Identification of IRAK1 as a risk gene with criticalrole in the pathogenesis of systemiclupus erythematosusChaim O. Jacoba,1,2, Jiankun Zhub,1, Don L. Armstronga,c,1, Mei Yanb, Jie Hanb, Xin J. Zhoub, James A. Thomasb,Andreas Reiffa,d, Barry L. Myonese, Joshua O. Ojwangf, Kenneth M. Kaufmanf, Marisa Klein-Gitelmang,Deborah McCurdyh, Linda Wagner-Weineri, Earl Silvermanj, Julie Zieglerk, Jennifer A. Kellyf, Joan T. Merrillf,John B. Harleyf, Rosalind Ramsey-Goldmanl, Luis M. Vilam, Sang-Cheol Baen, Timothy J. Vyseo, Gary S. Gilkesonp,Patrick M. Gaffneyf, Kathy L. Moserf, Carl D. Langefeldk, Raphael Zidovetzkic, and Chandra Mohanb,2

aThe Lupus Genetic Group, Department of Medicine, University of Southern California, 2011 Zonal Avenue, Los Angeles, CA 90033; bDepartments of InternalMedicine (Rheumatology), Pediatrics, Pathology, and Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390; cDepartment of CellBiology and Neuroscience, University of California, Riverside, CA 92521; dChildrens Hospital of Los Angeles, Los Angeles, CA 90027; eTexas Children’s Hospital,Baylor College of Medicine, Houston, TX 77030; fOklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, OK 73104; gChildren’sMemorial Hospital and Northwestern University, Chicago, IL 60614; hDepartment of Pediatrics, University of California, Los Angeles, CA 90095; iLaRabida Hospitaland University of Chicago, Chicago, IL 60649; JHospital for Sick Children, Toronto, ON, Canada M5G 1X8; kWake Forest University Health Sciences, Medical CenterBoulevard, Winston-Salem, NC 27157; lDivision of Rheumatology, Northwestern University, Feinberg School of Medicine, McGaw Pavilion, Chicago, IL 60611;mDivision of Rheumatology, University of Puerto Rico Medical Sciences Campus, San Juan, Puerto Rico 00917; nDivision of Rheumatology, Department of InternalMedicine and the Hospital for Rheumatic Diseases, Hanyang University, Seoul 133–791, Republic of Korea; oImperial College London, Hammersmith Hospital, DuCane Road, London W12 0NN, United Kingdom; and pMedical University of South Carolina, Charleston, SC 29425

Communicated by Ellen S. Vitetta, University of Texas Southwestern Medical Center, Dallas, TX, February 4, 2009 (received for review January 3, 2009)

A combined forward and reverse genetic approach was undertakento test the candidacy of IRAK1 (interleukin-1 receptor associatedkinase-1) as an X chromosome-encoded risk factor for systemic lupuserythematosus (SLE). In studying �5,000 subjects and healthy con-trols, 5 SNPs spanning the IRAK1 gene showed disease association (Pvalues reaching 10�10, odds ratio >1.5) in both adult- and childhood-onset SLE, in 4 different ethnic groups, with a 4 SNP haplotype (GGGG)being strongly associated with the disease. The functional role ofIRAK1 was next examined by using congenic mouse models bearingthe disease loci: Sle1 or Sle3. IRAK1 deficiency abrogated all lupus-associated phenotypes, including IgM and IgG autoantibodies, lym-phocytic activation, and renal disease in both models. In addition, theabsence of IRAK1 reversed the dendritic cell ‘‘hyperactivity’’ associ-ated with Sle3. Collectively, the forward genetic studies in human SLEand the mechanistic studies in mouse models establish IRAK1 as adisease gene in lupus, capable of modulating at least 2 key check-points in disease development. This demonstration of an X chromo-some gene as a disease susceptibility factor in human SLE raises thepossibility that the gender difference in SLE may in part be attributedto sex chromosome genes.

autoimmune disease � genetic association � SNP � inflammation �interferon

Systemic lupus erythematosus (SLE) is a debilitating multisystemautoimmune disorder affecting �0.1% of the North American

population, mainly females, characterized by chronic inflammationand extensive immune dysregulation in multiple organ systems,associated with the production of autoantibodies to a multitude ofself-antigens (1). The prevalence of SLE varies among ethnicpopulations (higher in non-Caucasians) and is likely attributable toethnic differences in genetic susceptibility. Despite many advancesin recent years, the pathogenesis of SLE remains largely unclear.

Genetic approaches have gained much power and popularity inidentifying the component mechanism(s) underlying the pathogen-esis of common human diseases. Forward genetic approaches, inwhich human populations are studied to identify the genes involvedin disease processes, have inherent shortcomings for the analysis ofcommon diseases involving multiple genes because each genecontributes modestly, often in interaction with environmental fac-tors. On the other hand, reverse genetic approaches—in which agene is characterized by perturbing it in an experimental system,and then elucidating its effect on the trait of interest—have their

own significant limitations. Often, such experimental approachestake place in an oversimplified context where potential interactionsbetween the gene of interest and the genetic background or theenvironment are eliminated and data interpretation may be con-founded by the impact of the gene on cell and organismal devel-opment. In the present study, a combined forward and reversegenetic approach is pursued, resulting in the unequivocal identifi-cation of the gene IRAK1 as an important risk factor for SLE, witha critical role in disease pathogenesis.

ResultsWe have recently developed a set of programs that implement acombination of automated and manual approaches to maximizethe power of gene association studies by using prior informationto select and prioritize genes, to reduce the number of SNPstested resulting in higher power, and to increase the likelihoodof uncovering reproducible associations (2). We have previouslyused this bioinformatics-driven design for a custom-made plat-form incorporating �10,000 SNPs derived from �1,000 selectedgenes to genotype a sample of 753 subjects composed of 251childhood-onset SLE trios (SLE patient and both parents) (3).Family-based transmission disequilibrium test (TDT) and mul-titest correction analyses showed a significant association be-tween the IRAK1 gene on chromosome Xq28 and childhood-onset SLE (3).

In the present study, we have used a case-control associationapproach to test the hypothesis that IRAK1 is a candidate genepredisposing to SLE. To this end, we have tested an independentchildhood-onset cohort of 769 childhood-onset SLE patients, 5,337

Author contributions: C.O.J., J. Zhu, D.L.A., R.Z., and C.M. designed research; C.O.J., J. Zhu,D.L.A., M.Y., J.H., X.J.Z., J.A.T., A.R., B.L.M., J.O.O., K.M.K., M.K.-G., D.M., L.W.-W., E.S.,J. Ziegler, J.A.K., J.T.M., J.B.H., R.R.-G., L.M.V., S.-C.B., T.J.V., G.S.G., P.M.G., K.L.M., C.D.L.,R.Z., and C.M. performed research; D.L.A., M.Y., J.H., X.J.Z., J.A.T., A.R., B.L.M., J.O.O.,K.M.K., M.K.-G., D.M., L.W.-W., E.S., J. Ziegler, J.A.K., J.T.M., J.B.H., R.R.-G., L.M.V., S.-C.B.,T.J.V., G.S.G., P.M.G., K.L.M., and R.Z. contributed new reagents/analytic tools; C.O.J., J. Zhu,D.L.A., X.J.Z., C.D.L., R.Z., and C.M. analyzed data; and C.O.J., J. Zhu, D.L.A., R.Z., and C.M.wrote the paper.

The authors declare no conflict of interest.

1C.O.J., J. Zhu, and D.L.A. contributed equally to this work.

2To whom correspondence may be addressed. E-mail: jacob@usc.edu or chandra.mohan@utsouthwestern.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0901181106/DCSupplemental.

6256–6261 � PNAS � April 14, 2009 � vol. 106 � no. 15 www.pnas.org�cgi�doi�10.1073�pnas.0901181106

North American adult-onset SLE subjects, and 5,317 healthy con-trols, each group being composed of 4 ethnicities as detailed inTable S1. Childhood-onset SLE constitutes a unique subgroup ofpatients for genetic analysis because the earlier disease onset, themore severe disease course, the greater frequency of family historyof SLE, and a lesser contribution of sex hormones in diseasedevelopment (4, 5) may all translate to a higher genetic load or amore penetrant expression of this genetic load, and this mayfacilitate gene discovery relative to studies of the adult-onsetdisease. Therefore, we analyzed childhood-onset and adult-onsetgroups of SLE patients separately. To account for any potentialconfounding substructure or admixture, we performed principalcomponent analyses (PCA) (6), as detailed in Methods. Excludingthe outliers, the analyses resulted in low inflation factors in allethnicities except Hispanic Americans, with only the latter requir-ing additional principal component correction.

Fig. 1 shows the association of IRAK1 SNPs in four racial groupsof childhood- and adult-onset SLE. It is noteworthy that themajority of the significantly associated SNPs are within a relativelysmall interval of 3.3 kb between intron 10 and intron 13 of theIRAK1 gene. Most of these SNPs show significance in multipleethnicities, as is evident from Fig. 1. The classical Bonferronicorrection and similar procedures for controlling the family-wiseerror rate for multiple testing are both too strict and inappropriatein studies such as the present one because they assume that each testis independent, whereas in actuality a complex and unknownmutual dependence exists among SNPs on the same gene (3, 7).Therefore, for multiple test correction we calculated estimates ofthe false discovery rate (FDR) q values by using the Benjamini–Hochberg procedure (8) considering the total number of SNPstested and the 4 different ethnic groups (Table 1). Combined p

values were calculated from the per-ethnicity p value by using theFisher method. Table 1 shows that 5 SNPs out of the 13 testedwithin the IRAK1 gene showed significant association with SLE inmultiple ethnic groups after correction for multiple testing. Thereare a number of highly significant SNPs with combined p valuesreaching 10�10, and attaining 10�9 in individual ethnicities, corre-sponding to FDRs of 10�9 and 10�7, respectively.

Three of the 5 associated SNPs (rs2239673, rs763737, andrs7061789) overlap in both the childhood- and adult-onset SLEpatients, suggesting a similar involvement of IRAK1 in both adult-and childhood-onset SLE. The odds ratio (ORs) of all significantlyassociated SNPs are in the same direction (�1), implying that therewas no residual population stratification. It is also noteworthy thatORs for the associated SNPs, with the exception of rs5945174, are�1.5, a value that compares well with published associations in SLEand other similar complex human disorders (9–14).

The most significantly associated SNPs are in a linkage-disequilibrium block that extends from intron 10 to intron 13 of theIRAK1 gene. Haplotype analyses in different racial groups showthat the GGGG haplotype (defined as ‘‘G’’ at rs2239673, ‘‘G’’ atrs763737, ‘‘G’’ at rs5945174, and ‘‘G’’ at rs7061789) is significantlyassociated with disease in 3 of the 4 racial groups in adult-onset SLEand in 3 ethnicities in childhood-onset SLE (Table 2 and Fig. S1).The p values for association reach 10�5 in children and 10�6 inadults. On the other hand, the AAAA haplotype is clearly associ-ated with protection from disease.

Currently no human biological system is available that wouldallow one to ascertain an in vivo connection between IRAK1 andits biological relevance in SLE. To test this, we turned to thelaboratory mouse, as mice lacking IRAK1 function and mice proneto spontaneous lupus have both been described on the same

02

46

8

Adult AAAdult EAAdult HAAdult AsAAdult Combined

Childhood AAChildhood EAChildhood HAChildhood AsAChildhood Combined

02

46

8−

log 1

0 of

p v

alue

s

152932 152934 152936 152938Position on Chr X (kbp)

12345678910111213

Fig. 1. Association of IRAK1 SNPs with SLE in 4 ethnic groups (EA, European Americans; AA, African Americans; AsA, Asian Americans; HA, Hispanic Americans) inchildhood- and adult-onset SLE cases. The position of exons (green rectangles) and introns (connecting lines) are indicated in the bottom plot. The dotted horizontalline corresponds to P � 0.05. The exact numbers of subjects studied are detailed in Table S1.

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C57BL/6 (B6) genetic background (15–17). Recent studies havesucceeded in defining the genetic basis of lupus in the NZBxNZWderived NZM mouse models, and have uncovered Sle1 on chro-mosome 1 and Sle3 on chromosome 7 as 2 of the most criticalelements for disease in these models (16–20). By introgressing theseintervals onto the relatively normal C57BL/6 (B6) background, theimmunological properties of these 2 key loci have been elucidated(16, 17). Whereas a critical gene within the Sle1 interval, Ly108,breaches central B cell tolerance, resulting in anti-chromatin auto-reactivity and lymphocytic activation (19), the Sle3 gene(s) con-tributes to SLE by activating myeloid cells, including dendritic cells(DCs) (20). Importantly, the combined action of these 2 loci leadsto full-blown lupus and lupus nephritis, which is indistinguishablefrom the disease noted in the traditionally studied (NZBxNZW)F1and NZM mouse models (18).

Because Sle1 and Sle3 represent 2 key complementary loci forSLE development, we evaluated the role of IRAK1 in mediatingthe contributions of these 2 loci to SLE pathogenesis. B6.Sle1z mice(that were homozygous for the Sle1z allele) were bred toB6.IRAK1�/Y mice (15), to eventually derive B6.Sle1z.IRAK�/Y

mice. Because Sle1z leads to spontaneous anti-nuclear antibody

formation on the B6 background, notably anti-histone/DNA anti-bodies, splenomegaly, and spontaneous B cell and T cell activation(16), these phenotypes were first examined. Compared to age- andsex-matched B6.Sle1z control, B6.Sle1z.IRAK1�/Y mice exhibitedsignificantly reduced IgM and IgG autoantibodies to ssDNA,histone/DNA, and dsDNA (Fig. 2). Likewise, B6.Sle1z.IRAK1�/Y

mice also exhibited reduced spleen weights, total splenocyte counts,as well as total B cell and CD4-positive T cell counts, compared withthe controls with an intact IRAK gene (Fig. 3). In addition, theabsence of IRAK1 also dampened the number of B cell blasts (asgauged by forward scatter analysis) (Fig. 3E) and reduced thenumbers of activated CD4 T cells as assessed by surface CD69expression (Fig. S2). No differences were, however, noted in theexpression of surface CD86 or CD69 on B cells from both strains.Collectively, the above findings indicate that the absence of IRAK1significantly attenuated the serological and cellular phenotypesattributed to the lupus susceptibility locus, Sle1.

Next, we proceeded to examine the impact of IRAK1 in medi-ating the lupus contributions of the second locus, Sle3. In the B6background, Sle3z leads to low-grade anti-nuclear serological au-toreactivity, myeloid cell hyperactivity resulting in secondary acti-

Table 1. IRAK1 SNPs significantly associated with SLE in multiple ethnic groups after multitest-correction analyses

doohdlihCtludAsPNS 1KARI fo noitaicossA

SNP SNPLocation

Com

bine

dp

valu

esAd

ult

Com

bine

dFD

R A

dult

Com

bine

dp

valu

esC

hild

hood

Com

bine

dFD

RC

hild

hood

Eth

nici

ty

Freq

uenc

yC

ontro

l

Freq

uenc

yC

ase

OR p q

Freq

uenc

yC

ase

OR p q

AA 0.45 0.45 0.98 7.76E-1 5.32E-1 0.42 0.87 3.68E-1 3.24E-1EA 0.20 0.23 1.22 1.60E-4 1.28E-3 0.25 1.35 4.21E-2 9.24E-2HA 0.49 0.56 1.32 2.83E-2 9.28E-2 0.63 1.76 2.86E-3 1.55E-2rs2239673 Intron

13 2.56E-09 2.18E-8 5.61E-04 3.37E-3

AsA 0.76 0.84 1.62 1.77E-7 2.80E-6 0.83 1.51 2.32E-2 6.40E-2AA 0.45 0.44 0.97 6.58E-1 4.98E-1 0.42 0.89 4.57E-1 3.55E-1EA 0.19 0.22 1.19 1.05E-3 6.96E-3 0.24 1.32 6.67E-2 1.20E-1HA 0.47 0.56 1.41 6.45E-3 3.03E-2 0.61 1.79 2.37E-3 1.34E-2rs763737 Intron

12 5.04E-10 4.76E-9 1.40E-04 1.27E-3

AsA 0.75 0.83 1.68 2.29E-8 4.61E-7 0.84 1.77 2.56E-3 1.43E-2AA 0.79 0.80 1.05 6.04E-1 4.78E-1 0.88 1.90 8.28E-3 3.41E-2EA 1.00 1.00 NA 3.53E-1 3.71E-1 1.00 NA 5.98E-1 3.93E-1HA 0.97 0.99 2.89 1.75E-2 6.63E-2 0.99 3.71 8.96E-3 3.58E-2rs3027907 Intron

11 8.28E-02 1.85E-1 2.13E-04 1.65E-3

AsA 1.00 1.00 NA NA NA 1.00 NA NA NAAA 0.32 0.35 1.11 2.12E-1 2.91E-1 0.31 0.92 5.91E-1 3.90E-1EA 0.14 0.18 1.40 6.78E-9 1.41E-7 0.17 1.27 1.50E-1 2.04E-1HA 0.34 0.42 1.41 7.67E-3 3.50E-2 0.38 1.19 3.76E-1 3.26E-1rs5945174 Intron

10 1.37E-09 1.21E-8 1.67E-01 2.73E-1

AsA 0.48 0.50 1.08 2.82E-2 9.28E-2 0.45 0.88 8.80E-2 1.44E-1AA 0.47 0.46 0.95 4.63E-1 4.22E-1 0.42 0.82 1.84E-1 2.34E-1EA 0.20 0.23 1.23 9.84E-5 8.21E-4 0.25 1.38 2.62E-2 6.87E-2HA 0.48 0.56 1.38 8.91E-3 4.03E-2 0.62 1.78 1.89E-3 1.11E-2rs7061789 Intron

10 7.06E-10 6.43E-9 1.99E-04 1.58E-3

AsA 0.77 0.84 1.62 3.65E-7 4.95E-6 0.83 1.49 3.11E-2 7.69E-2

The values of p and q �0.05 are in bold. The SNPs with combined (FDR-corrected) q values of �0.05 were considered significant. Abbreviations of ethnicitiesare given in the legend to Fig. 1. NA, not applicable.

Table 2. IRAK1 haplotype block associated with SLE with P <0.05

Age of onset Ethnicity

SNP number

Casefrequency

Controlfrequency �2 P

1rs2239673

2rs763737

3rs3027907

4rs5945174

5rs7061789

8rs11465835

Adult AsA G G G G 0.486 0.444 5.757 1.64E-2Adult AsA G G A G 0.34 0.307 3.907 4.81E-2Adult AsA A A A A 0.162 0.231 23.304 1.38E-6Adult HA A A A A 0.435 0.511 7.53 6.10E-3Adult HA G G G G 0.479 0.377 13.561 2.00E-4Adult EA A A A A 0.766 0.8 17.821 2.43E-5Adult EA G G G G 0.217 0.182 21.035 4.51E-6Childhood AsA A A A A 0.174 0.231 4.399 3.60E-2Childhood AA G G A G G C 0.267 0.215 4.479 3.43E-2Childhood HA A A A A 0.378 0.511 16.912 3.92E-5Childhood HA G G G G 0.509 0.37 18.404 1.79E-5

Numbers of case and control samples are given in Table S1. Abbreviations of ethnicities are given in the legend to Fig. 1.

6258 � www.pnas.org�cgi�doi�10.1073�pnas.0901181106 Jacob et al.

vation of lymphocytes, and a modest degree of nephritis (17, 20).Compared to B6.Sle3z controls, B6.Sle3z.IRAK1�/Y mice exhibitedsignificantly reduced IgM and IgG anti-ssDNA and anti-dsDNAAbs (Fig. 4 A and B, D and E), as well as milder or negligible renaldisease, as evidenced by the reduced proteinuria and renal glomer-ular pathology (Fig. 4 C and F). Moreover, these mice had reducedsplenocyte numbers, including total T cells and B cells (Fig. S3). Acardinal feature associated with Sle3z, namely increased CD4:CD8ratios, were normalized by the absence of IRAK1 (Fig. 5F).

Because the above phenotypes had previously been attributed tothe intrinsic impact of Sle3z on myeloid cells (20), these wereexamined next. Although the strains did not differ in absolutenumbers of splenic myeloid cell subpopulations, interesting differ-ences in their activation and maturation status were observed. Inthe absence of IRAK1, Sle3z myeloid DCs and macrophagesexamined ex vivo from spleens showed reduced surface expressionof CD80, but not CD40 or CD86 (Fig. 5 A and B and Fig. S4). Thesedifferences became more pronounced when bone marrow (BM)-derived DCs were examined. Thus Sle3z BM-DCs deficient inIRAK1 exhibited reduced levels of several activation/maturationmarkers both basally and after TLR ligation using poly(I�C) or CpG(Fig. 5 C and D). The IRAK1-deficient B6.Sle3z DCs also producedreduced levels of proinflammatory cytokines, such as TNF-� (Fig.5E). Hence, all of the phenotypes previously attributed to the Sle3z

lupus susceptibility locus appear to be, at least partly, dependentupon IRAK1 function.

DiscussionIRAK1 (interleukin-1 receptor associated kinase 1) is a serine/threonine protein kinase involved in the signaling cascade of theToll/IL-1 receptor (TIR) family (21). The TIR family comprises theIL-1 receptor subfamily, recognizing the endogenous proinflam-matory cytokines IL-1 and IL-18, and members of the Toll-likereceptor (TLR) subfamily, which recognize pathogen-associatedmolecular patterns. A hallmark of the TIR family is the cytoplasmicTIR domain, which serves as a scaffold for a series of protein–protein interactions, which result in the activation of a unique andexclusive signaling module consisting of MyD88, IRAK familymembers, and Tollip. Subsequently, several central signaling path-ways of the innate and adaptive immune systems are activated inparallel, the activation of NF�B being the most prominent event ofthe inflammatory response (21). Particularly noteworthy is theobservation that IRAK1 is considered to be the ‘‘on-switch’’ of thesignaling complex by linking the receptor complex to the centraladapter/activator protein TRAF6, and also the ‘‘off-switch’’ of thecomplex because of its autoinduced removal from the complex (22).

+/+ −/−

0.05

0.15

0.25

0.35

IgM

ssD

NA

(O

D 4

50)

p = 8.3e−10

IRAK1

A

+/+ −/−

0.05

0.10

0.15

IgM

dsD

NA

(O

D 4

50)

p = 0.00082

IRAK1

B

+/+ −/−

0.0

0.1

0.2

0.3

0.4

0.5

IgM

DN

A/H

isto

ne (

OD

450

)

p = 3.7e−12

IRAK1

C

+/+ −/−

0.00

0.05

0.10

0.15

0.20

0.25

IgG

ssD

NA

(O

D 4

50)

p = 0.0048

IRAK1

D

+/+ −/−

0.00

0.10

0.20

0.30

IgG

dsD

NA

(O

D 4

50)

p = 0.051

IRAK1

E

+/+ −/−

0.00

0.05

0.10

0.15

IgG

DN

A/H

isto

ne (

OD

450

)

p = 0.41

IRAK1

F

Fig. 2. Reduced serum IgM and IgG autoantibodies in B6.Sle1z.IRAK1�/Y mice.B6.Sle1z mice (homozygous for the z allele of Sle1) either sufficient or deficient inIRAK1 (n � 15–20) were examined at the age of 9–12 months for serum levels ofIgM (A–C) and IgG (D–F) autoantibodies to various nuclear antigens. Shown dataare drawn from 2 independent experiments. The composite results are plotted asbox and whisker plots. IRAK1 knockouts (labeled as �/�) are indicated with grayfilled boxes; Sle1 sufficient for IRAK1 (�/�) are unfilled. The box contains theinterquartile range (Q1–Q3) with the median indicated as a thick black line; thewhiskers contain the observations within 1.5 times the interquartile range, andobservations outside this range are indicated with open circles.

+/+ −/−

100

150

200

250

Spl

een

Wei

ght (

mg) p = 0.006

IRAK1

A

+/+ −/−

23

45

67

Spl

een

CD

4 T

Cel

ls (

107 )

p = 0.0026

IRAK1

B

+/+ −/−

45

67

89

10S

plee

n B

Cel

ls (

107 )

p = 0.00017

IRAK1

C

+/+ −/−

1015

20To

tal S

plee

n C

ells

(10

7 )

p = 0.00010

IRAK1

D

+/+ −/−

8085

9095

100

Spl

een

mea

n B

−ce

ll si

ze (

FS

C)

p = 0.05

IRAK1

E

Fig. 3. Cellular phenotypes in B6.Sle1z.IRAK1�/Y mice. B6.Sle1z mice eithersufficient or deficient in IRAK1 (n � 7–10) were examined at the age of 4–6months for spleen weight (A) and cellularity (B–D), as well as the mean B cell size(as assessed from the forward scatter channel) (E). Data shown are drawn from 2independent experiments and are presented as in Fig. 2.

Fig. 4. B6.Sle3z.IRAK�/Y mice exhibit reduced serum autoantibodies and ne-phritis. (A, B, D, and E) B6.Sle3z mice (homozygous for the z allele of Sle3) eithersufficient or deficient in IRAK1 were examined at the age of 9–12 months forserum levels of IgM and IgG autoantibodies to various nuclear antigens (n � 17).(C) The 24-hr proteinuria as a measure of glomerulonephritis is assessed in bothstrains (n � 9–15). Shown data are drawn from 2 independent experiments. Datashown in A–E are presented as in Fig. 2. (F) Representative H&E staining (400�magnification) of kidney sections from an IRAK1-sufficient Sle3z mouse showingWorld Health Organization grade 3 glomerulonephritis and an IRAK1-deficientSle3z mouse with World Health Organization grade 1 glomerulonephritis.

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The extensive involvement of IRAK1 in the regulation of theimmune response renders its association with SLE a prime candi-date for careful genetic and functional analysis.

We envision the potential involvement of IRAK1 in at least thefollowing 3 immune cell functions that have been reported to beaberrant in SLE. First, IRAK1 is involved in the induction of IFN-�and IFN-�: the production of both types of IFN has been shown tobe aberrant in SLE (23, 24). Second, IRAK1 is a pivotal regulatorof the NF�B pathway. Abnormal NF�B activity in T lymphocytesfrom patients with SLE has been amply documented (25). Finally,a growing number of studies demonstrate a role for TLR activationin the pathogenesis of SLE, including the activation of anti-nuclearB cells and the subsequent immune complex formation (26). Themurine studies presented in this communication resonate well withthe earlier published literature on IRAK1.

The most significantly associated SNPs are in a linkage-disequilibrium block that extends from intron 10 to intron 13 of theIRAK1 gene, encompassing exons 11–13, which correspond to theC1 domain of IRAK1 (27). It has been shown that this domain isat least partially responsible for the interaction with signal trans-duction factors such as TRAF6 (28). Furthermore, a naturallyoccurring splice variant of IRAK1, IRAK1c, lacks exon 11 and mostof exon 12 (27). A previous report suggests that IRAK1c maysuppress NF�B activation and inhibit innate immune activation (29)and thus suppress chronic inflammatory responses. This regioncontains a putative nuclear localization sequence (amino acids503–508) as well as a nuclear exit sequence (amino acids 518–526).The absence of these sequences may explain IRAK1c’s stability andcytoplasmic localization and possibly its antiinflammatory role. It istherefore tempting to hypothesize that the SLE-associated haplo-type block may affect these activities of IRAK1. Clearly, thesepredictions warrant direct testing in future studies.

IRAK1 is located on chromosome Xq28, juxtaposed to a secondgene that has also been implicated in SLE susceptibility. A recentstudy by Sawalha et al. (30) reported the association of theneighboring gene, MECP2, and SLE in Korean and Europeancohorts. Given the physical proximity of IRAK1 and MECP2 onXq28, it is plausible that they are in linkage disequilibrium, and the2 independent studies possibly describe the same genetic associa-

tion. However, without further reverse genetic studies, it wouldhave been impossible to ascertain whether the disease-causativepolymorphism(s) exert their effect through changes in IRAK1 orMECP2. In this regard, the reverse genetic studies presented in thiscommunication shed light on this ambiguity, allowing us to confi-dently establish that the IRAK1 gene has a critical role in thepathogenesis of SLE. Whether MECP2 is also a causative gene forlupus awaits support from analogous experiments with that gene.Nevertheless, the results we present herein with IRAK1 exemplifythe power of combining forward genetic studies in patient cohortswith reverse genetic and functional studies in animal models toelucidate the genetic basis of complex diseases. This powerfulbipronged approach can be gainfully used in studies of other genesin SLE and yet other complex genetic diseases.

Although it is too early to suggest the mechanism(s) by which theIRAK1 polymorphisms may alter the disease process in humans,the murine studies presented in this communication suggest animportant role for IRAK1 at 2 key checkpoints in lupus develop-ment. The first step, which leads to benign serological and cellularautoreactivity, may be the consequence of a breach in centraltolerance in the adaptive arm of the immune system, whereas thesecond step, which leads to pathological autoimmunity, may bemediated by increased activity in the innate arm of the immunesystem (19, 20, 31). It is remarkable that IRAK1 significantlyimpacts both checkpoints in lupus development. The likely role ofIRAK1 in driving the second checkpoint, myeloid cell hyperactiv-ity, is quite apparent given the central role of IRAK1 in mediatingTLR signaling, and hence myeloid cell activation (27). In contrast,the potential role of IRAK1 and TLR signaling at various B cellcheckpoints is currently unknown and warrants careful analysis tobetter understand how IRAK1 might operate in the first checkpointof lupus development. Conditional deletion of IRAK1 in selectedcell types is clearly necessary to address this important gap in ourknowledge. Along these lines, future studies elucidating the mech-anistic role that IRAK1 might play at both these checkpoints areclearly warranted. The impact of IRAK1 deficiency on otherpolycongenic models of severe lupus nephritis also needs to beexplored.

Fig. 5. Cellular phenotypes in B6.Sle3z.IRAK1�/Y mice. (A–D) B6.Sle3z mice either sufficient (shown in pink) or deficient (shown in green) in IRAK1 were examinedat the age of 4–6 months for the surface expression of activation/maturation marker CD80 in comparison to an isotype control antibody (gray) (n � 8) F4/80hi,CD11bhi/medium, CD11clow splenic macrophages (A) and CD11b� CD11chi splenic myeloid DCs (B). (C and D) Bone-marrow-derived DCs from B6.Sle3z.IRAK1-sufficient and B6.Sle3z.IRAK1-deficient mice, stimulated with TLR ligand poly(I�C) (C) or CpG oligonucleotide (D). (E) TNF-� production by bone-marrow-derivedDCs 24 hr after stimulation with TLR ligand. (F) Ratio of CD4 to CD8 spleen T cells in B6.Sle3z.IRAK1-sufficient vs. -deficient mice. A–D are representativenormalized histograms of flow cytometry data. E and F are box and whisker plots as described for Fig. 2.

6260 � www.pnas.org�cgi�doi�10.1073�pnas.0901181106 Jacob et al.

Several autoimmune disorders are characterized by a strong sexbias, with females being afflicted by SLE almost 10 times morefrequently than males. Research efforts over the past 3 decadeshave implicated sex hormones as being responsible for the sexdifference in disease susceptibility. However, effects of sex hor-mones do not rule out a more direct effect of the X chromosome.Very little is known about whether genes on the sex chromosomescan directly influence SLE susceptibility. Recent reports in mousemodels have indicated that genes located on X/Y chromosomescould potentially influence lupus susceptibility (32–34). The presentreport constitutes the demonstration of a sex chromosome gene inhuman SLE. The data presented here provide clear evidence thatthe female predominance of the disease could be attributed, at leastin part, to IRAK1 gene dosage by virtue of its location on the Xchromosome. The challenge ahead is to fathom the degree to whichthe sex difference in SLE prevalence can be attributed to Xchromosome genes (such as IRAK1) versus hormonal differences.

MethodsRecruitment and Biological Sample Collection. Subjects were enrolled in theLupus Genetic Study Groups at the University of Southern California and theOklahoma Medical Research Foundation, in the PROFILE Study Group at theUniversity of Alabama at Birmingham, and from B.L.M., T.J.V., G.S.G., and S.-C.B.,using identical protocols. All patients met the revised 1997 American College ofRheumatology criteria for the classification of SLE (35). Ethnicity was self-reported and verified by parental and grandparental ethnicity, when known.Blood samples were collected from each participant, and genomic DNA wasisolated and stored by using standard methods. Cases were defined as childhood-

onset according to the criterion that the diagnosis of SLE was made before theage of 13 by at least 1 pediatric rheumatologist participating in the study. Allprotocols were approved by the institutional review boards at the respectiveinstitutions.

Genotyping, Statistical and Stratification Analyses, Immunophenotyping ofMice. For more information, please see SI Text.

Establishing IRAK1-Deficient Lupus Mice. All mice used were on the C57BL/6 (B6)background. B6.IRAK1�/Y, B6.Sle1z, and B6.Sle3z mice have been characterizedpreviously (15–18). B6.IRAK1�/Y mice were bred to B6-based Sle1z or Sle3z lupuscongenics to derive F1 hybrids. The F1 hybrids were intercrossed to generate F2

progeny that were then selected for mice that genotyped as B6.Sle1z.IRAK1�/Y orB6.Sle3z.IRAK1�/Y, both strains being homozygous at the respective lupus sus-ceptibility loci. Because IRAK1 is located on the X chromosome, male IRAK1�/Y

mice were used as IRAK1-deficient mice, whereas IRAK1�/Y males were used ascontrols in all experiments. All mice used for this study were bred and housed ina specific pathogen-free colony at the University of Texas Southwestern MedicalCenter Department of Animal Resources in Dallas, TX.

ACKNOWLEDGMENTS. We thank Drs. Yang Liu and Yong Du for their technicalassistance. This work was supported in part by National Institutes of Health GrantR01AR445650 and an Alliance for Lupus Research Grant 52104 (C.O.J.), theNational Institutes of Health Grant P01 AI 039824, and the Alliance for LupusResearch (C.M.), and by the University of Southern California Federation ofClinical Immunology Societies Centers of Excellence. Work at the OklahomaMedical Research Foundation was supported by National Institutes of HealthGrants (AI063622, RR020143, AR053483, AR049084, AI24717, AR42460,AR048940, AR445650, and AR043274). Work at the University of Alabama atBirmingham was supported by National Institutes of Health Grants P01-AR49084and P60-AR48095. S.-C.B. was supported by Republic of Korea Ministry for HealthGrant A010252.

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Supporting InformationJacob et al. 10.1073/pnas.0901181106SI TextGenotyping. Genotyping was performed using Illumina iSelectInfinium II Assays on the BeadStation 500GX system (Illumina).For analysis, only genotype data from SNPs with a call frequencygreater than 90% in the samples tested and an Illumina Gen-Train score greater than 0.7 were used. GenTrain scores mea-sure the reliability of the SNP detection based on the distributionof genotypic classes. The average SNP call rate for all sampleswas 97.18%. To minimize sample misidentification, data from 91SNPs that had been previously genotyped on 42.12% of thesamples were used to verify sample identity. In addition, at least1 sample previously genotyped was randomly placed on eachIllumina Infinium BeadChip and used to track samples through-out the genotyping process.

Statistical Analyses. Testing for association was completed usingthe freely available programs SNPGWA (www.phs.wfubmc.edu/web/public�bios/sec�gene/downloads.cfm) and PLINK (36). Foreach SNP, missing data proportions for cases and controls, minorallele frequency, and exact tests for departures from Hardy–Weinberg expectations were calculated. In addition to allelic testof association, the additive genetic model was used as theprimary hypothesis of statistical inference. Haploview version4.0 (37) was used to estimate the linkage disequilibrium betweenmarkers and haplotype structures in different ethnicities. Com-bined p values were calculated from the per-ethnicity p values byusing the Fisher method. q values were calculated for differentethnicities by using the q value package (available from http://cran.r-project.org), which implements the q value extension ofthe false discovery rate (FDR) (38). q values for combinedresults were calculated by using Benjamini–Hochman formalism(39). q values correspond to the estimation of proportion of falsepositives among the results. Thus q values �0.05 signify �5% offalse positives and is taken as a measure of significance.

Stratification Analyses. To account for potential confoundingsubstructure or admixture in these samples, principal component

analyses (PCA) were performed (6) using a large set of SNPs(18,446, which were genotyped on these subjects as part of alarger effort). Four principal components were identified thatexplained a total of �60% of the observed genetic variation.These were used to identify individuals who were geneticallydistant from other samples in the same ethnic subset, and thuscapable of introducing admixture bias. A total of 378 controlsand 569 adult SLE and 80 childhood-SLE cases were identifiedin this fashion and removed from further analysis as detailed inTable S1. After removing these genetic outliers, duplicates, andrelated samples, 5,457 independent SLE cases and 4,939 controlsremained for analysis. We then performed genomic controlanalysis to calculate the inflation factor � using the same set ofSNPs. This yielded a � of 1.13 in European-American samples,1.03 in Hispanic Americans, 1.08 in African Americans, and 1.04in Asian Americans. Only the Hispanic sample required a PCAcorrection to remove the final source of confounding via ad-mixture to obtain the inflation factor given above.

Immunophenotyping of Mice. The different strains were examinedat the age of 4–6 months or 9–12 months, as indicated in thefigure legends. The anti-dsDNA, anti-ssDNA, and anti-histone/DNA ELISAs were carried out as described before (16–18).Upon sacrifice, kidneys were fixed, sectioned, and stained withhematoxylin and eosin, and periodic acid Schiff. At least 100glomeruli were examined per section by light microscopy forevidence of inflammation and/or tissue damage, and gradedusing the World Health Organizaion scale, as described before(18), in a blinded fashion. For flow cytometric analysis, spleno-cytes were depleted of red blood cells by using Tris ammoniumchloride, and prepared as single-cell suspensions. The antibodiesused to define surface markers on various leukocyte subsets havebeen described previously (16–18). The mean linear units on theforward scatter channel (FSC) were used as indicators of cellsize. DCs were cultured from the BM of different strains andwere characterized as detailed elsewhere (20). Statistical com-parisons were performed by using the Student t test (SigmaStat,Jandel Scientific).

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Fig. S1. Schematic representation of the significant haplotype block structure in an IRAK1 gene among adult-onset Asian American SLE cases and inchildhood-onset SLE in African Americans. Blocks connecting SNP pairs are shaded according to the strength of the linkage disequilibrium between the SNPs,from 0.0 (white) to 1.0 (bright red) as measured by the disequilibrium coefficient D�. Shown block structures are similar to those constructed for the other ethnicgroups.

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+/+ −/−

1520

2530

3540

Spl

een

CD

69/C

D4

T C

ells p = 0.92

IRAK1Fig. S2. Box and whisker plot of the CD69/CD4 T cell ratios in B6.Sle1z mice either sufficient (white box) or deficient (gray box) in IRAK1, examined at the ageof 4–6 months (n � 7–10).

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+/+ −/−

100

150

200

250

Spl

een

Wei

ght (

mg) p = 0.039

IRAK1

A

+/+ −/−

510

1520

Tota

l Spl

een

Cel

ls (

107 )

p = 0.082

IRAK1

B

+/+ −/−

0.5

1.5

2.5

Spl

een

CD

4 T

Cel

ls (

107 )

p = 0.099

IRAK1

C

+/+ −/−

24

68

Spl

een

B C

ells

(10

7 )

p = 0.066

IRAK1

D

Fig. S3. B6.Sle3z mice either sufficient (white boxes) or deficient (gray boxes) in IRAK1 (n � 7–10) were examined at the age of 4–6 months for spleen weight(A) and cellularity (B–D). Data shown are drawn from 2 independent experiments and are presented as box and whisker plots.

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+/+ −/−

100

150

200

MF

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D80

on

Mye

loid

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s

p = 0.06

IRAK1

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+/+ −/−

4550

5560

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80 o

n M

acro

phag

es

p = 0.0024

IRAK1

B10

0020

0030

0040

00

IRAK1 +/+IRAK1 −/−

p = 0.00012

p = 0.038

C

CD40 CD54 CD80 CD86 CD40 CD54 CD80 CD86Poly IC CpG

Mea

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Inte

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Fig. S4. B6.Sle3z mice either sufficient (white boxes) or deficient (gray boxes) in IRAK1 were examined at the age of 4–6 months for the activation status ofCD11b� CD11chi splenic myeloid DCs (A) and F4/80hi, CD11bhi/medium, CD11clow splenic macrophages (B) as assessed based on surface CD80 expression (n � 7). (C)Bone-marrow-derived DCs from B6.Sle3z (white boxes) and B6.Sle3z.IRAK1�/Y (gray boxes) were stimulated with TLR ligands [CpG oligonucleotide or poly(I�C]and assessed for surface expression of several activation/maturation markers (n � 8). Data shown are drawn from 2 independent experiments and are presentedas box and whisker plots.

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Table S1. Cohorts of SLE subjects and controls used in the study

Cohort All Excluded from analysis Included in analysis

Samples 11,045 1,015 10,030AA 1,883 126 1,757EA 6,237 732 5,505HA 1,111 89 1,022AsA 1,814 68 1,746

Controls 5,317 378 4,939AA 914 88 826AsA 869 16 853EA 3,114 235 2,879HA 266 35 231

Cases 6,106 649 5,457AA 969 38 931AsA 945 52 893EA 3,123 497 2,626HA 845 54 791

Adult cases 5,337 569 4,768AA 831 28 803AsA 793 27 766EA 2,912 462 2,450HA 603 44 559

Childhood cases 769 80 689AA 138 10 128AsA 152 25 127EA 211 35 176HA 242 10 232

To account for potential confounding substructure or admixture in these samples, principal componentanalyses (PCA) were performed using a large set of SNPs (18,446, which were genotyped on these subjects as partof a larger effort). Four principal components were identified that explained a total of �60% of the observedethnic variation. These were used to identify individuals who were genetically distant from other samples in thesame ethnic subset and thus capable of introducing admixture bias. A total of 378 controls and 569 adult SLE and80 childhood-SLE cases were identified in this fashion and removed from further analysis as detailed. Afterremoving these genetic outliers, duplicates, and related samples, 5,457 independent SLE cases and 4,939 controlsremained for further analysis.

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