and wi h
he same disease-associa ed allele as p e iously epo ed (OR
= 1.26, P = 0.00085). The SNPs placed in he au osomes
showed simila esul s o hose ob ained in he uns a i ied
analysis. The e we e only less signi ican P alues due o he
smalle sample size, bu he e ec sizes (exp essed as ORs)
emained la gely unchanged. The BANK1 SNP was no asso-
cia ed in women, bu his was he SNP wi h ewe a ailable
samples because we ha e excluded om his analysis he
sample collec ions ha ha e p e iously been epo ed (powe
was 0.68 o P = 0.05 and OR = 0.78, which was p e iously
epo ed in Spanish samples) [5].
No p e ious de ailed in o ma ion o men wi h SLE has been
published o any o hese associa ed loci, al hough in a epo
Table 2
Combined analysis o allele equency di e ences be ween SLE cases and con ols o nine au osomal loci
Mino allele equency (%)aMan el – Haenszel analysis B eslow – Day es
SNP (locus) SLE cases Con ols OR (95% CI) P alue P alue
s1143679 (ITGAM) 23.2 (730/3,152) 15.1 (521/3,448) 1.70 (1.5 o 1.9) 1.1 × 10-16 0.5
s7574865 (STAT4) 32.8 (709/2,162) 23.2 (485/2,092) 1.62 (1.4 o 1.9) 2.4 × 10-12 1.0
s13277113 (C8o 13-BLK) 30.9 (874/2,824) 25.7 (776/3,024) 1.34 (1.2 o 1.5) 5.1 × 10-7 1.0
s2304256 (TYK2) 23.3 (733/3,152) 27.8 (960/3,450) 0.79 (0.7 o 0.9) 2.5 × 10-5 0.6
s10798269 (1q25.1) 27.3 (861/3,158) 31.8 (1,098/3,452) 0.81 (0.7 o 0.9) 0.00013 0.2
s17266594 (BANK1) 24 (526/2,192) 27.6 (624/2,260) 0.83 (0.7 o 0.9) 0.0062 0.4
s4963128 (KIAA1542) 30.3 (955/3,150) 34.0 (1,173/3,448) 0.84 (0.8 o 0.9) 0.0011 0.2
s6445975 (PXK) 27.3 (772/2,824) 24.2 (734/3,034) 1.19 (1.1 o 1.3) 0.0038 0.7
s509749 (LY9) 43.1 (1,359/3,154) 43.8 (1,513/3,452) 0.97 (0.9 o 1.1) 0.5 0.4
Loci o de ed by dec easing e ec size (odds a io (OR)). All esul s e e o he mino allele o each SNP, which is indica ed in Table 1. CI,
con idence in e al; SLE, sys emic lupus e y hema osus. aDa a p esen ed as pe cen age (numbe o mino alleles/ o al numbe o alleles).
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desc ibing associa ion o he ITGAM gene i was indica ed
ha esul s we e no di e en be ween women and men [7].
This lack o in o ma ion is p obably due o he a i y o men su -
e ing om SLE. In ou analysis, we ha e conside ed all male
da a oge he wi hou s a i ying o sample collec ion due o
he low numbe o men in each collec ion (Table 4). Resul s in
men we e simila o esul s in women, wi h he possible excep-
ion o he s1143679 in ITGAM (OR = 2.08 e sus 1.67; P =
0.03). Some SNPs we e no associa ed in men (in he TYK2,
1q25.1, BANK1 and LY9 loci), bu s a is ical powe o his
subg oup analysis was low, anging om 0.19 o
s17266594 in BANK1 o 0.25 o s2304256 in TYK2
among he nonassocia ed SNPs (powe was es ima ed o P
= 0.05 and OR = 1.2).
Table 3
Combined analysis o allele equency di e ences be ween SLE women and con ol women
Mino allele equency (%)aMan el – Haenszel analysis B eslow – Day es
SNP (locus) SLE cases Con ols OR (95% CI) P alue P alue
s1143679 (ITGAM) 22.3 (621/2,782) 15.1 (329/2,182) 1.67 (1.4 o 2.0) 2.0 × 10-11 0.6
s7574865 (STAT4) 33.1 (636/1,920) 24.0 (317/1,322) 1.60 (1.4 o 1.9) 8.4 × 10-9 0.8
s13277113 (C8o 13-BLK) 30.9 (777/2,514) 25.7 (475/1,848) 1.33 (1.2 o 1.5) 5.4 × 10-5 0.9
s2304256 (TYK2) 22.9 (638/2,782) 27.2 (592/2,180) 0.81 (0.7 o 0.9) 0.0022 0.2
s17435 (MECP2) 26.7 (744/2,784) 22.8 (498/2,186) 1.26 (1.1 o 1.4) 0.00085 0.7
s10798269 (1q25.1) 27.1 (754/2,786) 32.3 (705/2,182) 0.77 (0.7 o 0.9) 6.2 × 10-5 0.4
s17266594 (BANK1) 23.9 (464/1,938) 26.9 (386/1,436) 0.87 (0.7 o 1.0) 0.077 0.4
s4963128 (KIAA1542) 30.4 (845/2,778) 33.9 (741/2,182) 0.85 (0.8 o 1.0) 0.011 0.2
s6445975 (PXK) 26.7 (670/2,514) 23.5 (436/1,854) 1.22 (1.1 o 1.4) 0.0067 0.9
s509749 (LY9) 43.3 (1,206/2,784) 44.0 (962/2,184) 0.98 (0.9 o 1.1) 0.7 0.5
Loci o de ed as p esen ed in Table 2. All esul s e e o he mino allele o each SNP, which is indica ed in Table 1. CI, con idence in e al; OR,
odds a io; SLE, sys emic lupus e y hema osus. aDa a p esen ed as pe cen age (numbe o mino alleles/ o al numbe o alleles).
Table 4
Compa ison o SNP allele equencies be ween SLE men and con ol men
Mino allele equency (%)aMan el – Haenszel analysis
SNP (locus) SLE cases Con ols OR (95% CI) P alue
s1143679 (ITGAM) 27.9 (83/298) 15.7 (181/1,154) 2.08 (1.5 o 2.8) 1.2 × 10-6
s7574865 (STAT4) 31.7 (64/202) 21.5 (159/740) 1.69 (1.2 o 2.4) 0.0025
s13277113 (C8o 13-BLK) 31.0 (88/284) 25.1 (267/1,064) 1.34 (1.0 o 1.8) 0.045
s2304256 (TYK2) 26.8 (80/298) 28.8 (334/1,158) 0.91 (0.7 o 1.2) 0.5
s17435 (MECP2)b29.2 (42/144) 18.5 (105/568) 1.82 (1.2 o 2.8) 0.0046
s10798269 (1q25.1) 28.0 (84/300) 30.7 (355/1,158) 0.88 (0.7 o 1.2) 0.4
s17266594 (BANK1) 26.2 (56/214) 28.4 (227/798) 0.89 (0.6 o 1.3) 0.5
s4963128 (KIAA1542) 28.0 (84/300) 34.9 (403/1,156) 0.73 (0.6 o 1.0) 0.025
s6445975 (PXK) 32.1 (86/268) 24.8 (265/1,068) 1.43 (1.1 o 1.9) 0.016
s509749 (LY9) 40.6 (121/298) 42.9 (496/1,156) 0.91 (0.7 o 1.2) 0.5
Loci o de ed as p esen ed in Table 2. All esul s e e o he mino allele o each SNP, which is indica ed in Table 1. No s a i ied analysis by
sample collec ion was done due o he small numbe o pa ien s wi h sys emic lupus e y hema osus (SLE) in each collec ion. CI, con idence
in e al; OR, odds a io. aDa a p esen ed as pe cen age (numbe o mino alleles/ o al numbe o alleles). bThese esul s a e o ca ie analysis
because MECP2 is in he X ch omosome and he e is a single allele in each man.
A h i is Resea ch & The apy Vol 11 No 3 Sua ez-Ges al e al.
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Discussion
Ou aim has been o con ibu e o he de ini ion o consis en
SLE gene ic ac o s de i ed om ecen sound s udies: ou o
he associa ions ha e been desc ibed in a GWA s udy, bu no
in a second GWA s udy o any o he s udy; ano he wo asso-
cia ions we e iden i ied in la ge s udies, bu no in any o he
GWA s udies; and one associa ion is mo e con o e sial
(Table 1). Ou esul s a e highly eassu ing because all o he
associa ions, excep one om he g oup no ound in any
GWA s udy, we e eplica ed wi h cla i y and showed he same
disease-associa ed allele as o iginally epo ed. This high
deg ee o ep oducibili y is a undamen al change ha la ge
s udies ha e b ough o gene ic esea ch o SLE and o he
complex diseases [2,21]. This change allows a b igh u u e
o he in es iga ion o he gene ic componen o SLE.
The mos ema kable esul om he p esen s udy has p oba-
bly been he associa ion signal obse ed wi h he s2304256
nonsynonymous SNP o TYK2 (OR = 0.79) because his has
been a con o e sial SLE gene ic ac o . The s2304256 SNP
in oduces a aline o phenylalanine change in he Janus
homology domain 4 o TYK2 whose unc ional ele ance has
no ye been es ed. This nonsynonymous SNP showed he
s onges associa ion among he 11 TYK2 SNPs s udied in
Scandina ian amilies [15], bu was no associa ed in a s udy
o UK amilies [16]. This la e s udy, howe e , ound associa-
ion wi h ano he TYK2 SNP ( s12720270) ha was no asso-
cia ed in he Scandina ian s udy. Finally, he In e na ional
Conso ium o Sys emic Lupus E y hema osus Gene ics
(SLEGEN) GWA s udy excluded associa ion wi h he
s12720270 SNP ( he s2304256 SNP was no included in
he GWA panels) [3]. Ou esul s a e impo an in his con ex
because hey show a signi ican associa ion ha con i ms he
ole o he s2304256 nonsynonymous SNP. In addi ion, com-
bined analysis o all a ailable da a show a clea SLE associa-
ion (P = 2.10 × 10-11) ha is s onge han he equi ed o
genome-wide signi icance.
Tyk2 is a Janus- amily y osine kinase ha is bound o cy okine
ecep o s and becomes ac i a ed a e ligand binding. De i-
ciency o TYK2 leads o de ec s o mul iple cy okine pa hways,
including ype I in e e on, IL-6, IL-10, IL-12, and IL-23, and o
impai ed T-helpe ype 1 di e en ia ion and accele a ed T-
helpe ype 2 di e en ia ion [22]. Only u u e esea ch will indi-
ca e which o hese pa hways is c i ically a ec ed by he TYK2
isk allele.
Following in impo ance is he associa ion o MECP2 because
ou esul s p o ide eplica ion and indica e ha a p e ious
assump ion abou he ole o his gene ic ac o in con ibu ing
o he sex bias in SLE is ques ionable. Sawalha and col-
leagues conside ed he X-ch omosome me hyl CpG binding
p o ein 2 coding gene (MECP2) as a possible SLE gene ic
ac o based on wo ea u es: SLE p edominance in women
and abno mal egula ion o me hyla ion-sensi i e T-cell genes
in SLE [13]. MECP2 could be in ol ed in bo h phenomena
because his gene is in he X ch omosome and pa icipa es in
DNA me hyla ion. Sawalha and colleagues ound associa ion
wi h se e al SNPs in women om wo e hnic g oups, Ko ean
and Eu opean (OR o s17435 = 1.58 and 1.29, espec i ely)
[13]. The associa ion we ha e ound in women (OR = 1.26) is
e y simila o ha epo ed in hei Eu opean sample, p o id-
ing s ong con i ma o y e idence. This eplica ion is impo an
o he s a us o MECP2 due o he lack o associa ion signals
in he SLE GWA s udies.
In addi ion, we ha e ound ha he MECP2 SNP is also asso-
cia ed wi h SLE in men (OR = 1.82, P = 0.0046), which was
no p e iously known. This esul se iously unde mines he
hypo hesized ole o MECP2 in SLE gende bias. In e o-
spec , lack o sex speci ici y is cong uen wi h expe imen s
ha showed MECP2 is no exp essed in he inac i a ed X
ch omosome o women [23], which implies exp ession le els
in men and women should be equi alen . Fu u e esea ch
should aim o es ablish whe he any o he SLE-associa ed
SNPs in MECP2 has a unc ional e ec and o ind e idence
o he hypo hesized ela ionship be ween al e ed me hyla ion
o T-cell genes in SLE and MECP2. In addi ion, i is e en
unclea whe he he causal polymo phism a ec s MECP2
because SLE associa ion has also been epo ed wi h gene ic
a ian s in a neighbou gene, IRAK1, which is a key media o
in he signalling pa hways o Toll-like ecep o s/IL-1R [24].
The s10798269 SNP in he 1q25.1 locus, he s4963128
SNP in he KIAA1542 gene and he s6445975 SNP in he
PXK gene we e epo ed in he SLEGEN GWA s udy [3] wi h
P alues below 2 × 10-7, bu hey we e no epo ed in Hom
and colleagues' GWA s udy [4] and none o hem has ye
been eplica ed in any o he s udy. The h ee SNPs we e asso-
cia ed wi h SLE in ou s udy, wi h e ec sizes ha a e simila
o hose epo ed (OR = 0.81 e sus 0.82 o he 1q25.1
SNP, 0.84 e sus 0.78 o he KIAA1542 SNP, and 1.19 e -
sus 1.25 o he PXK SNP). None o hese h ee SNPs has any
p edic able unc ional e ec . In addi ion, he s10798269
SNP in he 1q25.1 locus is a om any known ansc ip and
he PXK and KIAA1542 genes a e o unknown unc ion. The
KIAA1542 gene, howe e , is abou 20 kb away om he IRF7
gene and in linkage disequilib ium wi h i , aising he possibili y
ha his associa ion could be ela ed wi h IRF7 unc ion [3].
Ou eplica ion o hese associa ions inc eases he need o
esea ch aimed o he iden i ica ion o hei unc ional e ec s.
We ha e also ound a signi ican associa ion wi h he
s17266594 in he BANK1 gene. This SLE gene ic ac o has
been iden i ied in a low- esolu ion GWA s udy in a Swedish
sample and eplica ed in o he Eu opean sample collec ions in
he same s udy [5], bu i was no ound in any o he high- es-
olu ion GWA s udies and has no ye been eplica ed by o he
g oups. Ou esul s p o ide his independen eplica ion,
al hough wi h a mo e modes e ec (OR = 0.83 in ou s udy
A ailable online h p://a h i is- esea ch.com/con en /11/3/R69
Page 7 o 9
(page numbe no o ci a ion pu poses)
e sus 0.70 in Kozy e and colleagues [5]). The causal poly-
mo phism can be he s17266594 SNP i sel , which seems o
al e splicing e iciency o BANK1, o wo BANK1 nonsynon-
ymous SNPs o possible damaging e ec . Linkage disequilib-
ium be ween hese h ee SNPs has p e en ed dissec ion o
hei ela ionship o SLE suscep ibili y [5]. BANK1 codes o a
B-cell sca old p o ein wi h anky in epea s ha is implica ed in
B-cell ecep o -media ed signalling.
The s509749 SNP o LY9 is he only SNP ha was no epli-
ca ed in ou s udy. We selec ed his SNP because i seems o
explain he 1q23 SLE-linked locus acco ding o a la ge amily-
based s udy [14]. 1q23 is one o he mos consis en ly
desc ibed SLE loci in linkage s udies (and i s syn enic egion
in he mouse lupus models) [1]. Examina ion o SNPs all along
his locus showed s onge associa ion wi h he s509749
SNP [14]. This SNP has a p edic able impac in p o ein unc-
ion and is associa ed wi h changes in he p opo ion o spe-
ci ic T-cell subse s [14]. All his e idence made he s509749
SNP a good candida e o eplica ion in ou iew, e en i he
le el o signi icance o he SLE associa ion was no ably lowe
han he epo ed o he o he nine SNPs s udied he e (P =
0.002). Lack o eplica ion o his SNP in con as wi h eplica-
ion o he o he nine SNPs p o ides suppo o he di ec ela-
ionship be ween e y low P alues ob ained in sound s udies
and he ep oducibili y o gene ic associa ion indings [21].
The mos associa ed SNPs in ou samples we e he h ee ha
we e al eady con i med p e ious o ou s udy. These h ee
SNPs we e associa ed wi h SLE in a leas h ee la ge s udies.
The la ges e ec was obse ed wi h a nonsynonymous SNP
in he hi d exon o he ITGAM gene ( s1143679, OR = 1.70)
[3,4,6,7]. This nonsynonymous SNP was he mos associa ed
in one o he p e ious s udies (wi h e y simila e ec , OR =
1.74) [7], and has been hypo hesized o dis u b ITGAM in e -
ac ion wi h i s ligands, bu s ill no unc ional e idence is a ail-
able. Ano he clea ly es ablished associa ion [3,4,6,8-12] was
he second s onges in ou s udy: SNP s7574865 in he
hi d in on o he STAT4 gene (OR = 1.62). This associa ion
seems s onge in pa ien s wi h a se e e pheno ype [12]; how-
e e , no unc ional polymo phism has been iden i ied in his
locus. The nex s onges associa ion (OR = 1.34) was wi h
he s13277113 SNP, which has been epo ed in he GWA
s udy o Hom and colleagues [4], wi h a simila e ec (OR =
1.39). This SNP is loca ed be ween C8o 13 (o unknown
unc ion) and BLK (B-lymphoid y osine kinase), wo genes
ha a e ansc ibed in opposi e di ec ions. No unc ional a i-
an has been iden i ied in his locus, bu he isk allele o his
SNP co ela es wi h low mRNA le els o BLK and high le els
o C8o 13, aising he possibili y ha ei he o hese wo
e ec s could be ela ed wi h SLE. G aham and colleagues
ound associa ion wi h a s ongly linked SNP in he BLK gene
[6], while he SLEGEN GWA s udy ound associa ion wi h an
unlinked SNP in his locus, sugges ing he possibili y o wo
independen gene ic ac o s [3].
In addi ion, we ha e ound ha mos examined SLE-associ-
a ed SNPs seem o be sha ed be ween women and men.
Resul s a e no de ini i e gi en he small numbe o men in he
pa ien g oup. This lack o di e en ial associa ion is impo an
because we do no know de ini i ely he causes o he emale
p e e ence o SLE. Lack o de ailed gende analysis in p e i-
ous gene ic epo s is eg e able because only agg ega ion o
da a om mul iple s udies will allow us o know whe he
gene ic ac o s con ibu e o his sex bias.
Conclusions
In summa y, ou s udy has p o ided independen eplica ion o
nine SLE-associa ed loci, six o hem o con i ma o y impo -
ance because hey ha e no ye been independen ly epli-
ca ed by o he g oups (1q25.1, MECP2, KIAA1542, PXK and
BANK1) o because hei associa ion was con o e sial
(TYK2). These esul s b ing he numbe o s ongly con i med
associa ed loci o 13. Replica ion in independen s udies is
indispensable o conside ing a gene ic ac o in his ca ego y,
al hough he common use o mul iple case–con ol se s inside
he same s udy o o la ge sample collec ions has inc eased
he chances o eplica ion [2]. Some o he p omising associa-
ions ha e been disco e ed [6,25], o awai su icien inde-
penden eplica ion [2], bu i is al eady ce ain ha he gene ic
componen o SLE is especially ich in gene ic ac o s wi h
e ec s abo e he de ec able le el wi h cu en s udies (OR =
1.15 o 1.25). We a e he e o e now in a phase o exci ing dis-
co e ies in his ield. The e s ill emain o midable challenges,
howe e , because i is necessa y o ans o m he in o ma ion
we ob ain in o use ul knowledge and, as has been discussed
abo e, we ha e e y ew clues ega ding he meaning o he
iden i ied SLE associa ions. Fu u e s udies should y o iden-
i y he causal a ian s and o de e mine hei e ec a molec-
ula , cellula and disease le els, including he assessmen o
hei ole in he di e en SLE pheno ypes and he p obable
simila e ec in women and men.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Au ho s' con ibu ions
MS-G pa icipa ed in design o he s udy, in geno yping he
samples, in in e p e a ion o he esul s and in w i ing he man-
usc ip . MC pa icipa ed in he s a is ical analysis and in he
in e p e a ion o esul s. EE, RP, JO-R, GDS, SR, MJS, CP,
MM, FNS, AS, FJB, SD'A, MB, PC, TW and SM pa icipa ed
in he acquisi ion o clinical da a and collec ion o samples and
in he analysis and in e p e a ion o esul s. JJG-R coo dina ed
he acquisi ion o clinical da a and collec ion o samples and
pa icipa ed in he analysis and in e p e a ion o esul s. AG
pa icipa ed in he design o he s udy and in he coo dina ion
o acquisi ion o clinical da a and collec ion o samples, and
supe ised geno yping, s a is ical analysis, in e p e a ion o
esul s and w i ing o he manusc ip . All au ho s ead and
app o ed he inal manusc ip .
A h i is Resea ch & The apy Vol 11 No 3 Sua ez-Ges al e al.
Page 8 o 9
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Au ho s' in o ma ion
O he con ibu o s o he Eu opean Conso ium o SLE DNA
Collec ions: A ila Ko acs (Albe Szen -Gyö gyi Medical and
Pha maceu ical Cen e, Uni e si y o Szeged, Hunga y);
Rudol Pullmann J (Ge on ology Resea ch Cen e , Na ional
Ins i u e on Aging, Bal imo e, MD, USA); E a Balada (Hospi al
Vall d'Heb on, Ba celona, Spain); C ibo Dos al (Ins i u e o
Rheuma ology, P ague, Czech Republic); Filipe Vinag e (Hos-
pi al Ga cia de O a, Almada, Po ugal and Ins i u o Medicina
Molecula , Faculdade de Medicina da Uni e sidade de Lisboa,
Po ugal); I is Kappou-Riga ou (E angelismos Hospi al, A h-
ens, G eece); Ra aella Sco za (Uni e si y o Milan and Fon-
dazione IRCCS Ospedale Maggio e Policlinico, Mangiagalli e
Regina Elena, Milan, I aly); Ma ia Ma oma i (A hens Uni e si y
Medical School, A hens, G eece); Ca men Gu ie ez (Hospi al
Uni e si a io Cen al de As u ias, Uni e sidad de O iedo,
Spain); Ignacio Rego (INIBIC-CH Uni e si a io A Co uña,
Spain); Nadia Ba izzone (Eas e n Piedmon Uni e si y,
No a a, I aly); Cees G Kallenbe g (Uni e si y Medical Cen e
G oningen, The Ne he lands); and Reinhold E Schmid (Han-
no e Medical School, Hanno e , Ge many).
No e
A epo published in A h i is & Rheuma ism a e publica ion
o his manusc ip p o ided u he con i ma ion o he associ-
a ion o MECP2 wi h SLE [26].
Addi ional iles
Acknowledgemen s
The au ho s hank Ca men Pena-Pena o p o iding ou s anding echni-
cal assis ance. MS-G is he ecipien o a FPU p edoc o al bu sa y o he
Spanish Minis y o Educa ion. The p esen wo k was suppo ed by
Fondo de In es igacion Sani a ia o he Ins i u o de Salud Ca los III
(Spain), g an s 04/1651 and 06/0620 ha a e pa ially inanced by he
Fondo Eu opeo de Desa ollo Regional p og am o he Eu opean Union,
by g an s om he Xun a de Galicia, and by BMBF KN Rheuma g an
C2.12 ( o TW).
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The ollowing Addi ional iles a e a ailable online:
Addi ional ile 1
A Wo d ile con aining Table S1 ha lis s he o igin and
emale pe cen age o he DNA sample collec ions, Table
S2 ha lis s he clinical cha ac e is ics o he pa ien s
wi h SLE, Table S3 ha lis s he p ime s and p obes
used o geno yping he 10 SNPs, Table S4 ha lis s he
geno ype coun s o each o he 10 SNPs de ailed o
each o he sample collec ions, and Table S5 ha lis s
he mino allele pe cen ages o each o he 10 SNPs o
each o he sample collec ions.
See h p://www.biomedcen al.com/con en /
supplemen a y/a 2698-S1.doc
A ailable online h p://a h i is- esea ch.com/con en /11/3/R69
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